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A microscopic look at dinosaur cartilage from roughly 75 million years ago has turned up a cluster of exquisitely-preserved cells, and they just might contain something rather familiar.

Dusting off the skulls of two juvenile duck-billed dinosaurs (Hypacrosaurus stebingeri), shelved after their discovery in the 1980s, researchers noticed a bunch of tiny circular structures at the back - some linked together, others standing apart, all of them frozen in time.

Looking closer, several of these circles contained a dark material reminiscent of a nucleus, and others held tangled coils resembling chromosomes.

"I couldn't believe it, my heart almost stopped beating," recalls vertebrate paleontologist Alida Bailleul from the Chinese Academy of Sciences.

In her shock, Bailleul told no one for several days, and even now, a decade later, the research team is cautious of saying too much.

Leading molecular paleontologist Mary Schweitzer, who joined the research after first seeing the skulls, has claimed in the past that Tyrannosaurus rex fossils can preserve protein cells for millions of years, and it was met with much controversy. Today, she chooses her words carefully.

"I'm not even willing to call it DNA because I'm cautious, and I don't want to overstate the results," Schweitzer told National Geographic.

"There is something in these cells that is chemically consistent with and responds like DNA."

If those hints turn into something more, it would mean genetic material can survive for much, much longer than we thought.

One of the many reasons the scenario of dinosaur resurrection in Jurassic Park is unbelievable, is because DNA is not thought to last that long - not even trapped in amber.

The half-life of this precious organic information has been calculated at about 521 years, so even under the best conditions, scientists predict it would only take about 5.3 millions years before the strands were completely unreadable.

Duck-billed dinosaurs were alive in Montana roughly 75 million years ago, which is 15 times longer than that; if their DNA is still around today, it would be astonishing.

Applying a couple of DNA stains to the fossilised cartilage cells, researchers now claim to have found several circular structures with potential.

Two of these examples were actually still linked, as though caught in the final stages of cell division.

(Bailleul et al., NSR, 2020)

All of the features observed were carefully summed up and compared to stained cartilage cells from emus, which showed similar intracellular contents, like proteins and nuclei.

To find out more, the team added antibodies of a dominant cartilage protein, known as Collagen II, to the cells. The way the organic matrix responded suggested a similar protein might be lurking inside.

"This immunological test supports the presence of remnants of original cartilaginous proteins in this dinosaur," Schweitzer explained.

But even if these ancient cartilage cells do hold remnants of intact dinosaur DNA, don't expect a real-life Jurassic Park to become any more viable.

In all likelihood, the information these cells might dish up would be too limited to sequence a whole genome. Currently, the oldest complete genome we've put together is only 700,000 years old.

But even a small dose of knowledge could tell us more than we ever knew about this long-extinct herbivorous dinosaur.

"These new exciting results add to growing evidence that cells and some of their biomolecules can persist in deep-time," Bailleul says.

"They suggest DNA can preserve for tens of millions of years, and we hope that this study will encourage scientists working on ancient DNA to push current limits and to use new methodology in order to reveal all the unknown molecular secrets that ancient tissues have."

This idea is still very much in its infancy, but it's true that recent studies have pointed towards a longer life for organic material than we thought possible.

In 2014, researchers in Sweden said they found fossilised nuclei and chromosomes in a 180 million-year-old fern. Last year, another study claimed to have found fossilised biomolecules in a now extinct creature over a half a billion years old.

And then, there's Schweitzer's own research on T. rex. While some critics in the past claim she mistook T. rex cells for bacteria or other forms of contamination, this time, she and her colleagues are adamant that's not the case.

"It is reasonable and logical to propose that fossil dinosaur bone contains contaminating microbial communities," they write in their new paper, "but the specific case that we present here... does not match the staining pattern of 'cell clusters' of contaminating biofilms."

Collagen II, for instance, is not produced in microbes, so the matrix shouldn't have reacted to that antibody. Plus, the comparisons to emu cells were done in a separate lab, so the risk of contamination from that source is also low.

Perhaps, the authors suggest, this ancient cartilage is simply better at preserving intracellular matter than bone. It's less porous, after all, and is exposed to less oxygen damage.

If they're right, there's a possibility this ancient tissue might be the carrier of unknown molecular secrets from long, long ago. The clue might be cartilage.

The study was published in the National Science Review.

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Ancient Dinosaur Remains Contain Something That Looks And Acts Shockingly Like DNA - ScienceAlert

Researchers in China and the US have found material in a dinosaur fossil that they claim looks like DNA. In a new paper in National Science Review, Alida Bailleul and colleagues report on their discovery of remarkably well-preserved cartilage from a Late Cretaceous dinosaur, Hypacrosaurus, from North America, dated at between 74 million and 80 million years old.

They highlight microstructures within the cartilage that they identify as nuclei and chromosomes from within its cells and also DNA. If accurate, this would be a hugely significant find. But can this report stand the scrutiny of a sceptical world? There are reasons to think not.

Co-author and supervisor of the new work, Mary Schweitzer of North Carolina State University, has previously reported similar findings from a variety of tissues from dinosaurs. There has been a strong negative reaction in the past to such reports, with other scientists claiming they couldnt replicate the results.

But the debates have been difficult because they hinge around particular specimens in particular laboratories. Researchers may be unable to replicate studies that claim to have found dinosaur biomolecules for all sorts of reasons. Schweitzer is quoted as saying that the sceptics can say what they want, but they need to come up with other explanations that fit the data better.

One such suggestion from a sceptic, Evan Saitta at the Field Museum in Chicago, is that the biomolecules that are being detected, including the tentatively suggested DNA, probably have nothing to do with dinosaurs or even with the Cretaceous period. They are more probably from modern microbes, as he showed in a recent paper.

Palaeontologists have been encountering similar problems for decades now. When Michael Crichton wrote about using dinosaur DNA preserved in amber to resurrect the prehistoric creatures in the original novel of Jurassic Park in 1990, he was drawing on real science.

A new technique called the polymerase chain reaction (PCR) method was allowing researchers to sequence and manipulate tiny quantities of DNA. Fact then followed fiction and a series of papers in 1992 and 1993 reported that scientists had been able to extract DNA from various fossils, including insects in amber and even from dinosaur bone preserved in sandstone.

Read more: Jurassic World: can we really resurrect a dinosaur?

But these suggestions of truly ancient DNA were rapidly debunked. What the researchers had been measuring was modern DNA contamination. The revolutionary properties of PCR were actually the downfall of these studies. It could clone such minute quantities of DNA that laboratory contamination, such as a molecule or two of modern insect DNA or a sneeze or flake of human dandruff, would provide convincing results.

Those studying what they believe to be ancient DNA are now careful to decontaminate their samples and work in antiseptic conditions. But we now also know that DNA molecules break down very easily and will typically survive only a few years. Hundred-year-old samples of DNA from museum specimens are massively fragmented and the breakdown of their molecular structure continues rapidly.

By using massive computing resources, DNA from fossils maybe 50,000 years old can be reconstructed from millions of short fragments. The oldest such samples are 700,000 years old a long way from the 66 million years of the last dinosaurs.

So, could it really be that the newly discovered microstructures in the dinosaur cartilage are ancient DNA? DNA molecules can be identified by staining them with propidium iodide. In their paper, Bailleul and colleagues note that they tested within the cells in the cartilage and identified stain responses. But they found no such responses in the general matrix of the bone, or presumably in surrounding sediment.

On the other hand, there are no tests in the paper to identify whether the reactive molecules are from a dinosaur or from a microbe. Its unlikely you could sequence the DNA to find out because its chains would have broken into such tiny fragments that youd probably be unable to extract useful information from them. If complete DNA could be extracted from the fossil, then it would much more likely be from a modern source than a creature that lived 80 million years ago.

Scientists are optimists. It would be fantastic if the authors of the paper are right and they have identified nucleic acid, or another biomolecule, from a dinosaur. Then the potential for cloning a long-extinct animal and a real-life Jurassic Park would be back in the frame. Unfortunately, we are probably a few steps away from an entirely convincing demonstration that these structures really are the cells of dinosaurs, or that the red-staining material is dinosaur DNA.

