Daily Archives: July 20, 2024

(July 18, 2024)Today, Attorney General Andrew Bailey of Missouri filed awrit of prohibition asking the Missouri Supreme Court to block theCircuit Court of St. Louis Countyfrom hearing the DNA evidence that proves Marcellus Williamss innocence. The circuit court had recentlyscheduled a hearing for August 21 to assess the clear and convincing evidence of actual innocence that led Prosecuting Attorney Wesley Bell to move to vacate Mr. Williamss wrongful conviction and death sentence. Despite overwhelming evidence of his innocence, Missouri has scheduled Mr. Williams for execution on September 24, 2024.

Below is a statement from Tricia Rojo Bushnell, one of Marcellus Williamss attorneys followed by case background:

Instead of using the offices time and resources to review the merits of Marcellus Williamss innocence claim, the Attorney General seeks to delay a court from even hearing the evidence until it is too late. Indeed, when the prosecutor filed a motion to vacate Mr. Williamss conviction in January, the Attorney General told the circuit court it was planning to file an oppositionbut waited four monthsuntil after an execution date was setto argue the court cannot hear the case. The evidence could have been heard in those four months. It can still be heard now. Instead of trying to prevent the circuit court from considering the DNA evidence that exonerates Mr. Williams, the Attorney General should join us in this truth-seeking process in Mr. Williamss case.

-Tricia Rojo Bushnell, attorney for Marcellus Williams

Background on Marcellus Williamss Innocence Case

DNA Evidence Proves Marcellus Williams is Innocent and the Prosecuting Attorney Seeks to Vacate His Wrongful Conviction, Yet Missouri has Scheduled His Execution for September 24

Marcellus Williams is scheduled to be executed on September 24 for a crime DNA proves he did not commit. The St. Louis County Prosecuting Attorney reviewed these DNA results and filed amotion to vacateMr. Williamss conviction because he believed the DNA results proved by clear and convincing evidence that Mr. Williams did not commit this crime. The circuit court has scheduled a hearing for August 21 to consider the exculpatory evidence and resolve the prosecuting attorneys motion.

A crime scene covered with forensic evidence contained no link to Mr. Williams

Mr. Williams has been seeking to prove his innocence throughout the 24 years he has spent on Missouris death row. On August 11, 1998, Felicia Gayle, a former reporter for theSt. Louis Post-Dispatch, was found stabbed to death in her home. The perpetrator left behind considerable forensic evidence, including fingerprints, a bloody shoeprint, hair, and trace DNA on the murder weapon, a knife from Ms. Gayles kitchen.None of this forensic evidence matches Mr. Williams.

A case built on snitch witnesses

The case against Mr. Williams turned on the testimony of two unreliable witnesses who were incentivized by promises of leniency in their own pending criminal cases and reward money. The investigation had gone cold when a jail inmate named Henry Cole, a man with a lengthy record, claimed that Mr. Williams confessed to him, while they were both locked up in jail, that he committed the murder. Cole directed police to Laura Asaro, a woman who had briefly dated Mr. Williams and had an extensive record of her own.

Both of these individuals were known fabricators; neither revealed any information that was not either included in media accounts about the case or already known to the police. Their statements were inconsistent with their own prior statements, with each others accounts, and with the crime scene evidence, and none of the information they provided could be independently verified. Aside from their testimony, the only evidence connecting Mr. Williams to the crime was a witness who said Mr. Williams sold him a laptop taken from the victims home, but the jury did not learn that Mr. Williams told the witness he had received the laptop from Laura Asaro.

New DNA testing confirms Mr. Williams is innocent yet no court has considered that evidence

As he has fought to prove his innocence, Mr. Williams has repeatedly faced imminent execution. To date, no court has given substantive consideration to the evidence exonerating him; the August 21 hearing will be the first time a court engages in that review.

Nine years ago, the Missouri Supreme Court stayed Mr. Williamss execution and appointed a special master to review DNA testing of potentially exculpatory evidence.This testing showed that Mr. Williams was not the source of male DNA found on the murder weapon.

However, in 2017, after the testing was completed but without conducting a hearing or making any findings based on the outcome of the testing, the appointed special master sent Mr. Williamss case back to the Missouri Supreme Court. That court, also without considering the DNA testing results, again scheduled Mr. Williamss execution.

Recognizing that the new evidence raised serious doubts about Mr. Williamss guilt, on August 22, 2017, mere hours before his execution and after his last meal, then-Governor Eric Greitens stayed the execution and convened a Board of Inquiry to investigate the case. Under Missouri law, the stay was to remain in place until the Board of Inquiry concluded its review and issued a formal report.

Yet in June 2023, while the Board of Inquirys review remained ongoing, Governor Mike Parson without warning or notice dissolved the Board without a report or recommendation from the Board. Missouri Attorney General Andrew Bailey then promptly sought a new execution date. Mr. Williams sued Governor Parson because the dissolution of the Board without a report or recommendation violated the law and Mr. Williamss constitutional rights. The Governor tried to dismiss the lawsuit, but a Cole County civil judge denied that request. The Governor then asked the Missouri Supreme Court to intervene. The Missouri Supreme Court agreed to do so and on June 4, 2024, it dismissed the lawsuit and immediately scheduled Mr. Williamss execution for September 24, 2024.

The St. Louis County Prosecuting Attorney has concluded that Mr. Williams is actually innocent and moved to vacate his conviction

After the exculpatory DNA evidence was brought to his attention, St. Louis County Prosecuting Attorney Wesley Bell appointed a special prosecutor to review Mr. Williamss case. The special prosecutor reviewed the findings of three independent DNA experts. All three concluded that Mr. Williamswas not the source of male DNA on the weapon,and therefore could not have killed Ms. Gayle. Mr. Williamss exclusion from the murder weapon is consistent with his exclusion from other forensic evidence collected from the crime scene including a bloody shoeprint and hairs found near the victims body.

Recognizing that new evidence suggests that Mr. Williams is actually innocent (p.3), in January 2024 the St. Louis County Prosecuting Attorney filed amotion to vacateMr. Williamss conviction. The motion explains: DNA evidence supporting a conclusion that Mr. Williams was not the individual who stabbed Ms. Gayle has never been considered by any court. This never-before-considered evidence, when paired with the relative paucity of other, credible evidence supporting guilt . . . casts inexorable doubt on Mr. Williamss conviction and sentence. (p.1) The Prosecuting Attorney urged the circuit court to begin the process of correcting this manifest injustice by [holding] a hearing on the newfound evidence and the integrity of Mr. Williamss conviction. (p.3)

The Missouri Attorney General continues its history of fighting innocence cases

Although the Prosecuting Attorneys motion remains pending and the law requires the circuit court to hold a hearing on it, as that court recognized, the Missouri Attorney General has taken the position that Mr. Williamss innocence does not matter, and the Missouri Supreme Court has scheduled his execution.

The Missouri Attorney Generals office has argued in other death penalty cases that even DNA evidence of innocence is not enough to stop an execution. In a2003 oral argument before the Missouri Supreme Court, Justice Laura Denvir Stith asked Assistant Attorney General Frank Jung, Are you suggesting even if we find that Mr. Amrine is actually innocent, he should be executed? That is correct, your honor, Jung replied. The Missouri Supreme Court ultimately disagreed, and Amrine was exonerated. But over 20 years later, the same arguments are still being made.

The Missouri Attorney Generals Office has opposed every innocence casefor the last 30 years,including every attempt made by a local prosecutorto overturn a conviction on the basis of innocence, as the St. Louis County Prosecuting Attorney is doing in Mr. Williamss case. In 2021 and 2023 Kevin Strickland and Lamar Johnson were exonerated despite the Attorney Generals attempts to thwart the prosecutors motions to vacate.

Incentivized informants are a leading cause of wrongful convictions

Jailhouse informant testimony like that leading to Mr. Williamss conviction is one of the leading contributing factors of wrongful convictions nationally, playing a role in15% of DNA exoneration cases.Eleven of the54individuals exonerated in Missouri were convicted with the use of informant testimony.

In capital cases, false testimony from incentivized witnesses is theleading cause of wrongful convictions, with informant testimony present in 49.5% of wrongful convictions since the mid-1970s (Source: Warden, R. 2005.The snitch system: How snitch testimony sent Randy Steidl and other innocent Americans to death row. Center on Wrongful Convictions.)

Racial bias contributed to Mr. Williamss wrongful conviction

Mr. Williams, a Black man, was wrongfully convicted of murdering a white woman. His jury was comprised of 11 white people and only one Black person. The prosecutor, whose institutional practice of racially discriminatory jury selection has been widelydocumented, successfully removed six of seven qualified Black prospective jurors with peremptory challenges. Arecent studyof 400 death-eligible cases in St. Louis County over a 27 year period also revealed racial disparity in the use of the death penalty based upon the race of the victim. Defendants were 3.5 times more likely to receive the death penalty if the victim was white, as in this case, compared to if the victim was Black.

Mr. Williams is devoutly religious and an accomplished poet

During his 24 years in prison, Mr. Williams has devoted much of his time to studying Islam and writing poetry. He serves as the imam for Muslim prisoners at Potosi Correctional Center and is known as Khaliifah. He has an exemplary prison record and is widely respected within the prison community and beyond.

Read more about Marcellus Williamss case atwww.savemarcellus.orgorwww.marcelluswilliams.org.

See original here:
Missouri AG Asks Missouri Supreme Court to Block Hearing on DNA Evidence Proving Marcellus Williams' Innocence - Innocence Project

Cas12 nucleases offer diverse yet complex opportunities for tracrRNA reprogramming

Type V CRISPR-Cas systems comprise numerous systems that involve tracrRNAs that could be amenable to tracrRNA engineering. Of the 14 subtypes of type V systems defined to-date, eight (associated with Cas12b, Cas12c, Cas12d, Cas12e, Cas12f1, Cas12g, Cas12k and Cas12l) exclusively rely on a tracrRNA for gRNA biogenesis (Fig.1b)5,6,11,34,35,36,37,38,39,40,41,42,43. For the remaining systems, the Cas12 nuclease directly recognizes and processes the transcribed repeat, as commonly demonstrated for Type V-A systems and its Cas12a nuclease7,11,44,45,46,47. Apart from collateral cleavage activity9,12,35,38,48, some or all of these DNA-targeting nucleases possess features distinct from more traditional Cas9 nucleases, such as pre-crRNA processing38,49,50, compact nucleases39,40,41,42,48,51, higher optimal temperatures9,36,52, crRNA-guided transposition37,53 and T-rich and C-rich PAM recognition6,11,43,49.

