Special Focus: A crisper picture with CRISPR

CRISPR/Cas9 gene-editing technology continues to make strides in life sciences

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Special Focus on CRISPR

A crisper picture with CRISPR
CRISPR/Cas9 gene-editing technology continues to make strides in life sciences
Even if you don’t know that it stands for clustered regularly interspaced short palindromic repeats, chances are that if you’re reading this magazine you know what CRISPR is, at least in general terms. Genome editing—or the promise of it, at least, before it became practical—has been a big draw for life-sciences research since even long before the Human Genome Project opened up so much of our own genetic information to study and decode. After all, when it comes to life sciences, it isn’t just about editing the human genome for health reasons, as making better animal models is a huge issue as well.
But getting back to CRISPR specifically, scientists in recent years have found out just how effectively they can take advantage of CRISPR’s natural ability to degrade sections of viral RNA and use CRISPR systems to remove unwanted genes from nearly any organism.
“Although CRISPR-Cas9 is the ‘celebrity’ CRISPR system, there are 19 different types of CRISPR systems, each of which may have unique advantages for genetic engineering. They are a massive, untapped resource,” according to biologist Gabriel C. Lander of The Scripps Research Institute (TSRI). “The more we learn about the structures of these systems, the more we can take advantage of them as genome-editing tools.”
As TSRI explains, for many bacteria, one line of defense against viral infection is CRISPR-Cas, which TSRI describes as “a sophisticated RNA-guided immune system” at the center of which is a surveillance complex that recognizes viral DNA and triggers its destruction.
And it is here that TSRI encourages you to envision bacteria and viruses locked in an arms race.
Because, as it turns out, viruses can strike back and disable this surveillance complex using “anti-CRISPR” proteins. The problem was that researchers had not been able to figure out exactly how these anti-CRISPRs work.
Researchers at TSRI, though, say that for the first time, they have solved the structure of viral anti-CRISPR proteins attached to a bacterial CRISPR surveillance complex, revealing precisely how viruses incapacitate the bacterial defense system.
The research team, co-led by Lander—the other half of that leadership being Blake Wiedenheft of Montana State University and the results having been published recently in Cell—discovered that anti-CRISPR proteins work by locking down CRISPR’s ability to identify and attack the viral genome. One anti-CRISPR protein even “mimics” DNA to throw the CRISPR-guided detection machine off its trail.
“It’s amazing what these systems do to one-up each other,” said Lander. “It all comes back to this evolutionary arms race.”
Using the high-resolution imaging technique called cryo-electron microscopy, the researchers discovered several important aspects of CRISPR and anti-CRISPR systems.
First, the researchers saw exactly how the CRISPR surveillance complex analyzes the genetic material of a virus to see where it should attack. Proteins within the complex wrap around the CRISPR RNA like a grasping hand, exposing specific sections of bacterial RNA. These sections of RNA scan viral DNA, looking for genetic sequences they recognize.
“This system can quickly read through massive lengths of DNA and accurately hit its target,” said Lander. If the CRISPR complex identifies a viral DNA target, the surveillance machine recruits other molecules to destroy the genome of the virus.
Next, the researchers analyzed how viral anti-CRISPR proteins paralyze the surveillance complex. They found that one type of anti-CRISPR protein covers up the exposed section of CRISPR RNA, thereby preventing the CRISPR system from scanning the viral DNA.
“These anti-CRISPR proteins keep the bacteria from recognizing the viral DNA,” Lander explained. He called these anti-CRISPR proteins “exceptionally clever” because they appear to have evolved to target a crucial piece of the CRISPR machinery that bacteria cannot mutate to avoid viral attacks because doing so would disable the entire CRISPR system.
They also noticed that another anti-CRISPR protein uses a different trick. Based on its location and negative charge, the researchers believe this anti-CRISPR protein acts as a DNA mimic, fooling CRISPR into binding this immobilizing protein.
The TSRI researchers believe this new understanding of anti-CRISPR proteins may eventually lead to more sophisticated and efficient tools for gene editing.