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Has dinosaur DNA been found? An expert explains what we really know - The Conversation UK

Marilynn Marchione, AP Chief Medical Writer Published 7:35 a.m. ET March 4, 2020 | Updated 1:46 p.m. ET March 4, 2020

Curious about your dog's family history, genes and medical risks? New DNA testing kits for pups will give you those answers. USA TODAY

Scientists say they have used the gene editing tool CRISPR inside someone's body for the first time, a new frontier for efforts to operate on DNA, the chemical code of life, to treat diseases.

A patient recently had it done at the Casey Eye Institute at Oregon Health & Science University in Portland for an inherited form of blindness, the companies that make the treatment announced Wednesday. They would not give details on the patient or when the surgery occurred.

It may take up to a month to see if it worked to restore vision. If the first few attempts seem safe, doctors plan to test it on 18 children and adults.

We literally have the potential to take people who are essentially blind and make them see, said Charles Albright, chief scientific officer at Editas Medicine, the Cambridge, Massachusetts-based company developing the treatment with Dublin-based Allergan. We think it could open up a whole new set of medicines to go in and change your DNA.

Charles Albright, executive vice president and chief scientific officer at Editas Medicine, a genome-editing company, in Cambridge, Mass., walks through the company's office on Jan. 8, 2020.(Photo: Rodrique Ngowi, AP)

Dr. Jason Comander, an eye surgeon at Massachusetts Eye and Ear in Boston, another hospital that plans to enroll patients in the study, said it marks a new era in medicine using a technology that makes editing DNA much easier and much more effective.

Doctors first tried in-the-body gene editing in 2017 for a different inherited disease using a tool called zinc fingers. Many scientists believe CRISPR is a much easier tool for locating and cutting DNA at a specific spot, so interest in the new research is very high.

The people in this study have Leber congenital amaurosis, caused by a gene mutation that keeps the body from making a protein needed to convert light into signals to the brain, which enables sight. They're often born with little vision and can lose even that within a few years.

Scientists can't treat it with standard gene therapy supplying a replacement gene because the one needed is too big to fit inside the disabled viruses that are used to ferry it into cells.

So they're aiming to edit, or delete the mutation by making two cuts on either side of it. The hope is that the ends of DNA will reconnect and allow the gene to work as it should.

It's done in an hourlong surgery under general anesthesia. Through a tube the width of a hair, doctors drip three drops of fluid containing the gene editing machinery just beneath the retina, the lining at the back of the eye that contains the light-sensing cells.

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"Once the cell is edited, its permanent and that cell will persist hopefully for the life of the patient," because these cells don't divide, said one study leader not involved in this first case, Dr. Eric Pierce at Massachusetts Eye and Ear.

Doctors think they need to fix a 10th to a third of the cells to restore vision. In animal tests, scientists were able to correct half of the cells with the treatment, Albright said.

The eye surgery itself poses little risk, doctors say. Infections and bleeding are relatively rare complications.

One of the biggest potential risks from gene editing is that CRISPR could make unintended changes in other genes, but the companies have done a lot to minimize that and to ensure that the treatment cuts only where it's intended to, Pierce said. He has consulted for Editas and helped test a gene therapy, Luxturna, that's sold for a different type of inherited blindness.

Some independent experts were optimistic about the new study.

The gene editing approach is really exciting. We need technology that will be able to deal with problems like these large genes, said Dr. Jean Bennett, a University of Pennsylvania researcher who helped test Luxturna at the Childrens Hospital of Philadelphia.

In one day, she had three calls from families seeking solutions to inherited blindness.

Its a terrible disease," she said. "Right now they have nothing.

Dr. Kiran Musunuru, another gene editing expert at the University of Pennsylvania, said the treatment seems likely to work, based on tests in human tissue, mice and monkeys.

The gene editing tool stays in the eye and does not travel to other parts of the body, so "if something goes wrong, the chance of harm is very small," he said. "It makes for a good first step for doing gene editing in the body.

Although the new study is the first to use CRISPR to edit a gene inside the body, another company, Sangamo Therapeutics, has been testing zinc finger gene editing to treat metabolic diseases.

Other scientists are using CRISPR to edit cells outside the body to try to treat cancer, sickle cell and some other diseases.

All of these studies have been done in the open, with government regulators' approval, unlike a Chinese scientist's work that brought international scorn in 2018. He Jiankui used CRISPR to edit embryos at the time of conception to try to make them resistant to infection with the AIDS virus. Changes to embryos' DNA can pass to future generations, unlike the work being done now in adults to treat diseases.

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Doctors altered a patient's DNA to treat blindness with controversial gene editing tool - USA TODAY

Abstract

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to GC base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

Base editing is a new genome-editing technology that enables the conversion of one base pair into another at a genomic locus of interest through the precise chemical modification of a target nucleotide (14). Base editors consist of two subunits: a catalytically impaired Cas9 subunit [Cas9 nickase (Cas9n)] that acts as a DNA binding module and a single-stranded DNA (ssDNA)specific editing enzyme subunit. The Cas9n binds to a preprogrammed genomic locus and opens the double-stranded DNA to expose a short stretch of ssDNA (5, 6). Subsequently, the ssDNA editing component carries out a chemical reaction to transform a target nucleobase into a noncanonical base (Fig. 1). Last, DNA replication or repair enzymes process the resulting mismatch into a canonical base pair to catalyze an overall base substitution reaction (1). Two types of base editors have been reported to date: cytosine base editors (CBEs), which rely on naturally occurring APOBEC enzymes (7, 8) to induce CG T:A mutations via a uracil intermediate (3), and adenosine base editors (ABEs), which use a modified version of the transfer RNA (tRNA) adenosine deaminase enzyme TadA to induce A:T GC mutations via an inosine intermediate (Fig. 1) (4). Both editors catalyze a deamination reaction at the target nucleobase and hence display considerable similarity between both the structure and mechanism of their enzymatic subunits.

(A) A schematic representation of base editing by ABEs. The ABEs studied as a part of the current work consist of a Cas9n fused to an evolved TadA* protein. The binding of Cas9n to the target genomic locus unwinds the DNA double helix and exposes a small region of ssDNA. TadA* acts on this ssDNA and deaminates adenine (A) to form inosine (I), which is subsequently converted to guanine (G) through DNA repair and replication. (B) Overall chemical reaction catalyzed by ABEs.

Since wild-type TadA (wtTadA) was unable to perform adenosine deamination chemistry on ssDNA, despite its structural similarity to several ssDNA modifying enzymes of the APOBEC family (9), the development of ABEs required extensive protein engineering and evolution efforts. Starting with the TadA enzyme from Escherichia coli (10), which deaminates the wobble position of tRNAArg, directed evolution (11) was used to achieve efficient editing on a ssDNA substrate. Seven rounds of directed evolution identified 14 point mutations that transformed TadA into ABE7.10, which displays both high editing efficiency and broad sequence compatibility (4).

Understanding the effects of the mutations identified in TadA during the initial rounds of evolution is critical, particularly considering that expansion of the current base editing arsenal would require similar protein engineering and evolution efforts. Evolving enzymes from zero initial activity is notoriously challenging, as it requires screening an enormous sequence space for a select few mutants that impart new activity upon the enzyme of interest; evolution projects that improve upon weak initial activity see higher success rates in contrast (12). Therefore, a molecular understanding of how the initial TadA mutations gave rise to nonzero DNA editing activity would be indispensable for aiding future evolution efforts.

While the wild-type TadA enzyme does not exhibit any enzymatic activity on ssDNA when fused to Cas9n, the first two rounds of identified mutations (Asp108Asn, Ala106Val, Asp147Tyr, and Glu155Val) are responsible for imparting experimentally detectable levels of DNA editing activity to TadA*-Cas9n (* indicates incorporation of mutations) (4). Atomistic understanding of these mutations that cause the onset of detectable activity is paramount to rationally guide the development of future base editors. In this study, we use a combination of molecular dynamics (MD) simulations complemented with experimental measurements to scrutinize the structural and functional implications of these initial mutations.

