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New gene editing tools transform disease models and future therapies CRISPR gene editing is taking biomedical research by storm. Providing the ultimate toolbox for genetic manipulation, many new applications for this technology are now being investigated and established. CRISPR systems are already delivering superior genetic models for fundamental disease research, drug screening and therapy development, rapid diagnostics, in vivo editing and correction of heritable conditions and now the first human CRISPR clinical trials.

The continuing patent battle for CRISPR-Cas9 licensing rights and the emergence of new editing systems such as Cpf1 has so far done nothing to slow the advance of CRISPR-Cas9 as the leading gene editing system. There are weekly press releases and updates on new advances and discoveries made possible with this technology; the first evidence is now emerging that CRISPR-Cas9 could provide cures for major diseases including cancers and devastating human viruses such as HIV-1.

The key to CRISPR-Cas9s uptake is its ease of application and design, with retargeting only a matter of designing new guide RNA. It has quickly surpassed TALENs (Transcription Activator-Like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) where editing, now possible with CRISPR, was previously prohibitively complex and time-consuming. As well as correcting gene mutations with scar-less modifications, with CRISPR-Cas9 it is possible to control the expression of entire genes offering longer term expression alteration compared to other methods such as RNAi.

LNA GapmeRs are highly effective antisense oligonucleotides for knockdown of mRNA and lncRNA in vivo or in vitro. Designed using advanced algorithms, the RNase H-activating LNA gapmers offer excellent performance and a high success rate.

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CRISPR-Cas9 systems, tools and basic methodology are very accessible as ready to go toolkits that anyone with lab space and an idea can pick up and start working with. This is thanks largely to the efforts of Addgene and commercial service and product providers. Alongside CRISPR research there are innovations in companion technologies and design software. In response to a growing need, companies such as Desktop Genetics have developed open access software to accelerate CRISPR experimentation and analysis.

It is not all about CRISPR-Cas9 though. Like Cas9, Cpf1 is a DNA-targeting CRISPR enzyme that is also recruited to the target site by sequence homology but with slightly different site requirements. Cpf1 has been reported to be efficient and highly specific in human cells, with low off-target cleavage suggesting a role for Cpf1 in therapeutic applications down the line. Cas13a is an RNA-targeting CRISPR enzyme which is showing promise as a rapid diagnostic tool. Unlike Cas9, the enzyme continues to cut after it has acted on its intended RNA target, a characteristic which has been exploited to develop diagnostic technology for the likes of Zika and Dengue virus. The group behind SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) combined this collateral effect of Cas13a with isothermal amplification and produced rapid DNA or RNA detection at attomolar sensitivity and with single-base mismatch specificity.

A particularly active area of CRISPR activity is the genetic manipulation of patient-derived stem cells to create models for diseases including Parkinsons, cystic fibrosis, cardiomyopathy and ischemic heart disease, to name but a few. With CRISPR it is now possible for researchers to correct disease-causing mutations in patient-derived pluripotent stem cells to create isogenic cell lines to differentiate to any cell type of interest for disease research. Generating these isogenic lines is making it possible, for the first time, to unambiguously show the contribution of gene mutations to a disease phenotype.

Dr Lise Munsie leads the pluripotent stem cell program at CCRM, a Canadian, not-for-profit organisation supporting the development of foundational technologies to support the commercialisation of cell and gene therapies, and regenerative medicine.

Gene editing technology now provides unlimited genetic flexibility to stem cell manipulation. You can target anywhere in the genome with relative ease and make it scar-less, saidDr Munsie.

Dr Munsies program is using CRISPR-Cas9 to produce reporter cell lines (for example with fluorescent protein inserted at a target gene) and isogenic lines from patient iPSCs. In stem cells, CRISPR-Cas9 is introduced with the Cas9 nuclease expressed from plasmid DNA or as purified Cas9 protein and the components are introduced into the cells by transfection or electroporation.

Dr Bjrn Brndl and his colleagues at the Lab for Integrative Biology at the Zentrum fr Integrative Psychiatrie, Universitatsklinikum Schleswig-Holstein, Germany, are also using stem cell gene editing to generate model systems for studying complex neurological disease such as Parkinsons and dyskinesia by correcting mutation in patient lines and introducing these mutations in control cells lines.