For example, anti-CRISPR proteins might factor into CRISPR systems as a way to move in to stop gene editing; conversely, degradation of anti-CRISPR proteins could trigger gene editing. In essence, this could allow for what amounts to an on/off switch for CRISPR gene editing.
Shutting it down
And with the TSRI team shifting from the arms race analogy to the on/off switch analogy, we can revisit a story we shared in the February issue, “A flick of the switch,” in which DDNews Managing Editor Kelsey Kaustinen wrote that researchers at the University of California, San Francisco (UCSF) had discovered a way to switch off the widely used CRISPR-Cas9 gene-editing system using newly identified anti-CRISPR proteins that are produced by bacterial viruses.
A large part of why this technique was pursued is for its the potential to improve the safety and accuracy of CRISPR applications both in the clinic and for basic research.
The work, published in Cell near the end of 2016, was led by Dr. Benjamin Rauch, a post-doctoral researcher in the laboratory of Dr. Joseph Bondy-Denomy, who is a UCSF Sandler Faculty Fellow in the Department of Microbiology and Immunology.
While many hope that CRISPR editing techniques will speed efforts to directly treat genetic disorders—in addition to its more general gene-editing utility—there is a problem: for the most part the technology has not yet proven quite precise enough for therapeutic use, sometimes making unintended edits along with the intended ones. As such, there is concern the very power and ease-of-use of CRISPR/Cas9 gene editing could combine to cause great harm in some cases, whether unintentionally or deliberately.
The anti-CRISPR proteins recently discovered by UCSF researchers—which are the first to work against the type of CRISPR-Cas9 system most commonly used right now—could help resolve both problems, Bondy-Denomy says, enabling more precise control in CRISPR applications but also providing a fail-safe to quickly block any potentially harmful uses of the technology.
“Cas9 isn’t very smart,” Bondy-Denomy said. “It’s not able to avoid cutting the bacterium’s own DNA if it is programmed to do so. So we looked for strains of bacteria where the CRISPR-Cas9 system ought to be targeting its own genome—the fact that the cells do not self-destruct was a clue that the whole CRISPR system was inactivated.”
Using a bioinformatics approach designed by Rauch, the team examined nearly 300 strains of Listeria, a bacterial genus famous for its role in food-borne illness, and found that 3 percent of strains exhibited “self-targeting.” Further investigation isolated four distinct anti-CRISPR proteins that proved capable of blocking the activity of the Listeria Cas9 protein, which is very similar to SpyCas9.
Additional experiments showed that two of the four anti-CRISPR proteins—which the researchers dubbed AcrIIA2 and AcrIIA4—worked to inhibit the ability of the commonly used SpyCas9 to target specific genes in other bacteria (such as E. coli) as well as in engineered human cells. Together, the results suggest that AcrIIA proteins are potent inhibitors of the CRISPR-Cas9 gene-editing system as it has been adopted in labs around the world.
“The next step is to show in human cells that using these inhibitors can actually improve the precision of gene editing by reducing off-target effects,” Rauch said. “We also want to understand exactly how the inhibitor proteins block Cas9’s gene targeting abilities, and continue the search for more and better CRISPR inhibitors in other bacteria.”
More options for CRISPR and molecular biology
Early this year, reflecting its drive to provide innovative solutions to the genomics community, Integrated DNA Technologies (IDT) said it had become the first genomics company to develop and bring to market a complete ribonucleoprotein (RNP)-based Cpf1 CRISPR system. The Alt-R A.s. Cpf1 CRISPR System, the company said, “inherits the optimized, efficient and cost-effective traits of IDT’s innovative Cas9-based system while taking advantage of Cpf1’s natural AT-rich target sequence preference and ability to make staggered cuts.”
In addition, IDT launched an associated range of CRISPR support tools to expand experimental options and capabilities for molecular biology researchers. The new tools extend the ease-of-use and performance of IDT’s Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.
Taken all together, the new expanded Alt-R range reportedly “breaks barriers to wider target spaces not addressable by Cas9 systems alone, and provides a level of flexibility in experimental design not previously possible.”