We initiated our investigations into the effects of the TadA mutations by studying their influence on the overall structure of the protein. As the first two generations of ABE complexes are composed of a TadA monomer fused to Cas9n (the wild-type enzyme acts on tRNA as a dimer), we furthermore focused our studies on monomeric TadA mutants. In addition, while the final generation ABE7.10 construct is composed of a wtTadA-TadA* dimer fused to Cas9n, we measured the A:T to GC base editing efficiency of the monomeric TadA7.10*-Cas9n construct at six different target As in human embryonic kidney (HEK) 293T cells and found no decrease in efficiency as compared to the dimer construct (Fig. 2A). These results suggest that the successive rounds of evolution performed on TadA have caused the enzyme to modify ssDNA as a monomer. Therefore, the TadA monomer is the most relevant model system with which to study the enzyme in the context of its interaction with ssDNA. Wild-type TadA consists of a five-stranded sheet core, with five helices wrapped around to form the active site. In addition, TadA displays a long-disordered loop (24 amino acids, residue numbers 118 to 142) that joins the 4 and 5 strands (Fig. 2, B and C) (10). We performed 500-ns all-atom MD simulations starting with the crystal structure of wild-type E. coli TadA (10) (TadA*0.1) to gain insights into the structural dynamics of the protein (see Materials and Methods). The simulations confirmed the highly fluxional nature of the 4-5 loop in the wild-type enzyme (Fig. 2, B and C). To observe the effects associated with the mutations on the structure and dynamics of TadA, we subjected the TadA*0.1 model to sequential mutations at residues 108, 106, and 147 and 155 to yield the TadA*1.1, TadA*1.2, and TadA*2.1 mutants, respectively. MD simulations of the four TadA* mutants reveal that the most substantial structural difference between TadA*0.1 and the higher-generation TadA*s occurs in this 4-5 loop. While TadA*0.1 displays high flexibility in this region, the first mutation (Asp108Asn) leads to restricted structural mobility of the loop, with the TadA*1.2 and TadA*2.1 following this same trend (Fig. 2B). The ubiquitous nature of this change is indicated by the reduced flexibility being observed for TadA*7.10, which harbors all the 14 mutation reported in the most evolved ABE protein (fig. S1A) (4).

(A) The A:T to GC base editing efficiency of the monomeric and dimeric ABE7.10 at six different target As in HEK293T cells. Values and error bars reflect the mean and SD of three independent biological replicates performed on different days. (B) Residue level flexibility of TadA* shown in terms of the root mean squared fluctuation (RMSF) of the C atoms of the peptide backbone. The 4-5 loop region is highlighted in blue, and each mutation is indicated with its respective location in the protein. (C) Representative clusters from the trajectory of TadA*0.1and TadA*2.1 superimposed on each other, with clusters color coded as indicated in (D). (E) Comparison of the secondary structure of zinc-dependent deaminases: TadA* and APOBECs. Helices and arrows denote the helices and strands, respectively. The 4-5 loop of interest in this study that interacts with the polynucleotide substrate is highlighted in both cases.

The suppression of the loop dynamics indicates that the replacement of Asp with Asn at residue number 108 of the protein is accompanied by a gain of structure. To quantify this effect in each TadA* mutant, we clustered all the conformations sampled by the 4-5 loop throughout the simulations into 10 structural groups representative of the conformational space. Comparison of these representative clusters reveals high variability among the loop conformations sampled by TadA*0.1 [average root mean square deviation (RMSD) = 1.75 ; table S1], while TadA*1.1 and higher display significantly smaller differences in the orientation of the 4-5 loop across the 10 representative structural groups (average RMSD = 0.74, 0.88, and 0.624 for TadA*1.1, TadA*1.2, and TadA*2.1, respectively; table S1). Our simulations also indicate that this decrease in the structural flexibility of the 4-5 loop of the TadA* mutants (Fig. 2) may be responsible for TadA* acting as a monomer to modify DNA, as it resembles the dynamics of the wtTadA dimer (fig. S2).

Next, we sought to understand the functional significance of the ABE mutations in the context of ssDNA binding. The lack of any reported structure of the entire ABE-DNA complex in the literature precludes the use of MD simulations on the entire ABE complex. Since the system of interest is only the evolving monomeric TadA enzyme and its ssDNA target and the TadA*-Cas9n complex has a size of more than 200 kDa, we reduced our molecular model to a series of TadA* mutants in complex with a 11-mer piece of ssDNA (5-GACTACAGACT-3). In lieu of including Cas9n and the full R-loop portions of the ABE complex, we have imposed constraints on the 5- and 3-terminal nucleotides of the ssDNA, keeping them 40 apart [based on Protein Data Bank (PDB) ID: 5y36 (13)] to maintain its R-loop conformation throughout the entirety of the simulations (Materials and Methods). We then carried out unbiased MD simulations in which we allowed each of the four TadA* mutants to interact with the constrained ssDNA for 500 ns and looked for changes in interactions between individual TadA* residues and the nucleic acid substrate among the four mutants. Experimentally, TadA*0.1 is not competent for base editing, but the three mutants (TadA*1.1, TadA*1.2, and TadA*2.1) are. We therefore specifically focused on identifying the interactions present in only TadA*1.1 and higher, with a particular emphasis on residue 108 (Asp in TadA*0.1 and Asn in all others), as this residue is responsible for imparting the enzyme with detectable base editing activity. To gain insights into the spatial extent of the interactions at play in the binding process, we projected the interactions between the target adenosine and its 5- and 3-adjacent bases (TAC) and the surrounding amino acids onto asteroid diagrams (Fig. 3, A to D). In these diagrams, we use a network representation in which these three nucleotides of the DNA are depicted as the central node and the TadA* residues are the peripheral nodes. As the typical donor atomdonor hydrogenacceptor atom distance is approximated to be 3.5 in globular proteins (14, 15), we defined the first interaction shell around the DNA as all amino acids within 4 of the three bases in the active site. The size of each node is proportional to the time individual residues spend within the 4- shell during the simulation. Hydrogen bonds between residues [defined as in the CPPTRAJ package (16, 17)] are depicted as arrows connecting the corresponding nodes, with the arrow size being proportional to the hydrogen-bond strength, which is defined as the number of times that the specific hydrogen bond is established (Fig. 3, A to D, and fig. S3). In the crystal structure of wild-type TadA in complex with its tRNA substrate [PDB ID: 2b3j (18)], Asp108 makes a hydrogen bond with the 2-OH group of the 5 flanking base. In contrast, when complexed with ssDNA, which lacks this hydrogen-bond donor, the repulsive electrostatic interactions between the negatively charged Asp108 and the phosphate backbone of the DNA favors a conformation in which Asp108 points toward the active site zinc ion (Fig. 3E). Mutating Asp108 to Asn neutralizes this repulsive interaction and causes the residue to flip into a more energetically favorable conformation in which it faces the DNA substrate and interacts with the base 5 to the target adenosine. This conformational change allows Asn108 to form a hydrogen bond with the carbonyl at position 2 of the 5 nucleobase when this base is a pyrimidine (Fig. 3F). This interaction between Asn108 and the 5 pyrimidine may explain the earlier generation ABEs strict sequence preference for a pyrimidine at this position. As subsequent mutations are introduced into TadA*, this hydrogen bond is progressively strengthened, and in the TadA*2.1 mutant, a second hydrogen bond forms between Asn108 and the phosphate backbone (Fig. 3F). We attribute this conformational switch to the hydrogen-bond donor nature of Asn as opposed to the hydrogen-bond acceptor nature of the negatively charged Asp. The Asp147Tyr and Glu155Val mutations, which are introduced as TadA*1.2 becomes TadA*2.1, do not lie within the first interaction shell, but rather cause structural rearrangements to the protein that strengthen the interactions between Lys110, Phe148, and Phe149 and the ssDNA and cause Arg153 to become a double donor (Figs. 3, D and F, and 4A).