TracrRNA reprogramming involves engineering the anti-repeat region to hybridize with an RNA-of-interest while maintaining the essential sequence and structural features of the natural repeat/anti-repeat (R/AR) duplex recognized by the Cas nuclease. While Cas9-associated RNA duplexes form a simple 2540bp stem typically interrupted by a small bulge54,55,56, Cas12-associated RNA duplexes adopt distinct and more complicated conformations (Fig.1c). In addition to a long repeat/anti-repeat (LR/AR) stem often containing an intervening bulge, the reported duplexes associated with Cas12b, Cas12f1, Cas12g and Cas12l also possess a pseudoknot that includes a 57bp short repeat/anti-repeat (SR/AR) stem6,15,43,57,58,59,60. For Cas12e and Cas12k, the reported RNA duplexes possess a 3-bp triple helix formed by two portions of the anti-repeat sandwiching the repeat in addition to the bulged LR/AR stem35,53,61,62 (Fig.1c and Supplementary Fig.1). Finally, for Cas12c, the reported RNA duplexes form three 4-7bp disjoint R/AR stems49 (Fig.1c). Given the diversity and complexity of these RNA duplexes, we explored the extent to which the RNA duplexes associated with these diverse tracrRNAs can be reprogrammed for RNA detection.

We started with the Bacillus hisashii Cas12b (BhCas12b) due to the relative simplicity of its RNA duplex comprising a 30-bp LR/AR stem with an intervening bulge, and a 5-bp SR/AR duplex between the LR/AR and the guide36,57 (Fig.2a). We sought to investigate the reprogrammability of both stems using a Cas12 cleavage assay conducted with a cell-free transcription-translation (TXTL) system63. As part of the assay, purified BhCas12b protein, a gRNA-expressing plasmid and a plasmid encoding the PAM-flanked dsDNA target upstream of a GFP reporter construct were added to a reaction, and we monitored fluorescence over time. Cleavage of the reporter construct leads to loss of GFP expression through rapid degradation of the linearized DNA (Fig.2b).

a Predicted tracrRNA-crRNA structure for BhCas12b based on its ortholog BthCas12b (PDB: 5WTI57). R/AR, repeat/anti-repeat. b Setup to assess Rptr functionality using cell-free transcription-translation (TXTL). Expressed Cas-guide RNA complex recognizes and cuts its dsDNA target, causing the degradation of target-encoding GFP reporter plasmid and resulting in lower fluorescence compared to a non-targeting guide control. c 16-hourendpoint fluorescence measurements in TXTL when changing the long and short RNA duplexes. NT, non-targeting guide; T, targeting guide; T-br, targeting crRNA with bulge removed. d Setup to reprogram tracrRNAs to sense a Campylobactor jejuni transcript CJ8421_04975 mRNA. The guide and target components are added in the form of DNA constructs, while the purified BhCas12b protein is used. mRNA(mut), mRNA with point mutations in the predicted seed region of the guide. Rptr(scr-LA), Rptr with the long anti-repeat sequence scrambled; Rptr(scr-SA), Rptr with the short anti-repeat sequence scrambled; Rptr(scr-LA&SA), Rptr with both long and short anti-repeat sequence scrambled. e 16-hourendpoint fluorescence measurements in TXTL when assessing Rptr-guided sequence-specific dsDNA targeting. Nucleotide changes in R/AR stems in c and d are indicated by gray boxes. Bars and error bars in c, e represent the mean and standard deviation from three independently mixed TXTL reactions. Dots represent individual measurements. ***p<0.001 based on a one-sided Students t-test with unequal variance (n=3). Source data are provided as a Source Data file.

To interrogate the reprogrammability of the crRNA-tracrRNA duplex, we began with the intervening bulge in the crRNA-tracrRNA duplex followed by the two stems. Previous studies showed that a bulge in the LR/AR duplex is necessary to maintain the dsDNA targeting activity for SpyCas9 and Sth1Cas955. However, removing this bulge from the LR/AR associated with BhCas12b did not impinge on GFP silencing (Fig.2c), likely due to the bulge falling outside of the nuclease binding region57. Using the bulge-removed variant as a baseline to interrogate programmability of the LR/AR and SR/AR RNA stems (Fig.2c), we found that both stems could be reprogrammed without impinging on GFP silencing, whether changing the lower or upper portion of the LR/AR stem (cr1-4) or the SR/AR stem (cr5-6). The crRNA-tracrRNA duplex could be similarly reprogrammed for the Bacillus thermoamylovorans Cas12b (BthCas12b)57, as changes in the LR/AR and SR/AR of a fusedsingle-guide RNA (sgRNA) were well tolerated (Supplementary Fig.2). Therefore, tracrRNAs associated with Cas12b nucleases are highly amenable to reprogramming.

We next explored the extent to which the BhCas12b Rptr could be applied for RNA detection. We started with the CJ8421_04975 mRNA previously used to evaluate Rptrs associated with different Cas9 nucleases31. Two Rptrs hybridizing at different loci of CJ8421_04975 mRNA were designed based on rules derived from our mutational analysis of the LR/AR and SR/AR (Fig.2c and Supplementary Fig.3). Strong GFP silencing was observed for both BhRptr1 and BhRptr2 when compared with the non-targeting crRNA control, and both Rptrs combined with the mRNA exhibited similar performance as their equivalent crRNA/tracrRNA pairs (Fig.2d, e). Furthermore, dsDNA targeting occurred specifically through the predicted guide sequence, as mutating the predicted seed region or scrambling the tracrRNA anti-repeat (long, short or both) fully inhibited GFP silencing. The one exception was scrambling the anti-repeat of the tracrRNA associated with locus 1 of the mRNA, which still maintained substantial targeting activity likely due to shifted base pairing in the short duplex (Fig.2d, e). Overall, the Cas12b tracrRNA can be reprogrammed to link an RNA-of-interest to sequence-specific dsDNA targeting.

Building on the reprogramming of Cas12b tracrRNAs, we turned to the Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1) from Type V-F CRISPR-Cas systems39,64. While its crRNA-tracrRNA duplex parallels that associated with BhCas12b (Fig.3a), AsCas12f1 is a much smaller protein and forms a homodimer when binding a single crRNA-tracrRNA duplex. Using the TXTL assay with plasmid-expressed AsCas12f1 and an sgRNA, we found that the intervening bulge was also dispensable and the LR/AR and SR/AR could be fully reprogrammed without impinging on GFP silencing (Fig.3b). The base-pairing in the SR/AR was crucial for dsDNA targeting, as deletion of the SR portion of the SR/AR or mismatches in the SR/AR substantially inhibited GFP silencing (Fig.3b). We further demonstrated that three Rptrs designed to hybridize to different loci in the CJ8421_04975 mRNA yielded GFP silencing with comparable performance as their equivalent crRNA/tracrRNA counterparts in TXTL (Fig.3c, d). As before, mutating the seed region in the predicted guide or scrambling the tracrRNA anti-repeat (long, short or both) fully inhibited GFP silencing.

a AsCas12f1 sgRNA structure (PDB: 8J1264). See the detailed information in Supplementary Fig.1. b 16-hourendpoint fluorescence measurements in TXTL when reprogramming the long and short RNA duplexes in theAsCas12f1 sgRNA. NT, non-targeting crRNA; T, targeting crRNA. c Setup to detect the Campylobacter jejuni transcript CJ8421_04975 mRNA using AsCas12f1 Rptrs in TXTL. d 16-hourendpoint fluorescence measurements in TXTL for Rptr-guided sequence-specific dsDNA targeting by AsCas12f1 in TXTL. e Structure of DpbCas12e sgRNA (PDB: 6NY3)35. In the triple-helix region, a cis Hoogsteen/Watson-Crick base pair is formed between the U.A and a cis Watson-Crick/Watson-Crick base pair between the A-U. f 16-hourendpoint fluorescence measurements in TXTL when assessing the changeability of the LR/AR region. Dpb_T-br, targeting sgRNA with thebulge and G.U wobble base pair removed. g 16-hourendpoint fluorescence measurements in TXTL when changing the RNA triple-helix region. h 16-hourendpoint fluorescence measurements in TXTL when changing the RNA triple-helix surrounding region. i, Setup to detect the Campylobacter jejuni CJ8421_04975 mRNA using DpbCas12e Rptrs in TXTL. j, 16-hour endpoint fluorescence measurements for Rptr-guided sequence-specific dsDNA targeting by DpbCas12e in TXTL. Rptr(scr-dplx), Rptr with a scrambled anti-repeat sequence; Rptr(scr-tplx), Rptr with the RNA triple-helix sequence scrambled; Rptr(scr-d&tplx), Rptr with the RNA duplex and triple-helix sequence scrambled. Nucleotide changes in AsCas12f1 sgRNA and DpbCas12e sgRNA in b, f, g and h are indicated by gray boxes. Bars and error bars in b, d, f, g, h, and j represent the mean and standard deviation from three independently mixed TXTL reactions. Dots represent individual measurements. No error bars are shown when only two replicates were successfully collected. *: p<0.05. **: p<0.01. ***:p<0.001 based on a one-sided Students t-test with unequal variance (n=3). Source data are provided as a Source Data file.

Deviating from Cas12b and Cas12f1, Cas12e nucleases rely on crRNA-tracrRNA duplexes containing an RNA triple helix instead of a pseudoknot (Fig.3e and Supplementary Fig.1)35,53,61,62, posing an even greater challenge for RNA detection with PUMA. We selected the previously characterized Deltaproteobacteria Cas12e (DpbCas12e)35 and evaluated the reprogrammability of the bulged stem as well as the triple helix. Paralleling BhCas12b and AsCas12f1, removing the bulge and a G-U wobble pair in the context of an sgRNA did not compromise GFP silencing, and the stem could be readily reprogrammed (Fig.3f). Turning to the triple helix, this helix is formed by two separate tracts of three uracils at the 5 end of the tracrRNA sandwiching three adenosines in the repeat (Fig.3e and Supplementary Fig.1)35. A cis Hoogsteen/Watson-Crick base pair forms between the U.A and a cis Watson-Crick/Watson-Crick base pair forms between the A-U, assigning the triple helix to the cWW/cHW triple family65. RNA triple-helix motifs are found in various functional RNAs, such as telomerase RNAs66,67, riboswitches68 and long noncoding RNAs69,70. Despite its diversified distribution, the changeability of RNA triple helix in these biologically important RNAs has not been systematically investigated.

We reasoned that other RNA triple helices in the same cWW/cHW family might preserve dsDNA targeting by DpbCas12e. Using the RNA Base Triple Database as a reference71, we tested all nine RNA triple helix combinations reported in existing functional RNAs (s11-s18, and the native U.A-U), three expected to form a triple helix but not observed to-date (s19-21), and two not expected to form a triple helix and not observed to-date (s22-23, Fig.3g). Among the 14 tested triple-helix combinations, two (C.G-U_s11, C.G-C_s18) yielded GFP silencing comparable to that of the native U.A-U. In addition, installing the combination of U.A-U and C.G-C base triples in the RNA triple-helix region (s24-26) also yielded comparable GFP silencing. As expected, disrupting the RNA triple-helix conformation in one of thethree triples in the triple-helix region abolished dsDNA targeting (s27-32, Fig.3g), indicating a stringent triple-helix conformation required by DpbCas12e.