As IDT noted, CRISPR is now one of the most widely used tools for genome modification, and the new Alt-R System is said to already be overcoming the limitations of using sgRNAs in the RNP complex by enhancing editing efficiency and lowering toxicity. Now, in developing a complementary Cpf1-based system, IDT says it has opened up options for targeting AT-rich sequences.
“In the initial development of our Alt-R product portfolio, we improved on the natural Cas9 system through chemical modification of the guide RNAs to create a more active, safer and cost-effective system, supported by our high-quality and high-throughput manufacturing capabilities,” said Mark Behlke, chief scientific officer of IDT. “Our work makes CRISPR more accessible, allowing researchers to work with a wider variety of genomes and to design more innovative studies that were simply not possible before now—and we will continue to support them as they do so.”
CRISPR and discovery work
But it’s not all about the editing. As Horizon Discovery Group plc and Fulcrum Therapeutics Inc. announced late last year, they have entered into a collaboration under which Horizon’s CRISPR-based screening platform will be used to identify novel targets for regulating gene expression. The program will initially focus on genetic diseases where no effective treatment options currently exist.
As Horizon noted generally, its CRISPR-based screening platform, sophisticated bioinformatics and cell line libraries provide a novel and highly efficient way to examine the regulation of genes and their role in disease; more specifically in this collaboration, Horizon will apply its platform and extensive know-how to identify novel gene regulation targets for further exploration by Fulcrum for the development of next-generation therapies.
“CRISPR-based screening promises to be a powerful tool for the identification of novel targets and the collaboration with Fulcrum demonstrates the potential for this technology to be used in areas outside of oncology,” said Dr. Darrin Disley, CEO of Horizon Discovery, adding: “This partnership confirms Horizon’s role as a preferred partner not only for established biotechnology and pharmaceutical companies, but also for start-ups looking for long-term collaborations rather than having to build in-house capabilities.”

On the business side
Mid-March saw eGenesis, a biotechnology company focused on utilizing genome-editing technology to make xenotransplantation a routine and lifesaving medical procedure, announce that it raised a $38-million Series A financing co-led by Biomatics Capital and ARCH Venture Partners, with participation from Khosla Ventures, Alta Partners, Alexandria Equities, Heritage Provider Network, Berggruen Holdings North America Ltd., Uprising and Fan Ventures.
Xenotransplantation is the process of grafting or transplanting organs or tissues to humans from other species, and it “holds vast potential for filling the tremendous medical need for healthy organs for transplantation,” eGenesis notes. In the United States alone, more than 118,000 people are in need of an organ transplant and 22 people die every day because an organ is not available, according the U.S. Health Resources and Services Administration.
“While some challenges remain, our founding team confidently aspires to create a world where patients don’t have to die waiting for an organ transplant,” said Dr. Luhan Yang, chief scientific officer and co-founder of the company along with Harvard Medical School geneticist Dr. George Church. “With this significant investment, we expect to leverage our powerful genome-editing platform to create a pathway toward developing and delivering a safe and effective xenotransplantation solution for patients in need. We hope to see xenotransplantation recognized as a viable resource in the medical community as soon as possible.”
To accomplish this goal, eGenesis is harnessing its CRISPR-based technology platform to deliver safe and effective human transplantable cells, tissues and organs, which are grown in pigs. The eGenesis approach is still in its early stages, but includes genomic engineering of pig cells, organ maturation and finally, successful organ transplantation.
CRISPR Therapeutics licenses cell engineering platform
In other business news of the CRISPR world, CRISPR Therapeutics and Casebia Therapeutics, the company’s joint venture with Bayer, have signed a licensing deal for a cell transfection platform from MaxCyte.
Under the terms of the agreement, CRISPR Therapeutics and Casebia will obtain non-exclusive rights to commercial use of MaxCyte’s Flow Electroporation technology for use in developing CRISPR/Cas9-based therapies for hemoglobin deficiencies and severe combined immunodeficiency.