Asteroid plots for (A) TadA*0.1-ssDNA, (B) TadA*1.1-ssDNA, (C) TadA*1.2-ssDNA, and (D) TadA*2.1-ssDNA complexes. Details of the conformational change of residue 108 when it is mutated from Asp (TadA*0.1) (E) to Asn (TadA*1.1and later) (F).

(A) The first and second interaction shell around the three nucleotides in the active site of the TadA*2.1-ssDNA complex. The size of the node corresponds to the time in which the amino acid resides in the first/second shell. First round mutations are red, and second round mutations are orange. (B) Structural overlay of average structure of TadA*0.1-ssDNA and TadA*2.1-ssDNA complexes. This 5 helix has been highlighted to depict its overall movement toward the active site upon Asp147Tyr mutation.

To better understand the effects of the second-generation mutations (Asp147Tyr and Glu155Val), which are located outside of the 4- primary interaction shell, we expanded our analysis of the TadA*-ssDNA simulations to include the secondary interaction shell, which encompasses all residues within 4 of the primary interaction shell residues. Analogous to Fig. 3, individual residues are represented by nodes whose sizes are proportional to the number of frames in the MD trajectory in which the residue lies within the specific shell, with hydrogen bonds between residues depicted as arrows between the interacting nodes, and the arrow size being proportional to the hydrogen-bond strength (Fig. 4A). We found that while Asp147Tyr and Glu155Val do not belong to the primary interaction shell, they do influence the manner in which the primary shell residues interact with the ssDNA. Mutation of Asp147 to Tyr abrogates a salt bridge between itself and Arg150 (primary interaction shell) that exists in TadA*0.1 (Fig. 2A). This lost interaction results in the movement of the entire 5 helix toward the active site (Fig. 4B), causing residues 150 to 153 to considerably spend more time within the primary interaction shell and increasing the strength of the hydrogen bonds between residues 148, 149, and 153 and the ssDNA (Figs. 3, A and D and 4A, and fig. S4A). Moreover, the Asp147Tyr and Glu155Val mutations, which convert negatively charged residues into neutral amino acids in the 5 helix, increase the positive charge density on the surface of the TadA*2.1 (fig. S4, B and C), potentially enhancing the electrostatic interactions of the TadA* with the negatively charged ssDNA.

After qualitatively observing the interactions between the TadA* residues and the ssDNA, we sought to quantify the thermodynamics of ssDNA binding by the four TadA* mutants. To this end, we performed umbrella sampling simulations to determine the potential of mean force (PMF) associated with the binding process. In this analysis, the PMF is calculated as a function of the relative distance between the centers of mass of the ssDNA and the TadA* mutants (, collective variable), which we vary from 10 to 30 (Fig. 5, A and B). The PMF profile describing the binding of TadA*0.1 to ssDNA has a minimum at = 20 and shows a relatively small (17 kcal/mol) dissociation energy as the ssDNA is moved away from the protein to = 30 . Once the Asp108Asn mutation in TadA*1.1 has been introduced, the PMF minimum slightly shifts toward the active site (to = 18 ), and we observe the free energy of binding increase to 42 kcal/mol as is increased to 30 (Fig. 5C). The PMF profiles calculated for the binding of TadA*1.2 and TadA*2.1 to ssDNA maintain this increased slope for larger than 20 , implying that the single Asn108 mutation is effectively responsible for increasing the binding free energy by 20 kcal/mol. For values less than 20 , the PMF profiles become sequentially more repulsive with subsequent generations, demonstrating a tighter binding of the ssDNA to the TadA*. We repeated the binding free energy calculations with a different sequence of ssDNA that lacks 5-pyrimidine (5-GTCAAGAAAC-3) and again observed mutation-dependent TadA*-ssDNA binding but to a lesser extent of only 10 kcal/mol for this substrate (fig. S5). These results are in agreement with experimental observations that these early generation ABE mutants had a strong preference for YAC (Y = pyrimidine) sequence motifs. These findings highlight the importance of the Asp108Asn mutation in imparting functional promiscuity to the TadA* enzyme toward ssDNA editing (4) through an increase in the free energy of binding. While the binding affinity is not a direct measure of the editing efficiency, our analyses of the TadA*-ssDNA complexes demonstrate that the initial Asp108Asn mutation, which plays a critical role in the onset of the DNA editing capability of the ABEs, leads to increased binding between the TadA* and the ssDNA substrate. We speculate that higher-generation mutations take advantage of this increased binding to improve the kinetics of base editing and broaden the substrate sequence scope.

(A) List of early generation mutations in TadA that were analyzed in this study. (B) The model of the TadA*-ssDNA complex simulated to determine the binding energy profile of the TadA* mutants. The binding-unbinding event was monitored using the collective variable () defined as the distance between the center of mass of the protein and DNA. (C) The free-energy profile of binding of the ssDNA to various TadA*s. For each TadA*-ssDNA complex, the average PMF is shown as a function of the continuously changing values. The shaded regions around individual curves depict the standard deviation for four independent replicates of the umbrella sampling simulations. The error bars associated with the mean PMFs indicate the error calculated using the block-averaging method.

To confirm the crucial role played by Asn108 in ssDNA editing by ABE, we subjected the higher generation of TadA* mutants (TadA*1.2 and TadA*2.1) to reversion analysis of this mutation. Specifically, by mutating Asn108 back to Asp108 in both TadA*1.2 and TadA*2.1, we generated two new TadA mutants, TadA*1.2(N108D) and TadA*2.1(N108D), respectively (Fig. 6A).

(A) ABE constructs created by reverting the Asp108Asn mutation in the higher generation ABEs. (B) RMSF of the C atoms of the TadA*1.2(N108D) and TadA*2.1(N108D) enzymes. (C) The free-energy profile of binding of the hybrid TadA*s to ssDNA. The shaded regions around individual curves depict the SD for four independent replicates of the umbrella sampling calculations. The error bars associated with the mean PMFs indicate the error calculated using block-averaging method. (D and E) A:T to GC base editing efficiencies in HEK293T cells by the various ABEs at six different target As. Fold-decrease values upon reversion analysis of the Asp108Asn mutation are indicated above the bars. Values and error bars reflect the mean and SD of three independent biological replicates performed on different days.

To disentangle the structural contribution of Ala106Val, Asp147Tyr, and Glu155Val from that of Asp108Asn, we monitored the structural flexibility of TadA*1.2(N108D) and TadA*2.1(N108D) (Fig. 6B). We observed the maintenance of the 4-5 loop stabilization, suggesting that the Ala106Val mutation is also sufficient to induce this change in structural flexibility (fig. S6). We also observed a slight increase in the flexibility of the 2 helix due to this mutation, but upon introduction of the round two mutations, this is lost. To complement these structural studies, we also characterized the binding free energy in the TadA*1.2(N108D)-ssDNA and TadA*2.1(N108D)-ssDNA complexes. Unlike the structural results and despite having respectively one and three mutations that were experimentally found to be favorable for ssDNA editing, TadA*1.2(N108D) and TadA*2.1(N108D) produced PMF profiles that are significantly different from those of their parent mutants (Fig. 6C). In particular, both PMFs closely follow the corresponding profile obtained for TadA*0.1 for values larger than 20 yet are considerably more repulsive for values smaller than 20 . We performed analogous reversion analysis for the TadA*7.10 (which contains all 14 identified mutations) and observed qualitatively similar trends for the TadA*7.10(N108D) (fig. S7A).

