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Posts Tagged ‘CRISPR/Cas’


Translational Gene Editing – June 16-17, 2016 in Boston, MA

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Learn More | Sponsorship & Exhibit Details | Register by April 29 & SAVE up to $200!

IMPROVING CRISPR FOR BETTER FUNCTIONAL SCREENING

Optimized sgRNA Libraries for Genetic Screens with CRISPR-Cas9
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT

Optimizing CRISPR for Pooled Genome-Wide Functional Genetic Screens
Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.

CRISPR-Cas9 Whole Genome Screening: Going Where No Screen Has Gone Before
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center

Cross-Species Synthetic Lethal Screens and Applications to Drug Discovery
Norbert Perrimon, Ph.D., Professor, Department of Genetics, Harvard Medical School and Investigator, Howard Hughes Medical Institute

Interactive Breakout Discussion Groups with Continental Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Topic: CRISPR/Cas9 System for In vivo Drug Discovery
Moderator: Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

  • Impact of CRISPR/Cas9 system on in vivo mouse models
  • Application of the CRISPR/Cas9 system in in vivo screens
  • Technical limitations/safety issues

Topic: Getting Past CRISPR Pain Points
Moderators: John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MITStephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School

  • Challenges and solutions for CRISPR gRNA design
  • Methods for detecting engineered changes

Topic: Cellular Delivery of CRISPR/Cas9
Moderator: Daniel E Bauer M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

GENE EDITING FOR SCREENING DISEASE PATHWAYS AND DRUG TARGETS

Scouring the Non-Coding Genome by Saturating Edits
Daniel E. Bauer, M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University

BUILDING THE CRISPR TOOLBOX

Beyond Cas9: Discovering Single Effector CRISPR Tools
Jonathan Gootenberg, Member, Laboratories of Dr. Aviv Regev and Dr. Feng Zhang, Department of Systems Biology, Harvard Medical School, and Broad Institute of Harvard and MIT

CRISPR-Cas9 Genome Editing Improves Sub-Cellular Localization Studies
Netanya Y. Spencer, M.D., Ph.D., Research Fellow in Medicine, Joslin Diabetes Center, Harvard Medical School

TECHNOLOGY PANEL: Trends in CRISPR Technologies
Panelists to be Announced

This panel will bring together 2-3 technical experts from leading technology and service companies to discuss trends and improvements in CRISPR libraries, reagents and platforms that users can expect to see in the near future. (Opportunities Available for Sponsoring Panelists)

APPLICATIONS OF CRISPR FOR DRUG DISCOVERY

Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.

Application of Genome Editing Tools to Model Human Genetics Findings in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Genetics and Pharmacogenomics, Merck & Co. Inc.

In vivo Application of the CRISPR/Cas9 Technology for Translational Research
Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

DEVELOPING TOOLS FOR BETTER TRANSLATION

Improving CRISPR-Cas9 Precision through Tethered DNA-Binding Domains
Scot A. Wolfe, Ph.D., Associate Professor, Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School

Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel G. Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Translating CRISPR/Cas9 into Novel Medicines
Alexandra Glucksmann, Ph.D., COO, Editas Medicine

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Gene-Silencing and Gene-Disabling in Pharmaceutical Development

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Down and Out with RNAi and CRISPR

http://www.genengnews.com/gen-articles/down-and-out-with-rnai-and-crispr/5619/

 

RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.

 

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.

RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.

RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.

“CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”

A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.

In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.

Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.

For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.

Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.

Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.

Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.

 

 

“RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”

RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.

 

Arrayed CRISPR Screens

Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.

 

In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.

To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.

GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.

“We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.

“These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”

 

Tool Selection

The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.

“Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.

RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.

 

Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.

 

Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.

Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.

To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.

Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.

The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.

“Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”

 

Converging Technologies

The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.

“From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”

Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.

The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.

Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.

 

Validating Antibodies with RNAi

Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.

The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.

The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.

“In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”

 

Down and Out with RNAi and CRISPR

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

RNA-Based Therapeutics and Vaccines

RNA-based biopharmaceuticals, which includes therapeutics and vaccines, is a relatively new class of treatment and prophylactic for a number of chronic and rare diseases, including cancer, diabetes, tuberculosis, and certain cardiovascular conditions. The field holds great promise in the prevention and treatment of these diseases as demonstrated by early-phase clinical trials as well as significant investment by the drug development community.