One of the biggest contributions of CRISPR to research is the ability to create isogenic stem cell lines. With these, we can create relevant disease models with near-perfect negative controls with the same genomic context varying only in the region of interest. Our goal is to compare disease patient lines with corrected lines by differentiating the induced pluripotent stem cells into neurons and studying differences in the phenotypes. In the biomedical field, we currently have a reproducibility crisis, so with clean and effective tools like isogenic pluripotent stem cells lines, we can improve the reproducibility and validity of our findings. One of the biggest challenges is working with the stem cells which are delicate and much more sensitive to the manipulations required for successful gene editing compared to standard cell lines.

CRISPR has completed upended how cell biology is approached. Being able to copy/paste DNA into the genomes has introduced a lot of ways of thinking about a problem. Genome editing has introduced engineering into the cell biology toolbox. saidDr Brndl.

An alarming number of bacteria are now resistant to our most effective antibiotics. The antibiotic resistance crisis has been given more of the attention it deserves thanks to initiatives from the WHO, UN, NICE and others but, in truth, the situation has been critical for over a decade. No new antibiotics have come out of pharma companies in the last 10 years and interest in their development has waned. Pharma companies are reluctant to invest the large sums required to develop new antimicrobials because of the inevitable resistant strains that will quickly follow and subsequent restrictions on their usage to preserve efficacy.

In short, we need a miracle, but the answer could come from CRISPR. Companies such as Nemesis Bioscience and Eligo Bioscience are developing antimicrobial technology and treatments made possible by CRISPR technology. Both technologies use modified bacteriophage as delivery vehicles for CRISPR-Cas9 gene editing systems that target and inactivate either virulence genes or the resistance genes themselves, leaving the rest of the microbiome intact.

Nemesis Bioscience employs CRISPR to target known bacterial resistance genes to deactivate them in situ and re-sensitise virulent bacteria making existing antibiotics effective again. Dr Frank Massam, CEO at Nemesis Biosciences explains, Killing bacteria stimulates resistance mutations we reasoned it would make more sense to inactivate bacterias ability to resist antibiotics and therefore make existing antibiotics work again. This approach would also mean that newly developed antibiotic assets could be protected from resistance, thereby increasing pharmas ROI and so making antibiotic development attractive again.

Nemesis Biosciences Symbiotics are based on modified CRISPR-Cas9 which enables highly multiplexed guide RNA targeting. Our first expression cassettes encode the S. pyogenes Cas9 plus a CRISPR array encoding guide RNAs that can target for inactivation members of 8 families of beta-lactamase genes. We call them the VONCKIST families, these are: VIM, OXA, NDM, CTX-M, IMP, SHV and TEM. The beta-lactamases encoded by these families are able to degrade >100 different types of beta-lactam antibiotics saidDr Massam.

The symbiotics are delivered by phage Transmids delivery vehicles based on phage architecture that deliver the DNA and then drop off. Once the Symbiotic is inside the bacteria, it can then spread further by conjugation from the edited bacteria to others it encounters, remaining invisible to the immune system. This provides both therapeutic applications as well as prophylactic ones in a probiotic delivery system to disarm the microbiome of antimicrobial-resistant bacteria. The technology is applicable to all bacteria, all antibiotic classes and all known resistance mechanisms and Nemesis have initially targeted resistant E. coli for in vivo testing.

Traditional small-molecule antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Eligo Biosciences flagship technology SSAMS eligobiotics, uses reprogrammed Cas9 targeted to bacterial virulence or resistance genes delivered by phagemids to produce selective killing of virulent and antibiotic resistant bacteria, leaving all other bacteria unaffected. The Eligo platform is being adapted for other microbial applications including in situ detection of specific live bacterial strains in complex microbiome samples and in situ expression of therapeutics protein to modulate and engineer host-microbiome interactions.

CRISPR-based therapies for human diseases could bring profound benefits to medicine, but there are many hurdles still to overcome. Despite the high degree of specificity of the CRISPR system, the induction of off-target mutations, at sites other than the intended target, is still a major concern especially in the context of therapeutic applications for heritable disease, and there are still considerable safety concerns about using CRISPR in humans. Assays for investigating the intended (on-target) and unintended (off-target) effects of CRISPR guides on in vitro and in vivo models are still in their infancy. The second major challenge is the development of safe carrier systems for CRISPR-Cas9 delivery to human cells in vivo.

Nonetheless, exciting progress is being made in the application of CRISPR gene editing to the treatment of heritable diseases for which there are only symptomatic treatments available, such as retinal myopathy where demonstrated recovery has been reported in a mouse model, and Duchenne muscular dystrophy, where the disease phenotype is reversed in mouse cells in vivo. We will also soon see the completion of the first clinical trials using CRISPR to try and correct genetic defects in vivo, the results of which are eagerly awaited.