These differences demonstrate weaker binding between the ssDNA and ABE mutants lacking the Asn108 mutation. To confirm our computational results, we generated the ABE1.2(N108D) and ABE2.1(N108D) constructs and experimentally measured their respective A:T to GC base editing efficiencies using high-throughput sequencing (HTS) alongside ABE0.1, ABE1.2, and ABE2.1 in HEK293T cells at six different targets. Reversion of Asn108 mutation to Asp led to an average decrease in the A:T to GC base editing efficiency of 22-fold (ranging from 6.5- to 42-fold) and 70-fold (ranging from 22.6- to 126-fold) for ABE1.2 and ABE2.1, respectively (Fig. 6D). It is notable that even the presence of all three Ala106Val, Asp147Tyr, and Glu155Val mutations was not sufficient to restore editing activity with Asp at position 108; both ABE1.2(N108D) and ABE2.1(N108D) induced average A:T to GC base editing efficiencies of 0.36 and 0.29% across all six editable As, as compared to 3.6 and 16.8% for their respective parental mutants. Reversion of the Asn108 mutation in the ABE7.10 background displayed a similar trend. Replacement of Asn108 with Asp in both monomeric and dimeric ABE7.10 decreased the A:T to GC base editing efficiency by an average factor of 146-fold (ranging from 67- to 176-fold) and 123-fold (ranging from 35-fold to 259-fold), respectively (Fig. 6E). This indicates that the presence of 13 higher generation mutations, independently of being installed in the monomeric or dimeric construct, cannot compensate for the loss of the Asn108 mutation. The importance of residue Asn108 in ABE7.10 was also recognized in the experimental study by Rees et al. (19), where radical substitutions of Asn108 with Phe, Trp, and Met were found to result in complete abolishment of any DNA editing activity at all target adenosines except when the target nucleobase was at position 5 within the protospacer. However, conservative substitutions of Asn108 with Gln, and Lys, resulted in decreased DNA editing efficiencies for these mutants, albeit in a sequence-dependent manner and to a much smaller extent than the substitution with Asp (19). The results of this study thus provide further support of the hydrogen-bonding analysis presented here, which emphasizes the requirement of a positive charge density, either in the form of a hydrogen-bond donor as Asn (Fig. 4) or Gln (19) or a positively charged residue as Lys (19) for enabling the ssDNA activity of TadA*. Collectively, these data demonstrate the drastic effects a single atom substitution (from N to O) can have on protein function and highlight the complexity of protein sequence-structure-function relationships.

Enhancing our understanding of how an enzymes sequence influences its function will help increase the success of future directed evolution projects. Although the mutations discovered using directed evolution are exceptional at enhancing the particular enzymatic property being pursued, these mutations are difficult to predict and require considerable experimental resources. As the development of future base editors will likely involve additional directed evolution efforts (20, 21), maximizing our understanding of the outcomes of previous studies on this front will aid in these future studies. This work is an a posteriori study using a combination of computational simulations and experimental measurements to understand the mutations generated during the directed evolution of ABEs (4). We have additionally carried out MD simulations of TadA* and TadA*-ssDNA models to explore how the initial mutations accumulated during directed evolution give rise to ssDNA editing by the ABE enzyme. Installation of the Asp108Asn mutation in the TadA*0.1 to generate TadA*1.1 leads to a significant decrease in the flexibility of the 4-5 loop of the TadA (Fig. 2). This loop is known to both impart sequence specificity to the wild-type TadA enzyme through interactions with the nucleobases immediately upstream of the target A base and also serve as the dimerization interface between the individual TadA proteins (18). Our simulations indicate that the structural dynamics of TadA* mutants (Fig. 2) resembles that of the wtTadA dimer (fig. S2), which may explain how the TadA* enzymes are performing DNA base editing as monomers. The changes observed in the dynamics of the 4-5 loop therefore may help broaden the substrate scope of the TadA* enzymes to include both tRNA and ssDNA. In addition, as the TadA* mutants were evolved to function as monomers, this change in the dynamics may be increasing the enzymes affinity for ssDNA at the expense of protein dimerization. This is proven to be the case, as we experimentally observe that the TadA enzyme works as a monomer when acting on ssDNA, a finding that represents a key step in characterizing the mechanism of base editing by ABE (Fig. 2). This is an unexpected result that fundamentally changes our understanding of how ABEs function and will likely affect future ABE engineering and optimization studies.

Intriguingly, loss of conformational flexibility in the 4-5 loop of TadA* appears to make the overall structure of the protein more analogous to the APOBEC family of proteins (Fig. 2E). APOBEC enzymes are a class of proteins that have cytidine deamination activity on both ssDNA and ssRNA (7, 8) and were repurposed into the original CBEs. The inherent nature of the APOBECs to edit a broad range of nucleotide targets is preserved in the CBEs, which have been shown to exhibit considerable off-target DNA and RNA activities due to the APOBEC1 portion of the base editor (22, 23). This dual-substrate specificity of APOBECs has been attributed to specific conformations of the active site loop (1-1 loop, 2-2 loop, and 4-5 also referred to as the loop 1, loop 3, and loop 7, respectively) that interacts with the 5 flanking base of the substrate nucleotide using both experiments and simulations (2427). Both TadA and the APOBEC enzymes share a core five-stranded sheet structural element surrounded by helices. The 4-5 loop serves the same functional purpose in both enzymes, but the length of this loop is substantially longer in TadA, and in the APOBECs, it assumes a definite -helical secondary structure (Fig. 2E and fig. S8).

The gain in structure of this loop in TadA may contribute to the gain of ssDNA editing capability by TadA* (7, 28), but it is not solely responsible for this activity. The TadA*1.2(N108D) enzyme retains reduced mobility in the 4-5 loop yet displays wild-type like ssDNA binding affinity according to our simulations and nearly undetectable base editing efficiencies in our experimental work. Note that the Ala106Val mutation causes a substantial gain in mobility of the 2 helix (Figs. 2A and 6B), which is canceled out when the Asp147Tyr and Glu155Val mutations are incorporated. The 2 helix of TadA aligns with the 2-2 active site loop of the APOBECs (Fig. 2E and fig. S8), which lacks secondary structure and has been shown to be responsible for sequence specificity of the enzymes.

Our simulations show that when wild-type TadA interacts with ssDNA, the absence of a hydrogen-bond donor (in the form of the 2-OH group of the ribose sugar in RNA) for Asp108 causes this residue to flip into an energetically unfavorable conformation away from the negatively charged DNA backbone. This unfavorable conformation is responsible for the lack of ssDNA editing by the wild-type enzyme, as the presence of all other 13 favorable mutations, and the favorable interactions they bring with them, is not enough to compensate for the strained configuration that Asp108 is forced to adopt when in the presence of DNA rather than RNA. However, upon neutralization of this negative charge when Asp108 is mutated to Asn (a single atom substitution from O to N), the residue can now rotate back into a more energetically favorable position, allowing for the enzyme to interact with ssDNA. This rotation toward the ssDNA substrate also allows for the formation of a hydrogen bond between residue 108 in TadA* and the ssDNA (the 1 nucleotide in Fig. 3, D and E). This hydrogen bond further strengthened in TadA*2.1, where Asn108 becomes a double hydrogen-bond donor, interacting with the phosphate backbone. The phosphate backbone is a structural element common to both DNA and RNA, suggesting that in the process of acquiring ssDNA editing capabilities, TadA* may not surrender its native RNA editing functionality. This has been confirmed by previous reports of off-target RNA editing by ABE enzymes (19, 23). Furthermore, it was recently found that removing wtTadA from ABE7.10 does not suppress its RNA deamination activity, which demonstrates that the Asp108Asn mutation supports RNA binding by TadA* (29).

