May 1, 2016 (Vol. 36, No. 9)
Meghaan M. Ferreira Ph.D. Contributor GEN and Clinical OMICs
More Genomes Are Going Under the CRISPR Knife, So Surgical Standards Are Rising
Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.
In the naturally occurring system, CRISPR-Cas9 works like a self-vaccination in the bacterial immune system by targeting and cleaving viral DNA sequences stored from previous encounters with invading phages. The endogenous system uses two RNA elements, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which come together and guide the Cas9 nuclease to the target DNA.
Early publications that demonstrated CRISPR gene editing in mammalian cells combined the crRNA and tracrRNA sequences to form one long transcript called asingle-guide RNA (sgRNA). However, an alternative approach is being explored by scientists at the Dharmacon subsidiary of GE Healthcare. These scientists have a system that mimics the endogenous system through a synthetic two-component approach that preserves individual crRNA and tracrRNA. The tracrRNA is universal to any gene target or species; the crRNA contains the information needed to target the gene of interest.
Predesigned Guide RNAs
In contrast to sgRNAs, which are generated through either in vitro transcription of a DNA template or a plasmid-based expression system, synthetic crRNA and tracrRNA eliminate the need for additional cloning and purification steps. The efficacy of guide RNA (gRNA), whether delivered as a sgRNA or individual crRNA and tracrRNA, depends not only on DNA binding, but also on the generation of an indel that will deliver the coup de grâce to gene function.
“Almost all of the gRNAs were able to create a break in genomic DNA,” said Louise Baskin, senior product manager at Dharmacon. “But there was a very wide range in efficiency and in creating functional protein knock-outs.”
To remove the guesswork from gRNA design, Dharmacon developed an algorithm to predict gene knockout efficiency using wet-lab data. They also incorporated specificity as a component of their algorithm, using a much more comprehensive alignment tool to predict potential off-target effects caused by mismatches and bulges often missed by other alignment tools. Customers can enter their target gene to access predesigned gRNAs as either two-component RNAs or lentiviral sgRNA vectors for multiple applications.
“We put time and effort into our algorithm to ensure that our guide RNAs are not only functional but also highly specific,” asserts Baskin. “As a result, customers don’t have to do any design work.”
Donor DNA Formats
Knockout experiments are a powerful tool for analyzing gene function. However, for researchers who want to introduce DNA into the genome, guide design, donor DNA selection, and Cas9 activity are paramount to successful DNA integration.
MilliporeSigma offers two formats for donor DNA: double-stranded DNA (dsDNA) plasmids and single-stranded DNA (ssDNA) oligonucleotides. The most appropriate format depends on cell type and length of the donor DNA. “There are some cell types that have immune responses to dsDNA,” said Gregory Davis, Ph.D., R&D manager, MilliporeSigma. The ssDNA format can save researchers time and money, but it has a limited carrying capacity of approximately 120 base pairs.
In addition to selecting an appropriate donor DNA format, controlling where, how, and when the Cas9 enzyme cuts can affect gene-editing efficiency. Scientists are playing tug-of-war, trying to pull cells toward the preferred homology-directed repair (HDR) and away from the less favored nonhomologous end joining (NHEJ) repair mechanism.
One method to achieve this modifies the Cas9 enzyme to generate a nickase that cuts only one DNA strand instead of creating a double-strand break. Accordingly, MilliporeSigma has created a Cas9 paired-nickase system that promotes HDR, while also limiting off-target effects and increasing the number of sequences available for site-dependent gene modifications, such as disease-associated single nucleotide polymorphisms (SNPs).
“The best thing you can do is to cut as close to the SNP as possible,” advised Dr. Davis. “As you move the double-stranded break away from the site of mutation you get an exponential drop in the frequency of recombination.”
Another strategy to improve gene-editing efficiency, developed by Thermo Fisher, involves combining purified Cas9 protein with gRNA to generate a stable ribonucleoprotein (RNP) complex. In contrast to plasmid- or mRNA-based formats, which require transcription and/or translation, the Cas9 RNP complex cuts DNA immediately after entering the cell. Rapid clearance of the complex from the cell helps to minimize off-target effects, and, unlike a viral vector, the transient complex does not introduce foreign DNA sequences into the genome.
To deliver their Cas9 RNP complex to cells, Thermo Fisher has developed a lipofectamine transfection reagent called CRISPRMAX. “We went back to the drawing board with our delivery, screened a bunch of components, and got a brand-new, fully optimized lipid nanoparticle formulation,” explained Jon Chesnut, Ph.D., the company’s senior director of synthetic biology R&D. “The formulation is specifically designed for delivering the RNP to cells more efficiently.”
Besides the reagent and the formulation, Thermo Fisher has also developed a range of gene-editing tools. For example, it has introduced the Neon® transfection system for delivering DNA, RNA, or protein into cells via electroporation. Dr. Chesnut emphasized the company’s focus on simplifying complex workflows by optimizing protocols and pairing everything with the appropriate up- and downstream reagents.
From Mammalian Cells to Microbes
One of the first sources of CRISPR technology was the Feng Zhang laboratory at the Broad Institute, which counted among its first licensees a company called GenScript. This company offers a gene-editing service called GenCRISPR™ to establish mammalian cell lines with CRISPR-derived gene knockouts.