Ready, Aim, CRISPR (or RNAi)

Recent progress in probing gene function via the RNAi and CRISPR methods were a strong theme of the Discovery On Target conference. Both methods enable researchers to impair the function of a targeted gene.

Masked RNAi Drug Slips through Membrane, Sheds Guise within Cell

For small interfering RNA, approaching a cell is like walking up to the door of an old speakeasy. Such doors were heavily reinforced and had a narrow, built-in sliding panel at eye level, and if the eyes peering out though the open panel didn’t like the look of you, well, you were not getting inside. Failing to gain entry is something that happens all too frequently to small interfering RNAs, which admittedly are anything but “life of the party” types.

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unchecked spread of engineered genes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Reining in Gene Drives

Researchers have developed two methods to avoid the unchecked spread of engineered genes through wild populations.

By Karen Zusi | Nov 18, 2015    http://www.the-scientist.com//?articles.view/articleNo/44501/title/Reining-in-Gene-Drives/

“Gene drive” is a phenomenon that causes a gene to be inherited at a rate faster than Mendelian principles would dictate. It relies on genes that can copy themselves onto a corresponding location in a paired chromosome, thereby overriding typical allele inheritance patterns. In conjunction with CRISPR/Cas9, gene drives can be created with almost any DNA sequence, raising questions about the risk of engineered genes spreading quickly through a population. But a team of researchers from Harvard University published a study this week (November 16) in Nature Biology that offers some safety constraints on the system.

description of the first CRISPR/Cas9 gene drive system was published in March by a team at the University of California, San Diego, and showed rapid spreading of a normally recessive phenotype inDrosophila. Other labs are researching the system’s potential to wipe out insect-borne diseases such as malaria by spreading mutated genes throughout a mosquito population. But the strategy carries the risk of accidental contamination of wild populations.

“We have a responsibility to keep our experiments confined to the laboratory,” Kevin Esvelt, an evolutionary engineer and coauthor on the paper, told Nature. “The basic lesson is: if you don’t have to build a gene drive that can spread through a wild population, then don’t.”

Esvelt’s team developed safety protocols—ways to prevent or reverse a released gene drive—using the yeast Saccharomyces cerevisia. One technique genetically separates the components necessary to create a gene drive, putting one half directly in the yeast genome and the other half on an external strand of DNA. The researchers also developed a method that uses one gene drive to overwrite the effects of another.

While these techniques are intended to stem the potential for gene drives in light of safety concerns, Esvelt told Nature that he hopes the scientific community will thoughtfully evaluate gene drives rather than dismiss them. “Should we use gene drive to eliminate malaria? Should we use it to replace broadly toxic insecticides? These questions all have to be considered separately,” he told Nature. “This paper is really about making sure we don’t blow it in the meantime and obviate the chance to talk about all of this.”

Safeguarding CRISPR-Cas9 gene drives in yeast

James E DiCarloAlejandro ChavezSven L DietzKevin M Esvelt & George M Church

Nature Biotechnology(2015)       http://dx.doi.org:/10.1038/nbt.3412

RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations could be used to address environmental and public health problems. However, the possibility of unintended genome editing occurring through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeastSaccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.

 

Figure 1: Mechanism and population-level effect of endonuclease gene drives.close

Mechanism and population-level effect of endonuclease gene drives.

(a) Homing endonucleases cut competing alleles, inducing the cell to repair the damage by copying the endonuclease gene. (b) By converting heterozygous germline cells into homozygotes containing two copies (teal), gene drives increase

 

Figure 3: Gene drives and cargo genes remain intact upon copying and can spread by targeting both nonessential and essential genes.close

Gene drives and cargo genes remain intact upon copying and can spread by targeting both nonessential and essential genes.

(a) The ADE2-targeting gene drive was modified to carry URA3 as a cargo gene. (b) Diploids produced by mating wild-typeURA3 haploid yeast with haploids encoding the gene drive carrying URA3 were allowed to sporulate and tetrads dissec…

 

CRISPR Chain Reaction

A powerful new CRISPR/Cas9 tool can be used to produce homozygous mutations within a generation, but scientists call for caution.