While one may expect only residues in the first shell (that interact directly with the ssDNA) to be primarily responsible for enhancing the thermodynamics and kinetics of ssDNA editing by TadA*, 6 of the 14 overall mutations accumulated during directed evolution actually reside in the second shell of the enzyme (fig. S1). In addition to electrostatic contributions, through our simulations, we observed that the Asp147Tyr and Glu155Val mutations, both of which reside in the 5 helix (fig. S4B), cause structural rearrangements in the protein, effectively initiating a chain reaction that strengthens the interactions between a variety of primary shell residues and the ssDNA substrate. Note that nearly half (6 of 14) of the ABE7.10 mutations are located in the 5 helix, highlighting the significance of understanding its role in ssDNA editing. These enhanced hydrogen-bonding interaction between the TadA* residues and the ssDNA, caused in aggregate by all four mutations, and the now-favorable conformation of the residue 108 when it is Asn, also translate into an increased free energy binding of the TadA*s to ssDNA (Fig. 5 and fig. S5). Upon reversion of Asn108 to Asp, however, even in the presence of the three other advantageous mutations (Ala106Val, Asp147Tyr, and Glu155Val), we observe a marked decrease in the binding affinity of TadA*1.2(N108D) and TadA*2.1(N108D) to ssDNA (Fig. 6C). On the basis of these observations, we speculate that the Asp108Asn mutation may play a bipartite role: It affords structural rigidity to the region of the enzyme responsible for sequence specificity and increases the binding affinity of the TadA enzyme to ssDNA through hydrogen-bonding interactions. However, the hydrogen bonds that Asn108 forms with the 5 nucleobase and the phosphate backbone are not its only contribution to the onset of DNA editing activity by ABEs. Simulations and experiments verify that reversion of Asn108 back to Asp from higher-generation ABEs leads to nearly complete loss in the base editing activities of higher ABE mutants (Fig. 6), despite the presence of up to 13 other beneficial mutations in TadA* that have created additional hydrogen-bonding interactions between TadA* and the ssDNA (Figs. 3D and 4). It is likely that the increased conformational strain imposed on the Asp108 residue when it must flip around to point away from the DNA backbone is energetically unfavorable enough to preclude ssDNA binding even with these additional favorable hydrogen-bonding interactions.

This study provides the first insights into the mechanism of base editing by ABEs, beginning with the observation that the TadA* enzyme acts a monomer to modify ssDNA. The results presented in this study additionally provide an explanation of the structural and functional roles of the initial TadA mutations identified in the evolution of ABE. We anticipate that this atomistic understanding of previous successful directed evolution experiments will enable the prediction of new mutations and lead to the rational engineering of future base editors.

The crystal structure of E. coli TadA enzyme (PDB ID: 1z3a) was used to define the initial coordinates for TadA*0.1 (10). The TadA*1.1, TadA*1.2, TadA*2.1, TadA*1.2(N108D), and TadA*2.1(N108D) mutants were prepared by inducing virtual mutations to the TadA0.1 structure using the mutagenesis plugin available in PyMOL (30). We then combined the crystal structure of E. coli TadA enzyme with the tRNA substrate from its structural homolog from Staphylococcus aureus [PDB ID: 2b3j (18)] to prepare the TadA*-ssDNA complexes. The remodeling of the tRNA structure by the removal of the 2 hydroxyl groups and all changes in the sugar pucker of the nucleotide backbone were carried out using the swapna command in the Chimera software (31). Moreover, since the tRNA structure was crystallized bearing nebularine, a nonhydrolyzable adenosine analog (18), we used the swapna command to substitute nebularine with adenine. To unpair the 3 and 5 ends of the hairpin loop, we used steered MD simulations using the exposed ssDNA nucleotides of the ternary complex of the cryoelectron microscopy structure of CRISPR-Cas9 [PDB ID: 5y36 (13)] as a reference structure (fig. S9). This yielded the TadA*0.1-ssDNA complex as illustrated in fig. S9. Similarly, the complexes of TadA*1.1, TadA*1.2, TadA*2.1, TadA*1.2(N108D), and TadA*2.1(N108D) mutants with ssDNA were developed using the mutagenesis plugin of PyMOL (30). All crystallographic water molecules within 3- distance of the protein/protein-ssDNA surface were preserved during the modeling process, and each of the systems was solvated using a truncated octahedral box of TIP3P water molecules (32). All titratable residues were assigned protonation states at pH 7 as predicted by the H++ server (33, 34). Varying number of Na+ ions were added to each system to maintain charge neutrality. The protein and the DNA atoms were represented using the Amber ff14SB force field and the bsc1 parameters, respectively (3537). All MD simulations were performed under periodic boundary conditions using the CUDA accelerated version of PMEMD implemented in Amber18 suite of programs (3840). The structures were first relaxed using a combination of steepest descent and conjugate gradient minimization. This was followed by a 1-ns heating to 298.15 K and 10-ns equilibration under harmonic restraints. Subsequently, we removed all restraints (except on the 5 and 3 termini of the substrate DNA sequence) and carried out 500-ns unbiased MD simulations for the six TadA* mutants and corresponding TadA*-ssDNA complexes. Additional details of this protocol can be found in Supplementary Materials and Methods. Table S2 summarizes all the simulations that were carried out during this study.

We calculated the free-energy binding profiles of the TadA*-ssDNA complexes along the collective variable corresponding to the distance between the centers of mass of the protein and the ssDNA substrate. For each TadA*-ssDNA complex, the PMF along this collective variable was calculated using umbrella sampling simulations. Starting from the equilibrated TadA*-ssDNA structures, we conducted four independent sets of umbrella sampling simulations for all of the six TadA*-ssDNA complexes, and the final PMFs were reconstructed using the weighted histogram analysis method (WHAM) algorithm (41). Additional error analysis was carried out using a custom block averaging script based on the method described by Zhu and Hummer (42).

The CPPTRAJ module implemented within Amber18 was used to analyze all the MD trajectories (16, 17). The root mean squared fluctuation of the ABE mutants and clustering of configurations from each MD trajectory were calculated, with respect to the C atoms of the protein backbone. We identified the primary and secondary interaction shells and the associated H-bonding network using the mask and hbond keywords of CPPTRAJ, respectively (see the Supplementary Materials for details). The PDB2PQR webserver, in conjunction with the APBS server, was used to calculate the electrostatic maps for the ABE0.1 and ABE2.1 models (43). The visualization of the MD trajectories was rendered using Chimera, and data were plotted using Matplotlib (44).

All ABE plasmids were constructed using USER cloning (45) with pCMV_ABEmax (Addgene plasmid no. 112095) as a template using Phusion U Hot Start Polymerase (Thermo Fisher Scientific). Complete sequences of ABEs are listed in the Supplementary Materials. All single guide RNA (sgRNA) expression plasmids were generated using blunt-end cloning (3) with pFYF1230 (Addgene plasmid no. 47511) as a template using Phusion High-Fidelity DNA Polymerase (New England BioLabs). Complete protospacer/protospace adjacent motif (PAM) sequences are listed in table S3. All DNA vector amplification was carried out using NEB 10- competent cells (New England BioLabs). All plasmids were purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research).

HEK293T cells (American Type Culture Collection, CRL-3216) were maintained in high glucose Dulbeccos modified Eagles medium supplemented with GlutaMAX (Thermo Fisher Scientific), 10% (v/v) fetal bovine serum (Thermo Fisher Scientific), and penicillin-streptomycin (100 g/ml; Thermo Fisher Scientific) at 37C with 5% CO2.

HEK293T cells were seeded in 48-well VWR Multiwell Cell Culture Plates at a density of 150,000 cells per well in 250 l of media without penicillin-streptomycin. Four hours after plating, 1000 ng of ABE plasmid and 250 ng of sgRNA plasmid were transfected using 1.5 l of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturers protocol.

Transfected cells were rinsed with phosphate-buffered saline (150 l/well; Thermo Fisher Scientific) 5 days after transfection. Cells were lysed on the plate by addition of 100 l of lysis buffer [10 mM tris (pH 7.5), 0.1% SDS, and proteinase K (25 g/ml)]. Lysed cells were then heated at 37C for 1 hour, followed by 80C for 20 min. Genomic loci of interest were polymerase chain reaction (PCR) amplified with Phusion High-Fidelity DNA Polymerase (New England BioLabs) according to the manufacturers protocol using the primers indicated in table S4, 1 l of genomic DNA mixture as a template, and 26 or fewer rounds of amplification. Unique forward and reverse combinations of Illumina adapter sequences were then appended with an additional round of PCR amplification with Phusion High-Fidelity DNA Polymerase (New England BioLabs) according to the manufacturers protocol using 1 l of round 1 PCR mixture as a template and 15 rounds of amplification. The products were gel purified and quantified using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Samples were then sequenced on an Illumina MiniSeq according to the manufacturers protocol.