“There are a lot of challenges with mammalian cells, and each cell line has its own set of issues,” said Laura Geuss, a marketing specialist at GenScript. “We try to offer a variety of packages that can help customers who have difficult-to-work-with cells.” These packages include both viral-based and transient transfection techniques.
However, the most distinctive service offered by GenScript is its microbial genome-editing service for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). The company’s strategy for gene editing in bacteria can enable seamless knockins, knockouts, or gene replacements by combining CRISPR with lambda red recombineering. Traditionally one of the most effective methods for gene editing in microbes, recombineering allows editing without restriction enzymes through in vivo homologous recombination mediated by a phage-based recombination system such as lambda red.
On its own, lambda red technology cannot target multiple genes, but when paired with CRISPR, it allows the editing of multiple genes with greater efficiency than is possible with CRISPR alone, as the lambda red proteins help repair double-strand breaks in E. coli. The ability to knockout different gene combinations makes Genscript’s microbial editing service particularly well suited for the optimization of metabolic pathways.
Pooled and Arrayed Library Strategies
Scientists are using CRISPR technology for applications such as metabolic engineering and drug development. Yet another application area benefitting from CRISPR technology is cancer research. Here, the use of pooled CRISPR libraries is becoming commonplace. Pooled CRISPR libraries can help detect mutations that affect drug resistance, and they can aid in patient stratification and clinical trial design.
Pooled screening uses proliferation or viability as a phenotype to assess how genetic alterations, resulting from the application of a pooled CRISPR library, affect cell growth and death in the presence of a therapeutic compound. The enrichment or depletion of different gRNA populations is quantified using deep sequencing to identify the genomic edits that result in changes to cell viability.
MilliporeSigma provides pooled CRISPR libraries ranging from the whole human genome to smaller custom pools for these gene-function experiments. For pharmaceutical and biotech companies, Horizon Discovery offers a pooled screening service, ResponderSCREEN, which provides a whole-genome pooled screen to identify genes that confer sensitivity or resistance to a compound. This service is comprehensive, taking clients from experimental design all the way through to suggestions for follow-up studies.
Horizon Discovery maintains a Research Biotech business unit that is focused on target discovery and enabling translational medicine in oncology. “Our internal backbone gives us the ability to provide expert advice demonstrated by results,” said Jon Moore, Ph.D., the company’s CSO.
In contrast to a pooled screen, where thousands of gRNA are combined in one tube, an arrayed screen applies one gRNA per well, removing the need for deep sequencing and broadening the options for different endpoint assays. To establish and distribute a whole-genome arrayed lentiviral CRISPR library, MilliporeSigma partnered with the Wellcome Trust Sanger Institute. “This is the first and only arrayed CRISPR library in the world,” declared Shawn Shafer, Ph.D., functional genomics market segment manager, MilliporeSigma. “We were really proud to partner with Sanger on this.”
Pooled and arrayed screens are powerful tools for studying gene function. The appropriate platform for an experiment, however, will be determined by the desired endpoint assay.
Detection and Quantification of Edits
Finally, one last challenge for CRISPR lies in the detection and quantification of changes made to the genome post-editing. Conventional methods for detecting these alterations include gel methods and next-generation sequencing. While gel methods lack sensitivity and scalability, next-generation sequencing is costly and requires intensive bioinformatics.
To address this gap, Bio-Rad Laboratories developed a set of assay strategies to enable sensitive and precise edit detection with its Droplet Digital PCR (ddPCR) technology. The platform is designed to enable absolute quantification of nucleic acids with high sensitivity, high precision, and short turnaround time through massive droplet partitioning of samples.
Using a validated assay, a typical ddPCR experiment takes about five to six hours to complete. The ddPCR platform enables detection of rare mutations, and publications have reported detection of precise edits at a frequency of <0.05%, and of NHEJ-derived indels at a frequency as low as 0.1%. In addition to quantifying precise edits, indels, and computationally predicted off-target mutations, ddPCR can also be used to characterize the consequences of edits at the RNA level.
According to a recently published Science paper, the laboratory of Charles A. Gersbach, Ph.D., at Duke University used ddPCR in a study of muscle function in a mouse model of Duchenne muscular dystrophy. Specifically, ddPCR was used to assess the efficiency of CRISPR-Cas9 in removing the mutated exon 23 from the dystrophin gene. (Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene and significant enhancement of muscle force.)
Quantitative ddPCR showed that exon 23 was deleted in ~2% of all alleles from the whole-muscle lysate. Further ddPCR studies found that 59% of mRNA transcripts reflected the deletion.
“There’s an overarching idea that the genome-editing field is moving extremely quickly, and for good reason,” asserted Jennifer Berman, Ph.D., staff scientist, Bio-Rad Laboratories. “There’s a lot of exciting work to be done, but detection and quantification of edits can be a bottleneck for researchers.”
The gene-editing field is moving quickly, and new innovations are finding their way into the laboratory as researchers lay the foundation for precise, well-controlled gene editing with CRISPR.