By Jenny Rood | March 19, 2015     http://www.the-scientist.com/?articles.view/articleNo/42504/title/CRISPR-Chain-Reaction/

A new genetic-editing technique based on integratingCRISPR/Cas9 technology into a Drosophila melanogaster genome can make homozygous mutants in half the time it would take using traditional crosses, according to a paper published today (March 19) in Science.

“The study is well done and also very elegant,” said Ji-Long Liu of the University of Oxford who was not involved in the research, but helped to develop CRISPR/Cas9 in Drosophila. Liu called the method “a really clever way to . . . make the magic happen.”

 

A rare mosaic female fly, with a lighter left half mutated by MCR and a wild-type darker right half.
UCSD, VALENTINO GANTZ AND ETHAN BIER

 

Safety upgrade found for gene-editing technique

Tweak reduces chance of a mutation escaping into the wild, and can help to undo a mutation after it has spread.

Heidi Ledford          http://www.nature.com/news/safety-upgrade-found-for-gene-editing-technique-1.18799

http://www.nature.com/polopoly_fs/7.31406.1447687781!/image/1.18799.jpg_gen/derivatives/landscape_630/1.18799.jpg

A method that can spread genetic changes rapidly through populations could aid the fight against the malaria parasite, shown here infecting red blood cells.

A genome-editing method that could allow researchers to rapidly engineer entire populations has had an important upgrade. A US team has added safeguards to reduce the chances that such ‘gene drives’ will escape the laboratory, and found a way to erase the genetic mutations after they have spread.

Gene drives hold the potential to wipe out insect-borne diseases and can speed up some genetic studies in the laboratory. But if released into the wild — whether intentionally or not — gene drives could irrevocably scar entire ecosystems.

The safeguards, published today in Nature Biotechnology1, may calm some fears about the technology. One of the techniques provides a way of genetically separating the components that fuel a gene drive, so that the engineered mutation will not spread as rapidly through a population. Another is a molecular ‘undo’ button: sending a second gene drive out to undo the effects of the first.

“We have a responsibility to keep our experiments confined to the laboratory,” says Kevin Esvelt, an evolutionary engineer at the Wyss Institute for Biologically Inspired Engineering at Harvard University in Boston, Massachusetts, and an author of the paper. “The basic lesson is: if you don’t have to build a gene drive that can spread through a wild population, then don’t.”

New life

The concept of a gene drive is an old one that was given new life by the advent of a genome-editing technique called CRISPR–Cas9. It allows researchers to make targeted changes to a genome with unprecedented ease and versatility.

Esvelt and others quickly realized that this technique could be used to engineer a gene drive by incorporating the genes encoding the Cas9 enzyme, which cuts DNA, and the guide RNAs, which direct Cas9 to a specific site, into the genome. Once present on one chromosome, the system can copy itself and the desired genome modification to the other chromosome, thus racing more rapidly through a population than a mutation would normally spread.

The first demonstration of this was published in March2 by developmental biologists Valentino Gantz and Ethan Bier at the University of California, San Diego. The team used gene drives to speed up genetic studies in certain species of fruit flies. But the publication kicked off concerns that the gene drive might escape from the lab into the wild, and the US National Academies of Sciences, Engineering and Medicine tasked a committee with evaluating the benefits and risks of the technology.

Even so, some researchers have embraced the approach, particularly as a means to prevent the transmission of insect-borne diseases such as malaria, says Esvelt. George Church, a bioengineer also at the Wyss Institute and a co-author on the latest report, predicts that gene drives to wipe out malaria and the tick-borne Lyme disease will be developed within the next two years. Esvelt is also collaborating with tropical-disease specialist Paul Brindley of George Washington University in Washington DC to study the application of gene drive to wiping out schistosomiasis, a disease caused by parasitic trematode worms.

Safety measures

But Esvelt worries that an accident could undermine the technique before it has a chance to prove its worth. “If anyone messes up and a gene drive gets out into the wild, there will be a huge media circus,” he says. “The message will be that scientists cannot be trusted to deal with this technology, and we will be set back by years.”

So he and his colleagues decided to develop safety measures using the yeast Saccharomyces cerevisiae. The organism is easy to work with in the laboratory and unlikely to spread a gene drive into wild populations because of its infrequent sexual reproduction.

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