There are a growing number of researchers from many disciplines collaborating to bring ambitious CRISPR-based insight, technology and therapeutics into the clinic. As CRISPR continues to undergo technical improvements, the prospects for these applications continues to look promising and as they move rapidly towards reality.

References

1. Yin, C., Zhang, T., Qu, X., Zhang, Y., Putatunda, R., Xiao, X., ... & Qin, X. (2017). In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Molecular Therapy.)

2. Hough SH, Kancleris K, Brody L, Humphryes-Kirilov N, Wolanski J, Dunaway K, Ajetunmobi A, Dillard V. Guide Picker is a comprehensive design tool for visualizing and selecting guides for CRISPR experiments. BMC bioinformatics. 2017 Mar 14;18(1):167.

3. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., ... & Koonin, E. V. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3), 759-771.

4. Kleinstiver, B. P., Tsai, S. Q., Prew, M. S., Nguyen, N. T., Welch, M. M., Lopez, J. M., ... & Joung, J. K. (2016). Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature biotechnology, 34(8), 869-874.

5. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 13:eaam9321

6. Bikard, D., Euler, C. W., Jiang, W., Nussenzweig, P. M., Goldberg, G. W., Duportet, X., ... & Marraffini, L. A. (2014). Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nature biotechnology, 32(11), 1146-1150.

7. Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., & Yang, S. H. (2015). Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy-Nucleic Acids, 4, e264.

8. Yu, W., Mookherjee, S., Chaitankar, V., Hiriyanna, S., Kim, J. W., Brooks, M., ... & Swaroop, A. (2017). Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nature Communications, 8.

9. Long, C., Amoasii, L., Mireault, A. A., McAnally, J. R., Li, H., Sanchez-Ortiz, E., ... & Olson, E. N. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science, 351(6271), 400-403.

10. Nelson, C. E., Hakim, C. H., Ousterout, D. G., Thakore, P. I., Moreb, E. A., Rivera, R. M. C., ... & Asokan, A. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 351(6271), 403-407.

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CRISPR: Emerging applications for genome editing technology - Technology Networks

Euan Ashley and his collaborators used long-read genome sequencing to diagnose a rare condition in a Stanford patient. It's the first time the technique has been used in a clinical setting. Steve Fisch

Stanford scientists have used a next-generation technology called long-read sequencing to diagnose a patients rare genetic condition that current technology failed to diagnose.

When Ricky Ramon was 7, he went for a routine checkup. The pediatrician, who lingered over his heartbeat, sent him for a chest X-ray, which revealed a benign tumor in the top-left chamber of his heart. For Ramon, it was the beginning of a long series of medical appointments, procedures and surgeries that would span nearly two decades.

During this time, noncancerous tumors kept reappearing in Ramons heart and throughout his body in his pituitary gland, adrenal glands above his kidneys, nodules in his thyroid.

The trouble was, doctors couldnt diagnose his condition.

When Ramon was 18, doctors thought his symptoms were suggestive of Carney complex, a genetic condition caused by mutations in a gene called PRKAR1A. However, evaluation of Ramons DNA revealed no disease-causing variations in this gene.

Now, eight years later, researchers at the Stanford University School of Medicine have used a next-generation technology long-read sequencing to secure a diagnosis for Ramon. Its the first time long-read, whole-genome sequencing has been used in a clinical setting, the researchers report in a paper published online June 22 in Genetics in Medicine.

Genome sequencing involves snipping DNA into pieces, reading the fragments, and then using a computer to patch the sequence together. DNA carries our genetic blueprint in a double-stranded string of molecular letters called nucleotides, or base pairs. The four types of nucleotides are each represented by a letter C for cytosine and G for guanine, for example and they form links across the two strands to hold DNA together.

Illuminating a dark corner

Current sequencing technologies cut DNA into words that are about 100 base-pairs, or letters, long, according to the studys senior author, Euan Ashley, DPhil, FRCP, professor of cardiovascular medicine, of genetics and of biomedical data science. Long-read sequencing, by comparison, cuts DNA into words that are thousands of letters long.

This allows us to illuminate dark corners of the genome like never before, Ashley said. Technology is such a powerful force in medicine. Its mind-blowing that we are able to routinely sequence patients genomes when just a few years ago this was unthinkable.

The study was conducted in collaboration with Pacific Biosciences, a biotechnology company in Menlo Park, California, that has pioneered a type of long-read sequencing. Lead authorship of the paper is shared by Jason Merker, MD, PhD, assistant professor of pathology and co-director of the Stanford Clinical Genomics Service, and Aaron Wenger, PhD, of Pacific Biosciences.