The RNA triple-helix region is surrounded by one C-G base pair at the 3 end and three unpaired nucleotides (AUC) at the 5 end of the repeat that may also represent necessary sequence or structural features (Fig.3h). For the C-G base pair, we found that introducing a C.A mismatch (s33) fully abolished silencing, while changing the base pair to U-A (s34) only modestly reduced GFP silencing (Fig.3h). For the AUC at the 5 end, mutating the C to A, G and U (s35-s38) resulted in similar or even improved GFP silencing (Fig.3h). The U could also be replaced with other nucleotides (s39-s41) without compromising activity (Fig.3h). Changing the A to C or G (s42, s44) was also well tolerated, while changing the A to U (s43) substantially inhibited GFP silencing (Fig.3h). Together, the RNA duplex and triple-helix regions are reprogrammable, albeit with less flexibility for the triple-helix region (Supplementary Fig.2).

Based on the insights from the systematic mutational analyses to DpbCas12e sgRNA, we designed three Rptrs targeting different loci in CJ8421_04975 mRNA (Fig.3i). We observed substantial GFP silencing for all three designed Rptrs, with comparable performance to that of their equivalent crRNA:tracrRNA pairs. As before, mutating the seed region in the predicted guide or scrambling the tracrRNA anti-repeat inhibited GFP silencing (Fig.3j). Overall, Rptrs could be extended to different Cas12 nucleases with varying tracrRNA-crRNA structures.

In contrast to Cas9, Cas12 non-specifically cleaves ssDNA upon target recognition, enabling signal amplification as part of CRISPR-based diagnostics2. We therefore reasoned that combining Rptrs, dsDNA targets, and ssDNA reporters would couple RNA detection by Cas12 to an amplified readable outputthe basis of PUMA. To assess the collateral effects of BhCas12b, we devised an in vitro collateral cleavage assay using purified BhCas12b protein, in vitro-transcribed sgRNAs or sensed RNAs and Rptrs, linear dsDNA targets and a ssDNA fluorophore-quencher reporter (Fig.4a). Upon recognition and cleavage of its dsDNA target, the nuclease non-specifically cleaves the fluorophore-quencher reporter, resulting in an increase in fluorescence.

a Schematic ofthe in vitro trans-cleavage assay. The assay includes purified aCas12 nuclease, anin vitro transcribed Rptr, and alinear dsDNA target. The Cas12-guide RNA ribonucleoprotein (RNP) recognizes and cleaves its dsDNA target, which triggers non-specific cleavage activity on ssDNA. Specifically, cleavage of the non-target strand (NTS) occurs before cleavage of the target strand (TS). F, fluorophore; Q, quencher. Yellow circle, PAM; b Impact of unprocessed or processed targets on in vitro trans-cleavage activity by BhCas12b. TS cleavage is the rate-limiting step. Red arrow, cleavage site. The cleavage site of TS is set as position 0. -, truncating the target sequence on NTS or TS. +, adding an overhang on NTS or TS. The PAM is in brown and the target is in blue. c, Direct detection of the full-length CJ8421_04975 mRNA by BhCas12b based on in vitro collateral cleavage activity. Yeast RNA is added in the same mass amount as the 1000nM sensed mRNA, and the best-performing dsDNA target NTS-6: TS-2 is used. d, Impact of unprocessed or processed targets on in vitro collateral cleavage activity by DpbCas12e. e Direct detection of the full-length CJ8421_04975 mRNA by DpbCas12e based on in vitro collateral cleavage activity. Yeast RNA is added in the same mass amount as the 1000nM sensed mRNA, and the best-performed dsDNA target NTS-8: TS-4 is used. 16h end-point values were used to make theplots in c and e. See Supplementary Figs.6b and 10a for the complete time courses. Curves in b and d represent the mean from two independent collateral assays. Bars anddots in c and e represent the mean andindividual measurements, respectively, from two independent collateral cleavage assays. Light blue bars indicate the limit-of-detection (LOD) conservatively estimated as the lowest concentration yielding an average fluorescence exceeding 50% of that of the no-RNA control. Source data are provided as a Source Data file.

We began with an sgRNA and a 334-bp linear dsDNA containing a 27-bp PAM-flanked target, with the resulting in vitro reaction conducted at 37C (Supplementary Fig.4a). We observed slight background fluorescence without the dsDNA target and monotonically increasing fluorescence with the dsDNA target that plateaued after 12hours (kobs=0.03h1, Supplementary Fig.4b, Supplementary Data1), in line with cis-cleavage of the dsDNA target triggering multi-turnover collateral cleavage of the fluorescent ssDNA reporter by BhCas12b. The activity exhibited by BhCas12b was weaker compared to that by FnCas12a (kobs=0.11h1), DpbCas12e (kobs=0.65h1) and LbCas12a (kobs=2.1h1) under equivalent conditions (Supplementary Fig.4b, Supplementary Data1). Elevating the temperature from 29 to 42C increased the reaction rate by 1.7-fold (kobs=0.19h1 at 42C) (Supplementary Fig.4c, Supplementary Data1), in line with higher temperatures yielding optimal cleavage activity for Cas12 nucleases9,36,52.

With an in vitro collateral cleavage assay in place, we next turned to the dsDNA target. Standard Cas12-based diagnostics have little control over the composition of the dsDNA target without extensive manipulations. In contrast, the dsDNA targetis provided as part of PUMA, granting complete control over its sequence, length, and chemistry. This control in turn could be leveraged to enhance the reaction. As a start, we evaluated the impact of using targets encoded on shorter linear DNA, perceivably by reducing the search time for the target sequence. In line with this rationale, we observed a 5.6-fold increase in collateral cleavage activity at 37C when shortening the dsDNA target length from 334bp (kobs=0.03h1) to 94bp (kobs=0.17h1). However, collateral cleavage activity decreased when shortening the DNA length to 60bp (kobs=0.12h1) or to 48bp (kobs=0.08h1) (Supplementary Fig.4d, Supplementary Data1). We also tested ssDNA targets, which exhibited at least a 2-fold increase in collateral activity than dsDNA targets of equivalent size (Supplementary Fig.5), in line with circumventing PAM recognition and DNA unwinding. We continued to use dsDNA targets though due to their more stringent and specific target recognition2.

The observed impact of DNA length on signal production led us to explore a distinct aspect of the dsDNA target: the extent of cleavage by Cas12 nucleases. Upon target recognition, Cas12 nicks the non-target strand followed by the target strand of the dsDNA target through the nucleases RuvC domain72,73, leading to a cleaved dsDNA target with a 5 overhang (Fig.4a). Complete cleavage of the dsDNA target normally precedes collateral cleavage72, with target strand cleavage posing the rate-limiting step36,73,74,75. We therefore hypothesized that using a dsDNA target with a processed target strand would increase the observed rate of collateral cleavage. In line with this hypothesis, a dsDNA target with a processed non-target strand yielded similar collateral cleavage rates to that of an unprocessed dsDNA target (kobs=0.06 - 0.16h1) at 37C for dsDNA lengths ranging between 45 and 94bp (Fig.4b, Supplementary Fig.6a, b, and Supplementary Data1). In contrast, a dsDNA target with a processed target strand yielded increased collateral cleavage rates (kobs up to 0.46h1 for NTS+55: TS+0), in line with target strand cleavage posing the rate-limiting step (Fig.4b and Supplementary Fig.6a, b). A similar collateral cleavage rate (kobs=0.44h1) was observed for a dsDNA target with both strands processed (NTS-6: TS+0) (Supplementary Fig.6a, b). Finally, trimming the target strand by two additional nts towards the PAM can further enhance the observed collateral cleavage activity (NTS-6: TS-2, kobs=0.56h1) (Supplementary Fig.6a, b). With conditions established for enhanced RNA detection using BhCas12b, we turned to detecting the full-length CJ8421_04975 mRNA using BhRptr4 in vitro. Under the optimal conditions with the shortest and processed dsDNA target (NTS-6:TS-2) at 42C, the sensed mRNA was detected at 1M in 45minutes and at 10nM in 16hours based on endpoint measurements compared to a no-RNA control (Fig.4c and Supplementary Fig.6c).

The optimized experimental setup with BhCas12b allowed us to assess how the ability of the sensed RNA and Rptr to hybridize impacts collateral cleavage activity. One potential factor is the formation of internal secondary structures that hinder hybridization. To test this factor directly, we introduced extensions to the 5 extensions to the ncrRNA associated with BhRptr4 (Supplementary Fig.7). The hairpins reduced collateral cleavage activity, with an internal hairpin inhibiting more strongly than a flanking hairpin. In the absence of these structures, introducing an annealing step did not enhance collateral cleavage activity (Supplementary Fig.8). Of note, collateral cleavage activity resulting from pairing of thepartial CJ8421_04975 mRNA fragment and BhRptr4 was higher than that obtained with the equivalent sgRNA, indicating that hybridization between a sensed RNA and Rptr is not necessarily a bottleneckto RNA detection.

With factors influencing RNA detection with BhCas12b established, we asked whether increasing the reaction temperature and truncating the dsDNA target also apply to DpbCas12e, which exhibited much higher collateral cleavage activities (Supplementary Fig.4). We tested DpbCas12e with DpbRptr1 against the full-length CJ8421_04975 mRNA along with different-sized dsDNA targets at different temperatures (Supplementary Fig.9ac). Similar to BhCas12b, DpbCas12e exhibited increased activity when elevating the temperature (kobs=0.07h1 at 29C, 0.43h1 at 37C and 0.67h1 at 42C) (Supplementary Fig.9b) and when shortening the length of the dsDNA target (kobs=0.43h1 for 331bp and 0.54h1 for 44bp) (Supplementary Fig.9c). Moreover, introducing a processed dsDNA target increased the collateral cleavage rate (for 91-bp target, kobs=0.46h1 for unprocessed strands, 0.78h1 for processed target strand) (Fig.4d and Supplementary Fig.10a). Finally, under the optimized conditions using the double-strand processed 38-bp dsDNA target at 42C, the sensed mRNA could be detected at a concentration of 1M in 9minutes and at a concentration of 0.1nM in 16hours (Fig.4e and Supplementary Fig.10b). Therefore, different tracrRNA-dependent Cas12 nucleases can be co-opted for direct, PAM-independent RNA detection in vitro.

