The CRISPR-Cas9 system has made gene editing accessible to many more researchers, being both cheaper and easier to use than its predecessors. With increased demand, many more sources and forms of molecular tools are emerging. The two main molecular players are the enzyme Cas9 and the guide RNA (gRNA), a sequence that binds to a specific gene target site and directs Cas9 to act there. This article will discuss commercially available gRNA sources for CRISPR-Cas9 gene editing, and give advice on designing and optimizing gRNA sequences.
Designing gRNAs
Scientists have quickly learned that following some basic tenets of gRNA design can tip the balance toward a more successful experiment. Brett Robb, scientific director at New England Biolabs, strongly recommends that scientists design and use multiple gRNAs because “testing multiple guides is better than optimizing just one,” giving a higher chance of the best gRNA. “Also, as we test gRNAs in vitro and in cultured cells, we find that they can differ in their abilities to make a null allele, or cut with high efficiency, even if they are similar,” he adds.
Louise Baskin, senior product manager of Dharmacon products at GE Healthcare, also recommends care in choosing gRNA target sites. “One pitfall to avoid is restricting all of your gRNA target sites to the earliest exon,” she says. “While conventional wisdom may indicate that this is the most likely way to cause downstream disruption of the coding region, it is also possible that a secondary start site for the gene will recover function.”
Twist Bioscience offers custom DNA oligo pools for CRISPR library generation, in varying pool sizes for different screening scales. “These customized oligo pools for CRISPR screening allow for complete flexibility in the library design, enabling researchers to perform the best possible screens to address their specific experimental questions,” says Patrick Finn, vice president of sales and marketing at Twist Bioscience. Twist’s customers use their oligo pools in functional genomics screens for cancer research, target discovery and validation, and drug discovery. Twist Bioscience has partnered with Desktop Genetics to help researchers design and optimize gRNA sequences. “Not all guide designs are created equal,” says Finn. “The improved library quality will accelerate the pace and scope of CRISPR library screening and discovery.”
Making gRNAs
New England Biolabs offers tools to help researchers make single gRNAs quickly and inexpensively. “It’s our mission to enable every researcher to perform CRISPR-Cas9 editing,” says Robb. Most popular is their EnGen® sgRNA Synthesis Kit, which allows generation and purification of 5−20 micrograms gRNA in under an hour. “You just need a short, single-stranded DNA oligo with your targeting sequence in it,” explains Robb. “There’s no PCR, no cloning, no waiting for chemical synthesis of RNA oligos.” New England Biolabs also offers tools to change the targeting region of a sgRNA in the context of a larger plasmid. The NEBuilder® HiFi DNA Assembly Master Mix can change 20 bases in a 10 kb plasmid, and only requires a single-stranded oligo.
GE Healthcare offers both synthetic and expressed gRNAs with Dharmacon™ Edit-R™ tools. Synthetic RNAs are available as a two-part complex (mimicking the endogenous bacterial system), or as a single synthetic gRNA (preferred, for example, in developing therapeutics). Guide RNAs are available in arrayed libraries as gene families, custom gene collections, or a whole human genome.
Baskin says their most popular gRNA reagent is the synthetic two-part gRNA, because it transfects easily with high efficiency into many cell types. However, for cells that are more difficult to transfect, the lentiviral gRNA is helpful, as viral transduction often overcomes this problem. For CRISPR-mediated knockouts, she recommends their predesigned synthetic or lentiviral gRNA reagents. “They have been designed by a validated algorithm that was trained on quantitative knockout data, so these reagents are more likely to cause a double-strand break that will lead to functional protein knockout,” explains Baskin. “There are many examples of double-strand breaks and subsequent repair that do not actually cause protein disruption.”
Guide RNA plasmids and libraries
Libraries of gRNA are essential tools for fast and efficient high-throughput screening. Cellecta offers pooled gRNA libraries and viral constructs for screening applications, individual constructs that target specific genes, and custom gRNA libraries. Their CRISPR Human Genome Knockout Libraries are available in plasmid and pre-packaged lentivirus forms. Cellecta’s tracrRNA HEAT design modifies the Cas9-binding region of gRNAs to make gene knockouts more effective. “CRISPR libraries made with these modified gRNA constructs generate stronger and more robust results,” says Paul Diehl, COO at Cellecta. While Cellecta offers screening services, many of their customers “want to set up a pipeline to do screens for target validation and discovery, and mechanism discovery,” he explains.
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Addgene, a nonprofit plasmid repository, offers over 2,000 “validated gRNAs” in lentiviral plasmids that depositors have previously used in experiments. Additionally, Addgene offers gRNA libraries at the genome-wide and sub-pool levels, as well as “empty backbones” for various experimental systems so that researchers can clone in the gRNA(s) for their gene of interest.
According to Addgene scientist Mary Gearing, their most popular plasmids are empty gRNA vectors, PX458 and PX459, which contain SpCas9 and selectable markers EGFP and PuroR, respectively. “These tools deposited by Feng Zhang give researchers working in mammalian systems an easy way to make modifications using SpCas9 and then subsequently select for modified cells,” she says. “Lentiviral gRNA backbones like lentiCRISPR v2, also from the Zhang lab, are popular as they allow researchers to create stable cell lines.”
Agilent SureGuide CRISPR libraries focus mainly on pooled libraries for functional genomics and target discovery, including custom gRNA libraries, and libraries with predefined content such as their GeCKO and CRISPR a/i libraries. According to Benjamin Borgo, senior global product manager at Agilent Technologies, Agilent developed a workflow to help researchers maintain library quality through multiple steps of gRNA preparation. “Each of these steps, if done incorrectly, can negate the quality of the underlying library,” says Borgo. “A good workflow for optimizing guide RNAs also makes it easier to detect off-target effects or other anomalies.”
MilliporeSigma offers a wide variety of CRISPR tools, including plasmid DNA, lentiviral tools for screening, and synthetic gRNA for single-gene projects. According to Martha Rook, head of gene editing and novel modalities at MilliporeSigma, researchers want CRISPR tools that cut as close as possible to their target with high efficiency. This is important because it maximizes SNP generation or tag insertions. “In mammalian cells, there is an exponential drop in mutation frequency as the distance between the cut site and mutation site increases,” she says.
Researchers want CRISPR tools that cut as close as possible to their target with high efficiency
MilliporeSigma’s new proxy-CRISPR method helps researchers cut more closely to their target by co-localizing CRISPR systems, for example by using FnCfp1, which has a more flexible PAM sequence. “For researchers attempting to target SNPs in AT-regions, FnCpf1 would have a major advantage over AsCpf1, which has a less flexible PAM sequence,” says Rook.
Looking more broadly at gRNA use in research, Borgo advises scientists to remember that reference genomes are not one-size-fits-all. Using the correct genetic background from the beginning of gRNA design is especially important when studying human diseases. “The functional result of both off-target and epistatic effects of a single knockout can vary significantly from one unique genome to another,” he says. “As CRISPR is used more and more to create new solutions to problems in human health, I expect it will become more and more important.” Even though CRISPR’s recent development as a technique has been rapid, it’s likely that there is much more to come.
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