Sequencing reads were demultiplexed in MiniSeq Reporter (Illumina), and individual FASTQ files were analyzed using a previously reported MATLAB script (4).

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz2309/DC1

Supplementary Materials and Methods

Fig. S1. Asteroid plot for TadA*7.10-ssDNA.

Fig. S2. Comparison of the structural flexibility of the TadA monomer with TadA dimer.

Fig. S3. Percentage contact and the fractional H-bonding between the three nucleotides and the first interaction shell amino acids.

Fig. S4. Asteroid plot for TadA*0.1-ssDNA complex.

Fig. S5. Mutations lead to an increase in TadA* binding to the ssDNA (AAG).

Fig. S6. Structural importance of Ala106Val mutation.

Fig. S7. PMF of the TadA*-ssDNA complexes calculated using steered MD simulations.

Fig. S8. Comparison of the structural flexibility of the ecTadA with hAPOBEC3A.

Fig. S9. Modeling of TadA*-ssDNA.

Fig. S10. Umbrella sampling data and biased statistics.

Table S1. Comparison of RMSD of the representative clusters.

Table S2. Summary of the systems modeled and the types of simulations conducted in this study.

Table S3. DNA sequences used for simulations and in mammalian tissue culture experiments.

Table S4. First round genomic DNA PCR sequences.

Supplementary sequences

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

W. L. DeLano, The PyMOL Molecular Graphics System, Version 1.8 (Schrdinger LLC, 2015).

D. A. Case, I. Y. Ben-Shalom, S. R. Brozell, D. S. Cerutti, T. E. Cheatham III, V. W. D. Cruzeiro, T. A. Darden, R. E. Duke, D. Ghoreishi, M. K. Gilson, H. Gohlke, A. W. Goetz, D. Greene, R. Harris, N. Homeyer, S. Izadi, A. Kovalenko, T. Kurtzman, T. S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D. J. Mermelstein, K. M. Merz, Y. Miao, G. Monard, C. Nguyen, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D. R. Roe, A. Roitberg, C. Sagui, S. Schott-Verdugo, J. Shen, C. L. Simmerling, J. Smith, R. Salomon-Ferrer, J. Swails, R. C. Walker, J. Wang, H. Wei, R. M. Wolf, X. Wu, L. Xiao, D. M. York, P. A. Kollman, AMBER 2018 (University of California, 2018).

Acknowledgments: We thank M. Norman for a Director's Discretionary Allocation on the Comet GPU cluster at the San Diego Supercomputer Center. K.L.R. thanks C. Egan and E. Lambros for helpful discussions. Funding: This research was supported by the University of California San Diego and the NIH through grant no. 1R21GM135736-01. All computer simulations used resources of the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by NSF through grant no. ACI-1548562. Author contributions: The manuscript was written through contributions of all authors. K.L.R. performed all computer simulations. K.L.R., A.C.K., and F.P. conceptualized and designed the research. Competing interests: A.C.K. is a consultant of Pairwise Plants and Beam Therapeutics, companies that are developing and using base editing technologies. All other authors declare that they have no competing interests. Data and materials availability: HTS data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database under accession code PRJNA590028. All other data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors - Science Advances

Jerome Mosley(Photo: Provided/Hamilton County Sheriff's Office)

An Over-the-Rhine man has been charged in three separate robberies using facial recognition and DNA from a coffee cup.

Jerome Mosley, 64, is charged with multiple counts of aggravated robbery.

While investigating the Jan. 10 armed robbery of the Vine Street Market in Hartwell, officers captured a clear image of the suspect from surveillance footage, according to court documents

Using a system called "Mug Master" available through the Hamilton County Sheriff's Office, investigatorsautomatically compared the image to all of the mugshots in the system.

Cincinnati police Lt. Steve Saunders explained this system often gives officers a few different suspects they can look into.

With the facial recognition results and further investigation, officers were able to create a photo line-up and present it to witnesses, who identified Mosley, Saunders said.

Mosley has also been indicted on charges connected to the Jan. 21 armed robbery of a Gold Star Chili.

Mosley was arrested on Jan. 24.Since that arrest, his DNA matched him to a third robbery.

On the same day as the Gold Star Chili robbery, a Days Inn was robbed in Springdale, police said.

According to court documents, a suspect held the clerk at gunpoint threatening to kill him.

"A coffee cup left behind by the defendant was swabbed for DNA," Springdale police reported. "A CODIS hit was received on Jerome Mosley."

By the time Springdale police received the news, Mosley was already in jail. New charges were filed.

Mosley is being held at the Hamilton County Justice Center on a $750,000 bond awaiting trial.

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Facial recognition and DNA connect man to string of armed robberies - The Cincinnati Enquirer

Researchers at Princeton University have revealed the inner workings of a gene repression mechanism in fruit fly embryos, adding insight to the study of human diseases.

Led by graduate student Shannon Keenan, the team used light to activate chemical signals in developing fruit flies and traced the effects on a protein called Capicua, or Cic. Located in a cell's nucleus, Cic binds to DNA and performs the specialized task of silencing genes. The study, published in Developmental Cell and made available online March 5, reveals the dynamics of gene repression by this protein.

A fruit fly embryo is shown dotted with fluorescent lights, indicating DNA transcription in progress at various points along the abdomen. A recent Princeton study gave an unprecedented look at how gene repression works, revealing the dynamics of the system that prevents transcription along certain sections of genetic code.

Image courtesy of the researchers

In a complex piece of music, the silences running through the melody contribute as much to the score's effect as the sounded notes. The biological processes that control development rely on highly sophisticated temporal patterns of gene activation and repression to create life's beautiful symphonies. When a pattern is disrupted, it's like a wrong note in the music. In this case, Cic is a repressor protein that silences certain parts of the genome, allowing other genes to express in harmony with one another. Understanding how repressors like Cic work allows researchers to better conduct the orchestra.

"Signals that tell you not to do something are just as important as signals to do something," said Stanislav Shvartsman, professor of chemical and biological engineering and the Lewis-Sigler Institute for Integrative Genomics and a group leader at the Flatiron Institute at the Simons Foundation. While both Cic and its target genes were established, little was known about the dynamics of gene repression, according to Shvartsman, the study's principal investigator and Keenan's Ph.D. adviser. "Proteins that activate and repress genes can be likened to the gas pedal and brakes on a car," he said. "We know a lot about how the gas pedal works. This is the first time we are seeing the brakes in action."

The authors, including Squibb Professor in Molecular Biology Eric Wieschaus, winner of the 1996 Nobel Prize in Physiology or Medicine, focused on a critical enzyme, called ERK, that transmits signals from the cells surface to its nucleus. By controlling ERK with light, in precisely timed sequences, they could parse out how and when the enzyme engaged the Cic protein. They found that, after about five minutes of ERK activation, the Cic protein is inactivated, which allows its target genes (normally repressed) to turn on. Remarkably, repression was reinstated just as quickly, demonstrating a very fast response time.

In addition to offering an unprecedented look at the dynamics of gene repression, the study revealed a rich new territory for further exploration. Natural selection has acted over hundreds of millions of years to tune these processes. Their timing is no accident. According to Keenan, the mechanism's fast response time guards cells against biochemical noise that could otherwise introduce catastrophic errors.

Researchers led by graduate student Shannon Keenan, and professors Stanislav Shvartsman, right, and Eric Wieschaus, left, used light to control a protein that represses genes and is tied to the onset of disease and developmental disorders.