When comparing collateral cleavage activities across Cas12 orthologs (Supplementary Fig.4b), we noticed that the LbCas12a-gRNA complex produced substantial fluorescence even in the absence of its corresponding dsDNA target, reaching approximately 70% of the levels seen when its dsDNA target is present after 16hours of incubation (Supplementary Fig.4b). A high background activity was also reported for AsCas12a in previous studies76,77. To assess the prevalence of this background activity, we tested four BhCas12b sgRNAs (#1-4, with the #4 guide used with other Cas12 orthologs in FigureS4b) using processed dsDNA targets (NTS-6: TS-2). Substantial DNA target-independent activity was observed for sgRNA#1 and #2, with comparable fluorescence levels to those with the dsDNA targets after 16hours (Fig.5a). Intriguingly, sgRNA#1 exhibited high cleavage activity (kobs=1.02-1.16h1) regardless of the presence or absence of the dsDNA target. This phenomenon was not isolated, as 5 out of 10 additional sgRNAs we tested exhibited DNA target-independent collateral activity higher than that of sgRNA#4 (Supplementary Fig.11).

a, Measured in vitro collateral cleavage activity with BhCas12b and an sgRNA with or without a dsDNA target. b, Measured in vitro collateral cleavage activity with BhCas12b and a Rptr and a dsDNA target with or without the sensed RNA. c, Sensitivity comparison between sgRNA and Rptr. In a-b, 37-bp NTS-2:TS-2 processed dsDNA targets were used for both sgRNA and Rptr. In c, 334-bp DNA fragments containing the core PAM-flanking target were used with the sgRNAs and 37-bp NTS-2:TS-2 processed dsDNA targets were used with the Rptrs. In a-c, sgRNA#1 and sgRNA#4 share the same guide sequences as those generated by Rptr#1 and Rptr#4, respectively. Dots represent individual measurements from two independent collateral cleavage assays. Bars represent the mean of the dots. In a-b, values represent fluorescence measurements after reaction times of 2hours and 16hours. In c, values represent fluorescence measurements after reaction times of 16hours. Light blue bars indicate the limit-of-detection (LOD) conservatively estimated as the lowest concentration yielding an average fluorescence exceeding 50% of that of the no-RNA control. Source data are provided as a Source Data file.

This DNA target-independent collateral activity would reduce the sensitivity of nucleic-acid detection, making it more challenging to identify low-concentration biomarkers. In contrast, we hypothesized that any background activities would be greatly reduced using Rptrs, as the guide RNA is principally formed only in the presence of the sensed RNA. Supporting this hypothesis, combining a sensed RNA and Rptr for BhCas12b drove collateral activity even in the absence of the DNA target (Supplementary Fig.12). In the absence of the sensed RNA, each Rptr alone resulted in endpoint fluorescence levels 3.5-fold to 29.9-fold lower than those observed in the presence of the corresponding sgRNA (Fig.5b). Based on this difference, we directly compared the sensitivity of BhCas12b detecting dsDNA with an sgRNA or detecting the equivalent RNA with a Rptr. The limit-of-detection was around 10-fold lower using a Rptr than an sgRNA for one site (#4), while RNA detection (with a Rptr) but not DNA detection (with an sgRNA) was possible at another site (#1) (Fig.5c). Thus, the sensitivity of nucleic-acid detection with Cas12 nucleases can be enhanced by detecting RNA with Rptrs rather than detecting DNA with sgRNAs, at least depending on the nuclease and detected sequence.

Given the enhanced sensitivity when detecting RNA with PUMA versus dsDNA traditionally detected with Cas12 nucleases, we asked how PUMA compares to the two standard CRISPR-based diagnostic approaches DETECTR for DNA detection with Cas122 and SHERLOCK for RNA detection with Cas134 (Supplementary Fig.13). We chose to detect three loci within the CJ8421_04975 DNA/mRNA and used sensitivity as the basis of comparison. BhCas12b was used for both PUMA and DETECTR to ensure a direct comparison, while PbuCas13b was used for SHERLOCK78. No pre-amplification was included to directly gauge the sensitivity associated with each Cas nuclease. Two of the sites lacked the PAM recognized by BhCas12b, in line with the requirement for a PAM inherent to DETECTR. Of the detected loci, the three approaches performed similarly, with the measured limit of detection either at 1nM or 10nM. Thus, PUMA can perform similarly to DETECTR and SHERLOCK, at least with the tested Cas nucleases, with PUMA targeting a broader range of sites than DETECTR.

One core feature of Rptrs is that base pairing with a sensed RNA is somewhat flexible, whereas the flanking guide sequence should direct dsDNA targeting that is highly sensitive to mismatches31. To exploit this feature, we applied Cas12 Rptrs to differentiate bacterial pathogens based on their 16S rRNA79,80. Differentiating pathogens can be important to select appropriate courses of treatment for different indications such as acute sepsis, urinary tract infections, or sexually transmitted diseases. Traditional CRISPR diagnostics based on collateral cleavage by Cas12 or Cas13-based diagnostics have taken strides in this direction81, with one example using multiple guide RNAs to detect different bacterial pathogens82. In contrast, with this core feature of Rptrs, a single Rtpr could be designed to pair next to a variable region of 16S rRNA indicating the genus. The variable region would then be matched to a dsDNA target, with its cleavage and subsequent collateral activity indicating which pathogen is present.

To determine how to best design the Rptr, we began by evaluating the specificity of the three different Cas12b homologs (BthCas12b, BhCas12b and AacCas12b), with the goal of identifying at least one homolog exhibiting high guide-target mismatch sensitivity. We assessed collateral cleavage activity of each homolog using a Rptr-sensed RNA encoding the same guide sequence along with dsDNA targets containing two consecutive mismatches sliding through the guide-target region (Fig.6a). Among the three orthologs, BthCas12b was the most sensitive to guide-target mismatches, especially in positions 5-12 proximal to the PAM in which the mismatches reduced collateral cleavage activity between 102-fold and 105-fold (Fig.6a). We also evaluated the extent to which BthCas12b accepts mismatches between the sensed RNA and the Rptr (Supplementary Fig.14). Mismatches in the long or short repeat consistently reduced but rarely eliminated activation of collateral cleavage activity even with four consecutive mismatches. This flexibility lends to pairing with conserved 16S rRNA regions with some variability, even if unintended RNA duplexes bound by Cas12 could be generated in the process. We therefore proceeded with BthCas12b and aimed for sequence differences to fall within the most sensitive positions of the target.

a Tolerance of guide-target mismatches for three different Cas12b orthologs based on in vitro collateral cleavage activity. The DNA target is the same as the one used in Fig.4B (BhsgRNA4 DNA target). Heat maps represent the mean kobs valuesfrom two independent collateral assays. See the kobs values in Supplementary Data1. b Setup to differentiate 16S rRNA from five different pathogens using only one universal BthCas12b Rptr binding to a conserved region of 16S rRNA. A truncated long anti-repeat of 18 nts instead of the usual 31 nts is used in the universal Rptr. In the alignment, sequences that match the E. coli 16S rRNA are in black, while those that do not match are shown in red. c Detection of pathogen 16S rRNAs with a universal Rptr and corresponding dsDNA targets based on in vitro collateral cleavage activity with BthCas12b. Partial 16S rRNA fragments of different pathogens at a final concentration of 100nM were used. Values represent 36-minute reaction times. Values in c represent the mean and standard deviation from two independent collateral assays. Source data are provided as a Source Data file.

Following this approach, we designed a single BthCas12b Rptr that hybridizes to a conserved region of bacterial 16S rRNA, with the downstream variable region serving as the guide sequence. We specifically focused on five common bacterial pathogens, E. coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes (Fig.6b), where the sequence differences fall within the region of mismatchsensitivity for BthCas12b. As before, a PAM did not need to appear within the sensed RNA, as this was encoded within the dsDNA targets. We then assessed collateral cleavage activity for each 16S rRNA fragment and each dsDNA target. The fragment was introduced at a final concentration of 100nM, reflecting the output of isothermal pre-amplification and in vitro transcription2. The presence of the 16S rRNA fragment from one specific pathogen triggers fluorescence release only when paired with its corresponding dsDNA target (Fig.6c and Supplementary Fig.15). We noticed 16S rRNA from L. monocytogenes also gave rise to substantial fluorescence when pairing with the dsDNA target from S. aureus, likely due to the high similarity between their 16S rRNA fragments with only three mismatches present outside of the seed region (Fig.6b, c and Supplementary Fig.15). Thus, specific detection of different pathogens based on 16S rRNA can be achieved via a single Rptr.

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TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases - Nature.com

Launched in 1990, a major goal of the Human Genome Project was to sequence the human genome as fully as possible. In 2003, project scientists unveiled a genome sequence that accounted for over 90% of the human genome as complete as possible for the technology of the time. Darryl Leja, NHGRI/Flickr hide caption

Launched in 1990, a major goal of the Human Genome Project was to sequence the human genome as fully as possible. In 2003, project scientists unveiled a genome sequence that accounted for over 90% of the human genome as complete as possible for the technology of the time.

The Human Genome Project was a massive undertaking that took more than a decade and billions of dollars to complete. For it, scientists collected DNA samples from anonymous volunteers who were told the final project would be a mosaic of DNA. Instead, over two-thirds of the DNA comes from one person: RP11. No one ever told him. Science journalist Ashley Smart talks to host Emily Kwong about his recent investigation into the decision to make RP11 the major donor and why unearthing this history matters to genetics today.

Read Ashley's full article in Undark Magazine here.

Curious about other biology stories? Email us at shortwave@npr.org.

Listen to Short Wave on Spotify and Apple Podcasts.

Listen to every episode of Short Wave sponsor-free and support our work at NPR by signing up for Short Wave+ at plus.npr.org/shortwave.

Today's episode was produced by Berly McCoy and edited by Rebecca Ramirez. They both checked the facts. Kwesi Lee was the audio engineer.

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The dubious consent question at the heart of the Human Genome Project : Short Wave - NPR

Thank you for reading D.C. Witness. Help us continue our mission into 2024.

By Sonia Vazquez , Lindsay Mutzman - July 18, 2024 Court | Daily Stories | Non-Fatal Shooting | Suspects | Victims |

A shooting defendant waived his right to independently test DNA evidence recovered from a crime scene before DC Superior Court Judge Robert Salerno on July 17.

Jonathan Dawkins, 42, is charged with assault with a dangerous weapon, possession of a firearm during a crime of violence, unlawful possession of a firearm by a prior convict, and threat to kidnap or injure a person, for his alleged involvement in a shooting that took place on the 700 block of G Street, SE, on April 24, 2023. No injuries were reported.

In court, the prosecution alerted the court that, despite recovering multiple items including cartridge casings, a car, and two phones, they would not be conducting any DNA testing.