“It is important we prepare for the future by securing our access to the leading ex-vivo delivery solution for both clinical and commercial use,” CRISPR Therapeutics Chief Business Officer Samarth Kulkarni said in a statement.
MaxCyte’s technology enables transfection of a variety of cell types at high efficiency, the company said. CRISPR Therapeutics and Casebia said they will use the technology to transfect hematopoietic stem cells.
Exonics Therapeutics launches
Exonics Therapeutics Inc., a newly formed biotechnology company focused on developing gene-editing technologies like CRISPR/Cas9 to permanently correct a majority of mutations causing Duchenne muscular dystrophy and other neuromuscular diseases, announced earlier this year a commitment of $5 million in seed financing from CureDuchenne Ventures LLC, a subsidiary of the nonprofit CureDuchenne. The initial seed funding will allow Exonics to advance the preclinical research of its scientific founder and chief science advisor Dr. Eric Olson.
Olson’s laboratory has demonstrated the ability to use adeno-associated virus to deliver a payload based on CRISPR/Cas9 technology that can identify and correct exon mutations that prevent the production of dystrophin, a protein that helps stabilize and protect muscle fibers. Dystrophin is the key protein missing in boys with Duchenne, and published preclinical data suggest that this approach has the potential to permanently treat up to 80 percent of children suffering from the disease. Additional preclinical data is expected to be published in March 2017.
“This represents the next generation of potential Duchenne muscular dystrophy therapies. By leveraging the revolutionary CRISPR/Cas9 method to permanently correct errors in the DNA sequence, it is our hope that we can develop a one-time therapy that provides lifelong benefit to Duchenne patients,” said Olson.
Duchenne is a rare X-linked genetic progressive muscle disease affecting nearly 15,000 boys in the U.S. and more than 300,000 boys worldwide. There is no cure for Duchenne. Children with the disease start missing development milestones at age 3 and often lose their ability to walk by age 12. All Duchenne patients will suffer from reduced mobility and independence, and ultimately respiratory or cardiac failure results in a reduced life expectancy in the mid-20s.

Browsing the libraries
The beginning of this year brought news that Cellecta Inc. had received Phase II SBIR grant funds from the National Institutes of Health to advance development of CRISPR/Cas9 library technology—advances that could significantly improve the performance and increase the utility of genetic screens for disease studies.
The improved screening platform will include genome-wide human and mouse CRISPR libraries and incorporate innovative features, such as multiple expression of sgRNAs that result in more effective irreversible gene disruption, enhanced gene activation (CRISPRa), or gene inhibition (CRISPRi). These technology improvements will be made available to the research community and Cellecta expects this to lead to enhanced drug discovery in many therapeutic areas.
“The work we are carrying out supported by this Phase II grant will accelerate systematic identification of new drug and biomarker targets, as well as facilitate the development of more successful targeted therapeutics,” said Dr. Donato Tedesco, Cellecta’s director of research and development. “Functional genomics screening comes with its unique challenges. We look forward to meeting these challenges with a significant improvement in what is becoming a go-to technology—CRISPR/Cas9—which we hope will drive meaningful discoveries to benefit human health.”
The advanced CRISPR libraries reportedly will improve effectiveness of gene knockout in CRISPR knockout applications, will increase promoter activation in CRISPRa and repression in CRISPRi applications for greater efficiency and will reduce off-target effects for greater specificity. The toolset now in development includes more efficient, genome-wide pooled sgRNA human and mouse CRISPR lentiviral libraries; a greater breadth of CRISPR-based libraries that enable more diverse genetic screens in a wider range of cell models; and expert services for custom library design, functional genetic screening and hit confirmation/validation assays.
Agilent launches SureGuide
In older but still relevant CRISPR library news, Agilent Technologies Inc. in late 2016 launched a comprehensive offering of pooled CRISPR libraries for functional genomics. SureGuide harnesses Agilent’s industry-leading oligonucleotide synthesis platform to create CRISPR guide libraries, which are a critical component of the CRISPR/Cas genome-editing system. The flexibility of Agilent’s SureGuide platform enables CRISPR-based functional screening for any application, from genome-wide knock-outs to fully customized, user-designed libraries.