The type of long-read sequencing developed by the research teams collaborators at the company can continuously spool long threads of DNA for letter-by-letter analysis, limiting the number of cuts needed.

This is exciting, said Ashley, because instead of having 100-base-pair words, you now have 7,000- to 8,000-letter words.

Falling cost

Thanks to technological advances and increased efficiency, the cost of long-read sequencing has been falling dramatically. Ashley estimated the current cost of the sequencing used for this study at between $5,000 and $6,000 per genome.

Though the cost of short-read sequencing is now below $1,000, according to Ashley, parts of the genome are not accessible when cutting DNA into small fragments. Throughout the genome, series of repeated letters, such as GGCGGCGGC, can stretch for hundreds of base pairs. With only 100-letter words, it is impossible to know how long these stretches are, and the length can critically determine someones predisposition to disease.

Additionally, some portions of the human genome are redundant, meaning there are multiple places a 100-base pair segment could potentially fit in, said Ashley. This makes it impossible to know where to place those segments when reassembling the genome. With longer words, that happens much less often.

Given these issues, 5 percent of the genome cannot be uniquely mapped, the researchers wrote. And any deletions or insertions longer than about 50 letters are too long to detect.

For patients with undiagnosed conditions, short-read sequencing can help doctors provide a diagnosis in about one-third of cases, said Ashley. But Ramons case was not one of those.

The technique initially used to analyze Ramons genes failed to identify a mutation in the gene responsible for Carney complex, though Ashley said co-author Tam Sneddon, DPhil, a clinical data scientist at Stanford Health Care who browsed through the database of Ramons sequenced genome by hand, did notice something looked wrong. Ultimately, the long-read sequencing of Ramons genome identified a deletion of about 2,200 base-pairs and confirmed that a diagnosis of Carney complex was indeed correct.

This work is an example of Stanford Medicines focus on precision health, the goal of which is to anticipate and prevent disease in the healthy and precisely diagnose and treat disease in the ill.

An exceedingly rare condition

Carney complex arises from mutations in the PRKAR1A gene, and is characterized by increased risk for several tumor types, particularly in the heart and hormone-producing glands, such as ovaries, testes, adrenal glands, pituitary gland and thyroid. According to the National Institutes of Health, fewer than 750 individuals with this condition have been identified.

The most common symptom is benign heart tumors, or myxomas. Open heart surgery is required to remove cardiac myxomas; by the time Ramon was 18 years old, hed had three such surgeries. He is under consideration for a heart transplant, and having the correct diagnosis for his condition was important for the transplant team. Beyond the typical screening for a transplant, Ashley said the team needed to ensure there werent other health issues that could be exacerbated by immune suppressants, which heart transplant patients must take to avoid rejection of the donated organ.

Though it helps his medical team to have a confirmed diagnosis of Carney complex, Ramon has found it disheartening to face the fact that he cannot escape his condition. I was pretty sad, he said. It took me a while to come to terms with the fact that Ill have this until the day I die.

He tries not to dwell on it, though. Live one day at a time, he said. The bad days are temporary storms, and theyll pass.

His story is quite incredible, said Ashley, who said it was a privilege to be working on Ramons team. To have such a burden on such young shoulders, and to decide whether or not he wants a transplant, requires incredible courage.

Because he couldnt wait any longer for a transplant, Ramon recently underwent his fourth surgery to remove three tumors in his heart. Joseph Woo, MD, professor and chair of cardiothoracic surgery, performed the operation at Stanford Hospital. It is exceedingly rare to have tumors in the heart, said Ashley. It was a particularly heroic operation. Though Ramon is still under consideration for a transplant, the need is less urgent now.

Im in good hands, Ramon said of the Stanford team. Im glad to be here.

A future in the clinic?

Ashley said he and many other doctors believe that long-read technology is part of the future of genomics.

Now we get to see how to do it better, said Ashley. If we can get the cost of long-read sequencing down to where its accessible for everyone, I think it will be very useful.

This article has been republished frommaterialsprovided by Stanford University. Note: material may have been edited for length and content. For further information, please contact the cited source.

Reference

Merker, J., Wenger, A. M., Sneddon, T., Grove, M., Waggott, D., Utiramerur, S., ... & Korlach, J. (2016). Long-read whole genome sequencing identifies causal structural variation in a Mendelian disease. bioRxiv, 090985.

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Long-read Genome Sequencing Used for First Time in a Patient - Technology Networks