Likewise, Dominique Winters, Dawkins defense attorney, alerted the court of his intent to waive his right to independent DNA testing.

Parties are slated to return on Dec. 20.

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Shooting Defendant Waives Right to Independent DNA Testing - DC Witness

Further information about the future of the controversial University of Cambridge project is expected to be shared later in 2024, Liam ODell can reveal.

Spectrum 10K was paused by researchers in September 2021 in order to carry out a consultation with the autism community, with the three-stage process concluding in May 2023.

However, the final report from independent consultants Hopkins Van Mil (HVM) on the findings of the consultation survey remains unpublished more than a year later.

Spectrum 10K academics failed to meet three 2023 deadlines for the documents publication, and in January an update from HVM told stakeholders to exercise patience.

It read: We are now in the process of collating our responses to the consultation feedback and recommendations to explain what we have learnt, the changes we will make in how we conduct research studies and how and why we want to continue to engage with the autism community in a meaningful way.

We are aware that our consultation participants, and the autism community more broadly, are awaiting this information and we would like to ask for your patience while we finish this process.

The full independent report and our response will be published on both the Hopkin Van Mil website and the Spectrum 10K website in due course.

Except questions are still being asked of the studys future, after Spectrum 10Ks website went offline in March without explanation.

In that same month, following a Freedom of Information request being made to the University of Cambridge by Liam ODell, the institution said the report would be published in the coming months.

This is despite the university initially telling this website the publication was imminent.

Now, in a statement on Friday, the Health Research Authority (HRA) pointed to further developments coming later this year.

A spokesperson said: We know that the Spectrum 10k team are taking the time to review the project following their consultation.

We expect to receive an update from the team regarding next steps for the study later this year.

The watchdog, which oversees the ethics of research studies, continues to monitor the studys progress after concluding an investigation into the project in May 2022.

The regulator said its favourable opinion of Spectrum 10K still stands, but added some of the issues raised as part of the complaints process could have been considered during the original review.

While no payment is expected or necessary to access this content, if you would like to support Liams independent journalism, you can send a tip viathe donation form on the right.

This report is the latest in his series The Spectrum 10K Files, with the full story of the controversial autism project explored in his upcoming non-fiction book, Selling Out the Spectrum to be published in late 2024.

Read the previous articlesonline now:

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Spectrum 10K: Update on autism DNA study expected later this year - Liam O'Dell

Allison Brocato last saw her sister alive on the afternoon of January 13, 1995. It was a Friday, and Catherine Edwards, Allisons 31-year-old identical twin, had just gotten off work at Price Elementary School, in Beaumont. On her way home, Edwards stopped to pick up her beagle, whom Brocato had been dogsitting. She lingered a few minutes to chat and to play with Brocatos infant daughter. She seemed kind of sad that day, Brocato would later recall. I think she had had a fight with an ex-boyfriend the night before.

Brocato and Edwards considered themselves best friends. After graduating from Lamar University, they both got jobs as public school teachers and moved into a modest town house in west Beaumont, where they lived together until Brocato got married. The sisters looked so alikea bit shy of five feet tall, slim, with pale skin and shy smilesthat their high school yearbook had mixed up their photos. Later, as teachers, they would occasionally fool their students by pretending to be each other.

The two women spoke again by phone that evening, as they usually did before bed. Edwards had decided to break off all contact with her ex-boyfriend. The sisters both planned to be at the familys traditional Saturday lunch the next day at their parents house, but Edwards never showed up. When her parents drove to her town house to check on her, they found their daughters body in the second-floor bathroom, slumped over the tub. She was nude from the waist down, and her wrists were handcuffed behind her back. Her father sounded frantic when he told Brocato what theyd found. He said, Your sisters dead, your sisters dead.

The brutal murder made front-page news for days in Beaumont, where Edwards was known as a dedicated teacher and a lifelong Presbyterian. She volunteered at St. Elizabeth hospital and served as a mentor for the I Have a Dream Program scholarship. Neighbors remembered her walking her dog each evening. She was loved by everybody, recalled Steve Thrower, a now retired investigator for the Jefferson County District Attorneys Office who was assigned to assist Beaumont Police Department detectives with the case. Great family. Never had any kind of criminal issue. Usually that really shrinks your suspect pool.

Crime scene investigators saw no signs of forced entry at the town house. Either Edwards had left her door unlocked or she had let her killer in. During the autopsy, a forensic pathologist took a vaginal swab that collected semen, which was also found on the comforter of her bed. The assailant appeared to have raped Edwards and drowned her in the bathtub. Detectives collected DNA samples from dozens of potential suspects, including Edwardss ex-boyfriend and several of her colleagues at Price Elementary. None matched the perpetrators DNA. Nor did the DNA match anyone in the Federal Bureau of Investigations recently created Combined DNA Index System, a national database of genetic profiles from convicted criminals, forensic evidence, and missing persons.

As the investigation dragged on month after month without any progress, the case slowly went cold. No arrest was made. For the next quarter century, the semen collected from the Edwards murder sat in a series of evidence-room freezers. Encoded in its DNA was the identity of the killer. But unless he left behind evidence at another crime scene, it seemed unlikely he would ever be caught.

Then, in 2020, a Texas scientist and entrepreneur named David Mittelman approached Beaumont police with an intriguing offer. Two years before, Mittelman had opened a private DNA lab in The Woodlands, an affluent master-planned community north of Houston. He named the company Othram, after the defensive wall of a fortress in J.R.R. Tolkiens The Lord of the Rings. (We serve a public-safety function, so theres a bit of a loose connection there, Mittelman explained.) The lab specializes in forensic genetic genealogy, a powerful new investigative method that combines whole-genome DNA sequencing with traditional genealogical research based on archival documents such as birth and death records. The technique first came to widespread public attention in 2018, when California detectives used it to identify Joseph James DeAngelo Jr. as the Golden State Killer, a serial murderer and rapist who had eluded police for decades.

When Beaumont detective Aaron Lewallen received Mittelmans offer to assist with any unsolved crimes, he immediately thought of the Catherine Edwards murder. This was Beaumonts most high-profile homicide, recalled Lewallen, a laconic 26-year veteran who had developed a specialty in cold cases. With the original detectives ruling out so many of the people who were close to her in her life, it had really become a whodunit. At Lewallens request, local officials agreed to pay Othram about $10,000 to conduct new DNA testing. A few weeks later, a FedEx courier dropped off a Styrofoam box at the labs headquarters. Inside, chilled by an ice pack, was a piece of floral-print fabric from Edwardss comforter and a vaginal swab from the posthumous rape kit.

Unless he was dead or in jail, the man who killed Edwards remained at large. Perhaps he was still in Beaumont. Perhaps he had moved away and started a new life. He had concealed his crime for nearly three decades; surely, he must have thought, the police had given up on the case. There was no way for him to know that in the early 2020s, a small group of detectives and scientists had dedicated themselves to unmasking him.

On a cool gray morning last fall, I drove thirty miles north from Houston to tour Othrams lab. The company rents space in a four-story building beside a lake in a heavily wooded office park off the aptly named Technology Forest Boulevard. David Mittelman greeted me in the elegantly furnished lobby. Wearing jeans and a rumpled black polo shirt, with disheveled hair and beard stubble, the 43-year-old scientist looked like he had been up all night. He was joined by his 45-year-old wife, Kristen, Othrams chief development officer. A lean, angular brunette who has moonlighted as a competitive bodybuilder, she was dressed in stylish athleisure wear. The couple met at Baylor College of Medicine, in Houston, where they both earned doctoratesDavid in molecular biophysics and Kristen in biochemistry.

Together the two have turned Othram into arguably the worlds leading forensic genetic genealogy lab. Over the past six years, the company has been publicly credited with helping to solve nearly 350 cases, including murders, rapes, and unidentified bodies. That number represents only a fraction of the thousands of cases it has actually assisted on, David told me, because some law enforcement officials prefer not to disclose Othrams role in their investigations. (For comparison, Virginia-based Parabon NanoLabs, another well-known company in the field, says it has assisted in more than 315 cases.) Othram has ongoing relationships with agencies around the world, including the Australian Federal Police, the Royal Canadian Mounted Police, and the Texas Rangers.

Kristen led me through two sets of key cardoperated double doors and down a long corridor lined with floor-to-ceiling glass walls. On the left was a series of research labs, where Othram scientists were exploring new ways to extract and analyze DNA. On the right were the forensic labs, where masked technicians in paper gowns and hairnets bent over hooded lab benches, working on crime scene evidence. In one room, a femur, stained dark from many decades underground, sat on a sheet of butcher paper. Because of privacy concerns, the Mittelmans couldnt reveal anything about the case other than that the bone belonged to a child.

Multiple times a day, packages containing crime scene evidenceblood, bones, hair, nail clippings, teetharrive at the lab. An Othram employee photographs the packages and then uploads the images to the companys online portal, where law enforcement agencies can keep tabs on their evidence. Each step of the process is documented to maintain a chain-of-custody record for use in subsequent legal proceedings. Especially with cold cases, tracking the circuitous route that evidence takesfrom a crime scene to a police property room to a forensic-testing lab and back to the property roomis critical. To discredit DNA evidence, defense attorneys will pounce on any potential contamination.

Othram technicians determine whether there is sufficient DNA to build a profile from the forensic material they receive. Unlike the cheek swabs used by medical testing companies, crime scene evidence often contains genetic samples from multiple individuals, and it can include plant or animal DNA. It also deteriorates over time. No matter how well the investigators try to keep it, its organic material, David explained. There are things that can developbacteria and other kinds of things. So that makes it tougher to read the data.

The lab ends up rejecting about a third of the evidence it receives. Better to wait until forensic technology improves, the Mittelmans believe, than make a futile bid to obtain a genetic profile. Each test uses up a portion of scarce crime scene DNA. Some forensic labs have destroyed entire samples without obtaining a profile. In medicine, you would never treat a patient if you had no idea whether it would help or not, Kristen said. If youre running the DNA on assays that dont work, youre consuming it, which means youre consuming someones last chance at justice.

Over time, Othram researchers have developed proprietary methods for obtaining profiles from ever-smaller amounts of DNA. In 2021 the lab established a new milestone by using just 120 picograms of DNAabout fifteen human cells worth of genetic materialto help identify the man who raped and murdered a fourteen-year-old Las Vegas girl, Stephanie Isaacson.

In the Edwards case, Othram technicians determined that the semen found on the vaginal swab had a high likelihood of yielding a strong DNA profile. They used a technique called differential extraction to distinguish the suspects sperm from Edwardss skin cells and other foreign material. Then they ran the sample through an Illumina NovaSeq 6000, a million-dollar whole-genome sequencer about the size of an office copier. Othrams custom software combed through the data for genetic markersunique DNA sequences that could be used to identify the suspect. To build a useful profile, Othram investigators need to find hundreds of thousands of such markers. In the Edwards case, they found more than half a million.