As Agilent notes, the introduction of genome engineering tools based on CRISPR has rapidly accelerated research related to functional studies of complex diseases and drug discovery. Genetic screens using pooled libraries are typically performed to locate and identify genes that are involved in cellular response, such as in signaling pathways, or to discover the function of novel genes, the company adds, and the introduction of CRISPR-based tools has provided an opportunity to overcome the limitations of previous technologies used in functional screening.
As a key component in any CRISPR experiment, guide library quality and composition can affect all of the downstream segments of the workflow including screening effort, sequencing cost and false positive/negative identification—reportedly, SureGuide offers high-fidelity CRISPR guides alongside SureVector cloning technology, which enable an optimal distribution of guides to be maintained across even the most complex libraries.
“The new libraries represent the first of several planned products in a CRISPR-focused portfolio, supporting every aspect of the CRISPR screening workflow all the way through analysis and hit validation,” said Herman Verrelst, Agilent’s general manager of the Genomics Solutions Division and Clinical Applications Division.
First of its kind in CRISPR RNA
Fall 2016 saw the arrival of the Dharmacon Edit-R Human Druggable Genome crRNA Library from GE Healthcare’s Life Sciences business, which reportedly enables screening of nearly 8,000 individual targets with CRISPR-Cas9 gene knockout. The offering is said to be the first arrayed synthetic CRISPR RNA (crRNA) library of its kind, and the company says it helps provide in-depth insight into a range of wide-reaching biological questions, adding, “With an emphasis on characterized genes of interest in drug discovery, it offers a powerful screening resource to identify potential therapeutic targets.”
Arrayed screening lets researchers overcome the limitations of selectable or sortable assays that are required for pooled library screening. Edit-R Druggable crRNA Library delivers more detailed one-gene-per-well information by enabling high-content and multiparametric assays to easily characterize complex phenotypes.

Perturb-seq for CRISPR-based perturbations
CRISPR-based single-cell genetics platform developed by Broad Institute and UCSF scientists enables rapid analysis of critical gene networks
CAMBRIDGE, Mass. & SAN FRANCISCO—Researchers from the Broad Institute of MIT and Harvard and from the University of California, San Francisco (UCSF) have developed a new method for performing high-throughput functional screening of complex genetic interactions and resulting phenotypes in single cells, which they have dubbed Perturb-seq.
The findings could greatly speed scientists’ ability to map gene interactions and responses to environmental stimuli to advance understanding of healthy gene networks, and how they go awry in the context of disease.
Described in two co-authored papers in the Dec. 15 issue of Cell, one led by Broad Institute researchers and one led by UCSF researchers, the Perturb-seq platform uses single-cell RNA sequencing to measure the effects of many CRISPR-based perturbations on large numbers of cells. The method can be used in many experimental applications, such as exploring the functional impact of genetic risk factors from genomic studies more efficiently than has been previously possible, or looking at genes mutated in cancer cells.
Working collaboratively, the Broad and UCSF teams used Perturb-seq to make new discoveries about, respectively, the immune response in dendritic cells, a cell type that acts as a critical messenger within the immune system, and the unfolded protein response, a cellular stress pathway implicated in a number of neurodegenerative disorders, demonstrating the potential of this platform to yield insight on a variety of biological questions.
“In Perturb-seq, we combined pooled CRISPR screens with the information-rich readout of droplet-based single-cell RNA sequencing, to give us a powerful tool that dramatically increases the scope of what we can learn from functional genomic screens about how circuits are wired inside cells,” said Aviv Regev, senior author of the Broad-led study, a professor of biology at MIT, a core faculty member at the Broad and a Howard Hughes Medical Institute investigator. “In particular, we can understand how ‘the whole is greater than the sum of its parts,’ that is, why perturbing two different genes together gives an effect that is different than perturbing each of them alone. This will help us predict better which genes to target in disease as we develop therapies.”