"The fly embryo is a very powerful system," Keenan said, and the things we learn from guide our understanding and treatment of human diseases, including cancer, where Cic is commonly mutated."

Keenan came to chemical and biological engineering from an interest in solving problems in human health. She wanted to use whatever skills she had to help people, and she was good at math and science. "I never thought I'd be working with fruit flies," she said, laughing. "When I came to Princeton, I met with Stas [Shvartsman], and he showed me all these images of where genes were turned on in fly embryos. It was just very beautiful."

Other contributors to the study included Shelby A. Blythe, from Northwestern University, and Robert A. Marmion and Nareg J.-V. Djabrayan, from Princeton. The work was supported by the National Institutes of Health.

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Geneticists pump the brakes on DNA, revealing key developmental process - Princeton University

Authorities have taken another step towards potentially learning more about the person investigators call the man in the dunes a headless torso discovered on a Sandwich beach six years ago.

Massachusetts State Police are now releasing a DNA-generated composite photo of what officials say the mans face may have looked like in hopes of identifying him, WCVB first reported Wednesday.

The gruesome discovery at Town Neck Beach on June 4, 2014 left authorities with little information about the man who was killed, according to State Police Sgt. Matt Lavoie.

The limbs were removed in such a way to hinder identification, probably to get rid of tattoos, Lavoie told WCVB. We ran the victims DNA through the national databases, there were no hits. There wasnt much of a description to work on.

Police turned over DNA collected from the torso to Parabon Labs in Virginia, which created the image, according to the news station.

The man may have been between 5 feet, 8 inches and 6 feet tall, and may have weighed 230 pounds, WCVB reports. He had a surgical scar on his stomachs right side.

Lavoie said identifying the victim is an important step in the investigation.

We can find out where they were from, what kind of life they led, who they dealt with in their personal life and hopefully try and track down the last people they dealt with, if possible, and maybe get a suspect from that, he told the station.

Authorities said that even with the photo, the public should remember the victim may have looked slightly different in real life. Anyone with information related to the case is asked to call detectives at 508 790-5799.

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DNA-generated composite photo puts a face to headless torso found on Cape Cod - Boston.com

To the editor:

It never ceases to irritate me to read an article about scientists James Watson and Francis Cricks discovery of the double-helix structure of DNA. (Todays Highlight in History, The Sun Chronicle, Page A8)

Well, here is the real deal.

British scientist Rosalind Franklin brilliantly produced an X-ray of the molecular structure of DNA, using X-ray crystallography. At this point Watson and Crick hadnt a clue to its structure.

A sneaky male colleague in Franklins lab provided Watson and Crick a look-see at the DNA photo #51 and her unpublished data. Did she know? History says, not likely.

As a result, Watson and Crick published a paper regarding this info in 1953. Eventually, Watson and Crick and another scientist, Maurice Wilkins, shared the 1962 Nobel Prize for describing DNAs double-helix structure.

Franklin died in 1958, making her ineligible for a Nobel Prize. Watson and Crick got the credit.

Gail Boone

Wrentham

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Letter to the editor: A woman, not men, discovered DNA's double helix structure - The Sun Chronicle

Genetic data suggests that Washington state is facing an already substantial outbreak that has gone undetected for weeks because of testing failures.

Last updated on March 3, 2020, at 4:53 p.m. ET

Posted on March 3, 2020, at 2:19 p.m. ET

Health care workers transport a patient on a stretcher into an ambulance in Kirkland, Washington.

As the coronavirus touches down in the US, Washington state has so far been the hardest-hit, with at least 27 confirmed cases, including nine deaths, as of Tuesday.

Many more are likely to follow. If a new genetic analysis is to be believed, the virus has been circulating undetected throughout the state for weeks.

Preliminary analysis of a pair of cases, conducted late last week by the Fred Hutchinson Cancer Research Center in Seattle, supports the increasing likelihood that some people are now becoming infected within the US, as opposed to while abroad or from a known case.

The findings also underscore how badly diagnostic testing has lagged nationwide, allowing the pathogen to spread in American communities largely undetected. In the latest sign of just how unclear the scope of the outbreak is, the earliest known death in the US occurred in the Seattle area last week, but its connection to the coronavirus was not reported until Tuesday afternoon.

Caitlin Rivers, an epidemiologist at the Johns Hopkins Center for Health Security, said that what is happening in the Seattle area may be happening elsewhere, too. California has reported at least four cases spread by community transmission. There were more than 100 cases across the US as of Tuesday.

We should be thinking of this as our first warning, or an early warning, that we are starting to see a new chapter in the US outbreak, Rivers told BuzzFeed News.

And Columbia University virologist Angela Rasmussen said by email, This indicates that there are likely foci of community spread in other states, which means that it is going to be a lot harder, if not impossible, to contain this virus.

On Saturday, Trevor Bedford, a scientist at Fred Hutchinson, revealed that his team had sequenced the genomes of two coronavirus cases in Washington. One of them, reported Jan. 19, was the first case in the US: a man who had recently traveled back from Wuhan, China, the epicenter of the virus, to Snohomish County, Washington.

The second, reported last Friday, was that of a high school student, who was also in Snohomish County but had not traveled to a country affected by the outbreak, according to local public health officials.

Bedford said on Twitter that the second case turned out to be genetically similar to the first, with the two sharing a rare variant that made the similarity highly unlikely to be a coincidence. More likely was that the first infection had spread in the community before spawning the second.

This strongly suggests that there has been cryptic transmission in Washington State for the past 6 weeks, he wrote, adding that he believed the state was facing an already substantial outbreak. His initial models projected that the number of infections in the state was likely around a few hundred, and a blog post including updated case counts on Monday estimated that it was closer to 600. Bedford wrote that this matched his projections of the number of infections in Wuhan on January 1.

Outside experts stressed that the models, based on the small amount of data currently available, were preliminary. Bedford was not available for further comment.

Washington Gov. Jay Inslee declared a state of emergency over the weekend. The nine deaths due to coronavirus in the state included elderly residents of the Life Care Center nursing facility in Kirkland.

Jamie Nixon, a spokesperson for the states Department of Health, said that the agency was evaluating the Fred Hutchinson teams findings. It is definitely possible that COVID-19 has been circulating, with people experiencing mild symptoms just like the flu, Nixon said by email.

Only now are those cases starting to be caught, in Washington and elsewhere.

The Centers for Disease Control and Prevention has been widely accused of failing to make testing broadly available during the initial, crucial weeks when the disease entered the US. Critics blame the agency for shipping hundreds of faulty diagnostic test kits to local and state laboratories and imposing strict criteria on who could be tested.

The CDC widened the criteria last week, and has said it's come up with a fix for its original test, tossing out a defective part of the DNA test. On Tuesday, the agency said it would be sending out 75,000 tests to labs across the country by the end of the week. And the head of the FDA said that total will come closer to 1 million with tests from commercial providers. But only about 500 Americans had been tested as of Sunday, and many scientists believe these efforts are too late.

Everyone in public health has asked for more testing for like a month now, said Tara Smith, an epidemiologist at Kent State University. Weve been slow to roll that out, and thats a big problem.

As the tests roll out, a spike in cases will likely mean that more existing infections are being confirmed, not necessarily that new infections are happening.

Its not like were having some crazy explosion in the next few days, Rivers said. Were just catching up to understand the current picture. The early numbers might feel alarming, but you have to interpret them knowing that were now just getting visibility on things that have already been happening.

Azeen Ghorayshi contributed reporting to this story.

Mar. 03, 2020, at 21:53 PM

This story has been updated to include more information about Trevor Bedford's projected number of cases in the Washington coronavirus outbreak.

Mar. 03, 2020, at 21:00 PM

This story has been updated to include coronavirus case counts in Washington as of Tuesday afternoon.

Mar. 03, 2020, at 19:46 PM

This story has been updated to include more up-to-date numbers about coronavirus cases spread through community transmission in California.

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The Coronavirus Has Likely Been Spreading Through Washington For Weeks, Scientists Said - BuzzFeed News