The genetic profile they developed was then uploaded, in the form of a digital text file, to a website called GEDmatch. Founded in 2010, the site maintains a database of more than 1.5 million profiles voluntarily submitted by users around the world, many of them hoping to find distant relatives. About 30 percent of those users have consented to law enforcement using their personal information to identify violent criminals. The website says it has helped solve more than four hundred cases.

The closest genetic match to the suspect in the database was a woman living in Louisiana. Based on their quantity of DNA in common, they appeared to be second cousins, which meant they shared a pair of great-grandparents. The woman might never have met the suspect or even known of his existence, but she had just become an unwitting genetic informer for the Beaumont Police Department. The information she provided was about to break the Catherine Edwards case wide open.

David Mittelmans path to Othram began in 1997, when he landed an internship at the University of Texas Southwestern Medical Center, in Dallas, working on the Human Genome Projectthe thirteen-year, $3 billion effort to map our entire genetic composition. At the time, he was a precocious student at Pearce High School, in nearby Richardson. It just seemed like an exciting opportunity to learn more about what all these pieces of genetic information meant, Mittelman said. Intuitively, I could sense that this was going to be a real driver in changing how society works.

He continued working on the project while attending college at UT Dallas. His job was to build and program robots that would automate various lab processes. Genomics in the nineties was very labor-intensive, he explained. Back then, it was a lot of human stuff and a little bit of computer stuff. That has kind of flipped now. After earning a bachelors degree in neuroscience from UT Dallas and his doctorate, David took a professorship at Virginia Tech, where he won a National Institutes of Health grant to research the use of genetic data for medical diagnosis and treatment.

One day in the early 2010s, a representative from the FBI visited Virginia Tech to speak about the role of DNA in criminal investigations. Humans share roughly 99.9 percent of our genetic material, but all of us have DNA sequencestypically along portions of the genome whose functions arent yet well understoodthat feature enough mutations to distinguish us as individuals. By the mid-1980s, scientists had identified the location of thirteen such sequences, also known as markers, which became known as a DNA fingerprint. (The number has since expanded to twenty markers.) Because the odds of any two unrelated individuals sharing the same DNA fingerprint are infinitesimal, this test has become the standard in international law enforcement. But the method works only if police have a suspect from whom to collect a DNA sample. If the culprit remains at large, as in the Catherine Edwards murder, the method is all but useless.

During the discussion, David realized that the field of forensics was woefully behind the times. Rather than relying on twenty markers, scientists could now use whole-genome sequencing to obtain hundreds of thousands. Sequencing an entire genome would give police detailed information about a suspects ethnic background, eye color, sex, and skin pigmentation. It would be like going from monochrome to Technicolor. The FBI guy asked me how much it would cost, and I said thirty thousand to forty thousand dollars, David recalled. He laughed at me and left. At that time, the economics didnt make any sense.

David set aside the idea and continued working on the medical side of genetics. In 2012 he cofounded Arpeggi, a tech start-up that created software to help physicians sift through the avalanche of data produced by whole-genome sequencing. Within a year the company was acquired by Gene by Gene, the Houston-based parent company of the popular genetic testing website FamilyTreeDNA. David resigned from Virginia Tech and moved back to Texas to become Gene by Genes chief scientific officer. This gave him an introduction to the world of direct-to-consumer DNA testing, which had exploded in popularity. Although he soon left to work at a series of other biomedical companies, his experience at Gene by Gene planted the seed for Othram.

The costs were coming down on sequencing, he said. I had learned a lot at FamilyTreeDNA about how people interconnect genetically. It was, like, maybe the economics are where they need to be. The technology has largely been solved for medical diagnostics. So how can we take this powerful technology to do something good in the world? And thats what shifted my mindset back to the problem of forensics. David soon discovered that there were hundreds of thousands of unsolved murders and tens of thousands of unidentified bodies in the United States alone. Crime labs nationwide were struggling to keep up with new investigations, let alone decades-old cold cases. The more David learned, the more outraged he became. People do not understand the magnitude of this problem, he told me.

In 2018, with $4 million in seed money led by a San Franciscobased investment fund, David set up shop in a one-story office building in The Woodlands, a five-minute drive from his house. Kristen initially declined to take a job at the company because it seemed like such a long shot. I told him that he had lost his mind, she said. Who is going to give you evidence? Were medical people. We have no policing background whatsoever. And youre going to build a forensic lab?

Genetic genealogy is such a young field that few regulations or accreditation standards exist. David knew that he would have to win the trust of law enforcement agencies unfamiliar with whole-genome sequencing. He would have to convince them that such testing was worth the initial price tag of $10,000 per DNA sample. Perhaps most important, he would need to prove that his work would stand up in a courtroom.

Brandon Bess, a bluff, plainspoken Texas Ranger who lives in the small town of Anahuac, midway between Beaumont and Houston, visited the lab shortly after it opened. It was David and, like, three other people working there at the time, and it was kind of a dump, he recalled. My first impression of David was that he looked like a mad scientist. He had on a T-shirt that was too small and looked like he hadnt slept in about three days. He had hair going all over the place, a ripped-up pair of jeans, and a pair of tennis shoes. But he was very focused. And he could talk. (David told me Besss impression was accurateearly on at Othram, he would often work for days with little sleep.)

Whole-genome sequencers such as the NovaSeq 6000 were built to analyze fresh DNA obtained from cheek swabs. Othram had to design lab processes capable of extracting data from damaged and degraded genetic material, then create software to analyze it. David hired engineers and molecular biologists with experience pulling DNA from inhospitable media, such as formaldehyde and paraffin, which are known to scramble genetic information. We developed a number of tools, both in the laboratory and on the computer side, that allowed us to get reproducible and predictable success from forensic samples, he said.

In 2019 Othram built a DNA profile that enabled investigators to identify a skeleton discovered in an Idaho cave in 1979 as the remains of Henry Loveless, a bootlegger and counterfeiter who was murdered in 1916. Then, in 2020, the lab received a flood of publicity after helping to solve the 1974 murder of Carla Walker, a seventeen-year-old Fort Worth high school student.

The company soon began announcing new successes on an almost weekly basis. It helped identify the perpetrators of the 1974 abduction and murder of a five-year-old girl from Montana; the 1977 rape and murder of a 77-year-old North Carolina woman; and the 1984 rape and murder of a nine-year-old girl in Canada. There were also many John and Jane Does it helped identify. A victim of a traffic accident on a Pennsylvania road in 1987. A corpse fished out of a lake in Washington State in 1994. A woman who mysteriously drowned in a Pecos hotel swimming pool in 1966.

To publicize Othrams work, Mittelman capitalized on Americas fascination with true crime. He became a regular speaker at CrimeCon, the popular annual true-crime conference, and he consulted on a 2021 episode of Law and Order: Special Victims Unit that featured a forensic genealogy lab inspired by Othram. The company hosts a private genetic database where users can upload their DNA data in hopes of helping solve a crime, and it has launched dozens of crowdfunding campaigns to finance work on cold cases around the country. On social media, Othram advertises its successful investigations and solicits donations.

Among the companys earliest large donors was Carla Davis, a self-taught genealogist from Mississippi who came across one of Othrams fund-raising campaigns on LinkedIn in late 2020. She gave tens of thousands of dollars that helped fund the identification of several sets of remains found in Mississippi between 1977 and 2020. Law enforcement agencies there are so underfunded, Davis recently told me over Zoom from her home in Dubai, where she and her husband have a real estate investment company. I was just trying to help give my home state the technology to solve their cases.

After Davis continued to lend Othram both financial support and genealogical expertise, the company hired her two years ago to lead its thirteen-employee genealogy team. Working remotely, Davis and her team have assisted in more than four hundred cases. Its really exciting to be part of this moment, she said. Were going to see so many more companies using this technology and more cases being solved. I think were not going to have cold cases in the future.

According to the business-research platform Crunchbase, over the past six years Othram has raised nearly $36 million in venture capital. Among its biggest investors is Gigafund, the Austin-based firm best known for its stake in several of Elon Musks companies, including the Boring Company, Neuralink, and SpaceX. Our investors are interested in the decade-long transformational shift in the way forensic genetic testing is done, David said. They understand its a long game. Like many tech start-ups, Othram has spent its early years burning through money in pursuit of its goals. In 2022 it moved to another office building in The Woodlandswith quadruple the lab space and additional room for a staff that now numbers more than sixty. It currently charges law enforcement agencies only enough to cover the incremental costs of an investigation, though David said that wont remain the case as economies of scale bring those costs down. He also plans to reach profitability by licensing the companys software and processes to other forensic labs.

Othram has attracted criticism for what some consider its sharp- elbowed business practices. In January, the company announced an exclusive partnership with FamilyTreeDNA, one of only two major databases that grant law enforcement agencies access to their profiles. (The other is GEDmatch, which is owned by Netherlands-based biotechnology company Qiagen.) Labs hoping to use FamilyTreeDNA to identify a suspect now have to use Othram software. In the wake of the announcement, the nonprofits DNA Doe Project and Intermountain Forensics temporarily stopped using FamilyTreeDNA. They were seeking additional clarification, so I personally got on the phone with them to talk them through the partnership, Mittelman said. Both organizations have since resumed working with FamilyTreeDNA.

Meanwhile, the field of genetic genealogy has come under fire from civil liberties groups concerned about the privacy of users who upload their DNA information to websites such as GEDmatch, and from bioethicists who worry about the dearth of federal regulation. DNA is valuableto governments, to bioscience companies, and to the policeand genetic databases, like anything else that lives online, can be hacked. Mittelman says he shares some of these concerns but points out that users submit their DNA to these sites voluntarily and can opt out of law enforcement searches.

Forensic genetic genealogy relies on a certain degree of ethical ambiguity. After a lab such as Othram identifies a potential culprit, detectives must then collect the suspects DNAoften surreptitiouslyin order to match it to DNA found at the crime scene. Usually this is done by taking items from a suspects garbage, as in the Golden State Killer case. According to long-standing legal precedent, police typically do not need a warrant to obtain evidence from garbage that has been left on the curb. So far, courts have ruled that the same holds true for the DNA in that trash.

Some experts, including Teneille Brown, a law professor at the University of Utah who has written about forensic genetic genealogy, argue that courts should declare the furtive seizing of someones DNA to be a violation of the Fourth Amendments prohibition against unreasonable searches and seizures. Obtaining someones entire genetic profile, after all, constitutes a significantly greater invasion of privacy than merely rummaging through their food scraps. DNA is not garbage, so we shouldnt be treating it like that, Brown told me. The American Civil Liberties Union has filed several amicus briefs opposing warrantless DNA collection.