“Perturb-seq brings two technologic advances—CRISPR-based perturbations and massively parallel single-cell RNA sequencing—together in a way that we think will greatly speed our ability to understand how different genes that encode for the components of cells are normally wired together, and what goes wrong in human disease,” said Jonathan Weissman, a professor of cellular and molecular pharmacology at UCSF, a Howard Hughes Medical Institute investigator and senior author of the UCSF-led study.
“Functional genomics studies can shed light on the connection between genotype and phenotype, but we’d like to also understand the mechanistic relationships between the two,” added Atray Dixit, co-first author of the Broad-led study and a graduate student at MIT. “Looking at the RNA level is a great place to start.”
In the Broad-led experiments, researchers used CRISPR/Cas9 nucleases to cut DNA and inactivate genes for transcription factor proteins (TFs) involved in the immune response in dendritic cells, and to inactivate genes for TFs and cell cycle regulators in a cancer cell line. Perturb-seq accurately identified individual gene targets, gene signatures and cell states affected by the individual gene modifications and explored how these genes interact and depend on one another.
“In the past, functional screens have had to choose between either measuring simple phenotypic changes induced by many perturbations, or taking a rich, high-resolution look at a limited number of perturbations,” said Oren Parnas, co-first author of the Broad-led study, a former postdoctoral researcher at the Broad Institute, and currently a researcher at the School of Medicine of the Hebrew University of Jerusalem. “With Perturb-seq, we can scale up the experiment on both ends.”
In the UCSF-led experiments, researchers used CRISPR-based transcriptional interference (CRISPRi) to simultaneously repress up to three genes in a cancer cell line and to investigate the unfolded protein response (UPR), a well-known quality control pathway that senses stress in the endoplasmic reticulum (ER), where many of a cell’s key proteins are made. The UPR detects errors in cells’ protein-production machinery and ensures that impaired cells self-destruct.
After identifying hundreds of genes whose functions are monitored by the UPR in a genome-wide CRISPRi screen, the UCSF-led team applied Perturb-seq to interrogate a subset of these genes with single-cell resolution, allowing the researchers to systematically reveal the relationships between the genes and dissect the complex ways in which cells respond to ER stress.
“With the unbiased readout we get from single-cell RNA sequencing, we can potentially discover things about biological pathways without a prior hypothesis,” said Tom Norman, co-first author of the UCSF-led study and a postdoctoral researcher in the Weissman lab. “It opens up new possibilities that might not be evident from more targeted studies.”

Desktop Genetics, Vectalys and Horizon Discovery collaborate
LONDON—Desktop Genetics Ltd., a software company looking to  revolutionize the way researchers use CRISPR genome-editing technology, announced late last year a collaboration agreement with Vectalys SAS and Horizon Discovery Group plc to design, produce and commercialize custom-made CRISPR lenti-gRNA libraries.
The agreement will provide customers with access to fully licensed lentivirus and CRISPR libraries designed for their specific experiment and cell line. The libraries will allow researchers to perform precise edits to the genome of cells, including a variety of cell lines that might otherwise be out of reach with standard CRISPR delivery methods. Incorporating Desktop Genetics’ expertise in gRNA library design and selection, the customized libraries are predicted to generate fewer off-target events and therefore improved accuracy of results.
Under the terms of the agreement, Desktop Genetics will provide its experience and expertise in CRISPR guide library design and methods and algorithms for gRNA library design and selection. This will be combined with Vectalys’ expertise and technologies in the design and production of lentiviral particle libraries and Horizon Discovery’s expertise in lenti-gRNA library applications.
“This deal augments our already-powerful CRISPR library design technologies with the capability to deliver custom-designed guides into a wide range of cell models with high efficiency,” said Riley Doyle, CEO of Desktop Genetics. “We are excited to be working with globally-recognized experts in lentiviral manufacturing. Through this collaboration we can now provide our customers with even more flexibility for their CRISPR experiments and screens in their search for novel druggable targets.”

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