Mittelman professes not to concern himself with such constitutional questions, deferring to the legal experts. For him and most law enforcement agencies, what matters is the ability to identify a victim or a suspect. At that Othram has proved remarkably successful. During one week in late May, the lab announced its role in solving six cases, including one murder and five unidentified bodies. And it is increasingly called upon to assist in active investigations. Earlier this year, Othram helped identify Victor Antonio Martinez-Hernandez, a fugitive from El Salvador, as a suspect in the 2023 murder of Maryland mother Rachel Morin. Martinez-Hernandez was arrested in June and charged with murder and rape.

With genetic-testing technology improving rapidly, the biggest remaining hurdle to solving cold cases may be money. These cases move at the speed of funding, David told me. In the United States, the largest single source of law enforcement funding is the federal government. This is where Kristen Mittelman comes in. After earning her doctorate, she worked as an intellectual-property specialist at Houston law firm Baker Botts before moving with David to Virginia Tech, where she became the universitys director of grants and contracts. In 2021 Kristen joined Othram, where she spends much of her time in Washington, D.C., lobbying Congress for money. He was struggling with funding, she told me. And I knew I was good at getting federal funding.

Thanks in part to Kristens efforts, Republican congressman Kelly Armstrong, of North Dakota, introduced the Carla Walker Act in 2022. Named after the Fort Worth teenager whose murder Othram helped solve, the bill would provide $20 million in grants to law enforcement agencies for forensic genetic genealogy investigations. The bill has yet to make it out of committee. Last August, U.S. senator John Cornyn traveled to The Woodlands to meet the Mittelmans and tour Othram. During a press conference at the lab, he promised to introduce a version of the Carla Walker Act in the Senate. This sort of technology is critical to solving crime and protecting public safety, he declared.

Kristen is working with Cornyns office to draft the proposed legislation, which she says will include reporting requirements to assess the work of forensic genetic genealogy labs. The Mittelmans worry about the damage an unscrupulous lab could do to the reputation of their nascent industry. There are so many companies out there selling the quickest way to make a profile with fewer markers, Kristen said. Because there are currently no metrics of success in forensics, people are taking shortcuts. This needs to be done predictably. You need to start collecting metrics, find technologies that work, and fund the implementation of those technologies.

After identifying the likely second cousin of Catherine Edwardss killer, Othram passed its findings to Beaumont detective Aaron Lewallen, who was now working with Brandon Bess, the Texas Ranger. Othram has an in-house genealogy team, but some law enforcement agencies use their own genealogist. In this case, Lewallen happened to know one willing to work for free: his wife.

Tina Lewallen, a detective in the auto crimes division of Beaumont Police Department, first got interested in genealogy in the nineties, to explore her family history. When direct-to-consumer DNA testing became available, she submitted a cheek swab to AncestryDNA. The results ruled out the French heritage her family had always claimed. That fascinated me, because DNA doesnt lie, Tina recently told me in her windowless Beaumont office, which was decorated with mug shots of suspected car thieves. A pair of rose-pink handcuffs, a gift from her husband, were clipped to her belt.

Othram provided Tina with a list of the suspects closest relatives on GEDmatch, along with how much DNA each shared and their likely familial relationship. The listed email address for one of the relatives, Paul Thomas LaPoint, led to his daughter-in-law, a professional genealogist named Shera LaPoint who lives in the small town of Bunkie, Louisiana, about eighty miles northwest of Baton Rouge. Shera had submitted Paul Thomass DNA to GEDmatch years before. Stunned to learn that her father-in-law was related to a suspected killer, she volunteered to help the Beaumont detectives with the case. After vetting her, the Lewallens brought Shera onto the team, forwarding the list they received from Othram.

It was a bunch of Cajun names, many of them people that I knew, because a lot of them are also into genealogy, Shera told me. Forensic genetic genealogy investigations start by identifying the most recent common ancestor between the suspect and their closest genetic match. In the Edwards case, the search was complicated by the suspects Cajun ancestry. Cajuns descend from a small colony of French Canadians who were expelled by the British in the mid-1700s and found refuge in Louisiana. Over the centuries, some members of the tight-knit Catholic community engaged in intermarriage with close relatives, such as cousins. As a result, performing genetic genealogy in Cajun families can be notoriously complex. Shera and her father-in-law, for instance, share thirteen ancestors. In a perfect world, when you look at DNA matches for a person who has tested on AncestryDNA or FamilyTreeDNA, you should be able to separate their four grandparents lines, Shera said. But when youre looking at Cajun DNA, its very difficult, because your maternal grandmother may be related to your paternal grandfather. So it makes it very difficult to find the common ancestor youre looking for.

Using Ancestry.com, which bills itself as the worlds largest genealogy site, Shera and Tina built a family tree for the presumed second cousin, going back in time until they identified her eight great-grandparents. Then they started working back down, following branches of the tree in search of a descendant who lived in Beaumont when Catherine Edwards was killed. But that effort led to a dead end. Because the family was Cajun, they realized, the presumed second cousin might actually be the suspects third cousin. Shera and Tina were forced to go back another generation, to the womans great-great-grandparents. They ended up with a family tree of more than 7,400 names.

To narrow the search, they asked Aaron Lewallen and Bess to request DNA samples from living members of these families. The detectives spent days driving around Texas and Louisiana, collecting dozens of cheek swabs. I thought it was going to be difficult to talk people out of their DNA, Aaron recalled. But youd be amazed how many people are out there interested in helping out. Theres a lot of true-crime buffs. Each swab was sent to Othram, which sequenced the DNA and uploaded it to GEDmatch. The results let Shera and Tina rule out entire family lines.

About three months into the investigation, Sheras research led to a husband and wife whod lived in Beaumont in the sixties. Birth records indicated that the couple had two sons who would have been about the same age as Edwards. Shera texted Tina and Aaron, who ran the names of the couples sons through a criminal background search. One of the brothers came up clean, but the other had a record. In 1981, Clayton Bernard Foreman had pleaded guilty to aggravated assault in Beaumont. I was like, holy shit, Aaron recalled. Its him.

According to the case file, Foreman, then a 21-year-old Nabisco salesman, had been driving through Beaumont when he saw a young woman whod had car trouble. Foreman stopped to offer her a ride, claiming to be a cop. He drove the woman to a secluded area, threatened to cut her throat with a knife, tied her hands behind her back with a belt, and raped her. About two weeks later, the traumatized woman went to the police. Foreman readily confessed, explaining that he had been out drinking and just got carried away. In exchange for pleading guilty to aggravated assault, he received three years of probation. Aaron soon learned that Foreman and Edwards were three years apart at Forest Park High School. Edwards and her twin sister, Allison, had even been bridesmaids at Foremans 1982 wedding.

An online search revealed that Foreman, now 61, was living with his fiance in a suburb of Columbus, Ohio, where he worked as an Uber driver. An officer from the local police department was dispatched to collect bags of trash from outside Foremans house. Several of the discarded items, including dental floss and plastic tableware, were sent to the Texas Department of Public Safety crime lab in Houston for testing. Foremans DNA was a clear match to that of the semen from Edwardss vaginal swab.

On April 29, 2021, Aaron and Bess flew to Ohio, where they met Foreman face-to-face at the local police station. A video recording of the interrogation shows an obese man with a receding hairline, thick glasses, and a pronounced East Texas accent. At first the detectives act as though they are merely seeking information about Edwardss murder. Foreman says he vaguely remembers her being a bridesmaid at his wedding but denies having any other contact with her or even knowing that she is dead. Only after being told that his semen had been found in her body does Foreman stop talking and ask to see a lawyer.

Aaron and Bess allowed Foreman to leave the interrogation room unimpeded. On his way out of the station, he was stopped and handcuffed by police officers. Foreman may have recognized the handcuffs. They had been sitting in an evidence room for nearly three decades, until Aaron and Bess received special approval to take them to Ohio. They were the same pair that had been found on Edwards in 1995.

After a three-year delay caused in part by the COVID-19 pandemic, the Clayton Foreman trial began on March 11 in downtown Beaumont. The visitors gallery was packed. Foreman sat calmly beside his attorney at the defense table, wearing an inscrutable expression. He had pleaded not guilty; if convicted, he would face a mandatory life sentence. Prosecutors decided not to seek the death penalty.

One of the first witnesses was Allison Brocato, now sixty years old with shoulder-length hair and matronly glasses. She spoke about her closeness with her twin sister, noting that she and her husband named their second daughter Catherine. After the murder, she said, the family was never the same. I think my parents died a little bit, too, when she did, Brocato told the jury, through tears. Neither lived to see the arrest of their daughters killer.

The prosecution also called Foremans former wife, Dianna Coe, who testified that she had learned about Foremans rape charge just three weeks before their wedding. When she confronted him, he claimed that the arrest was a big misunderstanding, and that the charges had been dropped. Coe, who was nineteen and had been dating Foreman for three years, decided to go ahead with the wedding. In 1984 she and Foreman had a son, who later attended Price Elementary while Edwards was teaching there.

One day Foreman confessed to his wife that in high school he felt protective of Edwards and Brocato. He thought they were so cute because they were twins, Coe testified. I didnt think anything of it at the time. Sometime around 1987, Coe discovered a briefcase in the trunk of Foremans car. Inside it was a gun, a pair of handcuffs, and pornographic material. The couple divorced in 1993. Years later, when Coe learned that Edwards had been murdered, she called her ex-husband to tell him the news. He had no feeling whatsoever, she recalled. It dumbfounded me.

David Mittelman took the stand on the third day of the trial. Wearing a white shirt and blue blazer, with a relatively clean-shaven face and hair that looked recently barbered, he patiently walked the jury through Othrams role in the Edwards case. Although the labs work has been used to convict dozens of murderers, this was Mittelmans first time to testify before a jury. He explained that Othram had found more than half a million genetic markers in the crime scene DNA, far in excess of what is necessary to produce a workable profile.

After seven days of testimony, the case went to the jury. It had taken nearly three decades to identify a suspect in the murder of Catherine Edwards, but it took the jury less than an hour to find Foreman guilty. He received an automatic life sentence and will be eligible for parole in thirty years, when he is 93. After the trial, one of the jurors gave an interview to a Beaumont TV station. He said it was the DNA evidence that convinced the jury of Foremans guilt: You cant deny that.

In the wake of the verdict, Mittelman expressed pride in having helped to resolve the three-decade-long investigation. Unsolved cases take an immense toll on families, he told me. Knowing that our technology has played even the smallest role in bringing both answers and then justice is profoundly moving.

The future is already here, novelist William Gibson once observed. Its just not very evenly distributed. David and Kristen Mittelman believe that forensic genetic genealogy will one day become as commonplace as fingerprint analysis. For now, this investigative tool still suffers technical limitations, including the relatively homogeneous geographical origin of the DNA profiles on GEDmatch and FamilyTreeDNA, most of which come from Australia, Europe, and North America. The majority of forensic genetic genealogy teams are located in the United States.

Considerable need exists for this technology. As crime spiked around the country during the COVID-19 pandemic, law enforcement agencies were tasked with investigating a tide of new offensesmore than one million violent crimes a year in the U.S.with a steady or shrinking number of officers. Every crime that isnt solved adds to the growing number of cold cases. In addition to hundreds of thousands of unsolved murders in the U.S., there are countless rape kits sitting in evidence rooms nationwide. For a variety of reasons, including insufficient funding, many have not received any kind of DNA testing.

Even when a kit does get tested, nearly always for the standard twenty genetic markers in a DNA fingerprint, it often doesnt match any of the profiles in the FBIs Combined DNA Index System. In those cases, police must either wait for the rapist to strike againleaving DNA at another crime sceneor pay for forensic genetic genealogy. Although costs are coming down, the method still runs about $10,000 per case, in addition to the expense of hiring a skilled genealogist. America spends more than $100 billion every year on law enforcement, but little of that is earmarked for forensic genetic genealogy. The amount of money it costs to investigate a case using traditional methods is absurd, David said. And the vast majority of it goes to salaries.

Thats why Othram sees government support as essential. Only Congress has the resources to fund genetic genealogy work at scale, and only Congress has the power to encourage local police departments to make use of itperhaps by threatening to withhold federal grants. (In the eighties, the federal government compelled states to raise the drinking age to 21 by indicating it would hold highway funding hostage.) But to be eligible for such funding, genetic genealogy will likely need to emerge from its Wild West era and embrace regulation. David and Kristen told me that Othram would welcome such a change. For a technology to be successful, it has to be predictable, Kristen said. Thats how medical testing works. Thats how almost everything works that is funded by the government.

Like many start-up founders, David balances his frustration at present circumstances with a supreme confidence about whats yet to come. He predicted that the technology pioneered at his modest lab in The Woodlands will one day become standard in police departments throughout the world. There was a sentiment, especially in the early years, that cases like Catherine Edwardss were remarkable one-off successesthings that are extraordinary, he told me. Were trying to go from the extraordinary to the ordinary.

This article originally appeared in the August 2024 issue ofTexas Monthlywith the headline Decoding a Killers DNA.Subscribe today.

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Inside the Texas Crime Lab Thats Cracked Hundreds of Cold Cases - Texas Monthly

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By Shea Carlberg - July 18, 2024 Daily Stories | Homicides | Shooting | Suspects | Victims |

A homicide defendant waived his right to independent testing of DNA evidence recovered from a crime scene, before DC Superior Court Judge Maribeth Raffinan on July 17.

Franklin Dorn, 44, is charged with second-degree murder while armed, possession of a firearm during a crime of violence, and unlawful possession of a firearm by a convict, for his alleged involvement in the shooting of 28-year-old Antonio Brown. The incident occurred on Aug. 6, 2023, on the 1200 block of North Capitol Street, NW.

At the hearing, the prosecution went over the physical evidence that was recovered from the scene and had tested, including a firearm, a magazine for the firearm, a bloodstained shirt allegedly worn during the incident and clothes recovered from Brown during the autopsy.

Parties are scheduled to reconvene Jan. 9.

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Homicide Defendant Waives Right to Independent Testing of DNA Evidence - DC Witness

STONY BROOK, NY / ACCESSWIRE / July 18, 2024 / Applied DNA Sciences, Inc. (NASDAQ:APDN) (Applied DNA), a leader in PCR-based DNA technologies, today announced that the U.S. Defense Logistics Agency (DLA) exercised the first one-year option period of a three-year base contract with two one-year option periods entered into in May 2021. The contract supports DLA's counterfeit mitigation initiatives and product verification and testing programs specific to FSC 5962 microcircuits. The maximal value of the indefinite delivery contract is $1.04 million over an up to five-year performance term. The option-year exercise continues Applied DNA's support of DLA's counterfeit mitigation program, which has been running since 2014.

"We are pleased to enable DLA to maintain program continuity in service of the nation's defense capabilities and further reinforce the application of our forensic DNA mark as a secure, high-resolution taggant to track provenance and ensure authenticity," said Judy Murrah, chief operating officer of Applied DNA.

About Applied DNA Sciences

Applied DNA Sciences is a biotechnology company developing technologies to produce and detect deoxyribonucleic acid ("DNA"). Using the polymerase chain reaction ("PCR") to enable both the production and detection of DNA, we operate in three primary business markets: (i) the enzymatic manufacture of synthetic DNA for use in the production of nucleic acid-based therapeutics and, through our recent acquisition of Spindle Biotech, Inc. ("Spindle"), the development and sale of a proprietary RNA polymerase ("RNAP") for use in the production of mRNA therapeutics; (ii) the detection of DNA and RNA in molecular diagnostics and genetic testing services; and (iii) the manufacture and detection of DNA for industrial supply chain security services.

Visit adnas.com for more information. Follow us on X and LinkedIn. Join our mailing list.

Forward-Looking Statements

The statements made by Applied DNA in this press release may be "forward-looking" in nature within the meaning of Section 27A of the Securities Act of 1933, Section 21E of the Securities Exchange Act of 1934 and the Private Securities Litigation Reform Act of 1995. Forward-looking statements describe Applied DNA's future plans, projections, strategies, and expectations, and are based on assumptions and involve a number of risks and uncertainties, many of which are beyond the control of Applied DNA. Actual results could differ materially from those projected due to its history of net losses, limited financial resources, the unknown future demand by the U.S. Defense Logistics Agency for its supply chain traceability service, and various other factors detailed from time to time in Applied DNA's SEC reports and filings, including its Annual Report on Form 10-K, as amended, filed on December 7, 2023 and Quarterly Report on Form 10-Q filed on February 8, 2024, and May 10, 2024, and other reports it files with the SEC, which are available at http://www.sec.gov. Applied DNA undertakes no obligation to update publicly any forward-looking statements to reflect new information, events, or circumstances after the date hereof or to reflect the occurrence of unanticipated events, unless otherwise required by law.

Contacts:

Applied DNA Sciences Investor Relations contact: Sanjay M. Hurry, 917-733-5573, [emailprotected] Web: http://www.adnas.com Twitter: @APDN

SOURCE: Applied DNA Sciences, Inc.

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U.S. Defense Logistics Agency Exercises 1-Year Option Period in Applied DNA Counterfeit Mitigation Contract - AccessWire

Thanks to Jack Lovegrove for the answer!

James - Can you extract DNA from fossils? Thanks, Phil. You've struck upon the fabulous field of ancient DNA. To help me answer your question. We put in a call to Jack Lovegrove at the Natural History Museum

Jack - For the majority of fossils, the answer is no. Traditionally, when we think of fossils, we are thinking of things that are many million years old. As the fossil is trapped in the sediment and the sediment is squished down and lithified turned to rock, pore waters get into the fossil and replace the original bone with different minerals, and the bone is remineralised, in effect, turned into stone. However, we now have been able to extract DNA from some of the most recent fossils. So the oldest DNA that's been extracted directly from a fossil comes from just over a million years ago, which was from a mammoth tooth that was found in the permafrost. The actual oldest DNA that's been extracted from anywhere comes not directly from a fossil, but from the sediment itself. And that's from a site in Greenland and about 2 million years old.

James - That's interesting. So some of that biological material preserves for millions of years. Life on Earth though, as we know, has been going on for tens and hundreds of millions of years. What's the limiting factor to DNA hanging around for longer, perhaps? Is it, as Paul suggests on our forum, DNA is strung together by means of phosphate ether bonds, which are not the most stable of chemical bonds.

Jack - Paul is right. I'd say there are two main limiting factors, and one of them is that DNA is not a very robust molecule compared to some other organic molecules and organic components of living things. So for example, there have been some controversial studies that have reported finding tiny traces of organic material from much older fossils. So even up to about 190 million years old. But even then, they're not talking about DNA. They're talking about very degraded remnants of collagen and maybe individual amino acids. It's been suggested, have been found in egg shells, but not DNA because it does fall apart. And the second reason that we don't find DNA is that most fossils have been remineralised. So all the organic material has been replaced with inorganic minerals.

James - Your specialty at the Natural History Museum is dinosaurs. It's bad news for those who are helping one day, we might uncover dinosaur DNA. Or is it?

Jack - In terms of non-bird dinosaurs, I would say it's very unlikely we're going to get DNA from their fossils. Sort of last of the non-bird dinosaurs, things like triceratops, that's about 66 million years ago. And then the stuff I work on, the earliest dinosaurs, you're looking at at least 240 million years ago. So sort of an order of magnitude out.

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Can we get DNA out of fossils? - The Naked Scientists

After 24 years, a significant breakthrough has occurred in the cold case of Sherry Holtz, who was found murdered in Sanford in December 1999. The Sanford Police Department has confirmed the arrest of Gary Durrance, 73, on Thursday, formerly Holtz's boyfriend, in connection with her death, modern DNA evidence gleaned from a knife found near the victim's body connects Durrance to the crime, according to ClickOrlando.

Police recounted how Holtz's body was discovered with evidence of a cut-throat, blunt force trauma, strangulation, and sexual battery, her body was found lying on a concrete slab in a wooded area behind a South Orlando Drive business, and while a sexual assault kit initially did not yield DNA evidence, a lock-blade knife recovered at the scene did contain human blood though it could not be tested at the time due to the small sample size, so the knife was preserved for over two decades, updated testing has now positively identified Durrance's DNA on the knife handle and Holtz's blood on the blade, "This was, theres no other way to describe it but a brutal homicide, OK? She was found laying on her back on a concrete slab approximately 20 feet into the wood line. Her clothing was pulled off, exposing most of her body," Sanford police strategic communications manager Bianca Gillett stated, as detailed by ClickOrlando.

Durrance and Holtz had a turbulent relationship, marked by repeatedly documented domestic violence incidents. According to interviews, the night before Holtz was last seen, an argument led to her expulsion from their shared residence, she was subsequently seen at a local bar and never returned.

Sanford Police Chief Cecil Smith addressed the gravity of the murder and the relief brought by the arrest saying, "24 years ago, Sherry Holtz was brutally murdered and left behind in the woods as if her life had no value. This was someone's mother, someone's daughter. Durrance will now face justice for the horrible and despicable things that he did." With Durrance now facing a second-degree homicide charge, he remains in the Seminole County Jail as reported by WESH.

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Arrest Made After 24 Years in Sanford Cold Case Murder, Modern DNA Evidence Links Suspect to 1999 Crime - Hoodline