Genome editing is a powerful technique that is having a tremendous impact across many areas of biological research and model systems. The simple-to-use CRISPR-Cas9 system especially has seen remarkable adoption, from forward- and reverse genetic screening to gene therapy. Variations on the system now allow researchers to direct a variety of tools to specified genomic loci, to affect a host of applications from epigenomic alteration to imaging.
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Yet despite its simplicity, ensuring a CRISPR experiment is as efficient as possible requires effort in the planning, execution, and analysis. This article examines some of the tools and strategies that help optimize gene-editing workflows and maximize the likelihood of experimental success.
What is CRISPR?
Earlier techniques such as TALENs and zinc-finger nucleases (ZFNs) use an engineered nuclease to cut the genome at a specified location, with each nuclease designed for that specific locus. What the CRISPR-Cas system brings to the toolbox is the ability of a specific guide RNA (sgRNA) to direct a generic Cas nuclease to a locus of interest. sgRNA is far easier, faster, and less expensive to engineer and produce than ZFN or TALEN proteins. Unique sgRNAs can be pooled or placed into array formats to use for screening.
DNA cut by Cas will generally result in repair by endogenous processes (non-homologous end joining, NHEJ), either accurately or more likely with insertions or deletions, that may render the gene non-functional (create a knockout). If a donor template with homology to the sequence surrounding the break is available, it may be inserted into the break by the endogenous homology-directed repair (HDR) process in a small percentage of cells. As these are probabilistic occurrences, it is necessary to determine what actually has occurred.
Choices
In planning a CRISPR experiment, as most experiments, there are choices to be made.
Among these is how components are to be delivered. For example, Cas-expressing cell lines are readily available, giving the experiment a head start. The nuclease can also be introduced to the cells as Cas-encoding DNA, or mRNA, or protein along with, or separately from, the sgRNA (and template). Transfection, transduction, electroporation, and incorporation into ribonucleoparticles (RNPs), among other techniques, have all been explored.
The amount and timing of expression—as may be influenced by the choice between transient transfection and lentiviral transduction—can have an effect on the frequency and even the fidelity of editing, among other things. Different versions of Cas (with low- or high expression, from different species, that have different recognition requirements, or use inducible promoters) are options, as are vehicles allowing control of the sgRNA and template. Note that this article generally uses the term “Cas” rather than “Cas9” to acknowledge the range of CRISPR nucleases beyond Cas9.
Another choice that researchers doing screening are faced with is whether to pool the sgRNAs or arrange them in arrays. Pooled screens typically are more cost effective, require no special equipment, and can be used to interrogate the entire genome. They require a binary readout that can be obtained after physically separating out successfully edited cells exhibiting a given phenotype—such as antibiotic resistance, for instance, or a FACS-sortable marker—followed by genotyping. Arrayed screens, on the other hand, can be multi-parametric and are compatible with sophisticated high-content assays. Selection and data deconvolution are not required.
Designer sgRNA
Cas proteins bind to DNA near a specific sequence called a short protospacer-adjacent motif (PAM). A host of bioinformatic tools—both web-based and downloadable code—exist to aid researchers in designing sgRNAs that will direct the nuclease to cut at the desired locus.
Among the most important features of such tools is to identify homologous sequences in the genome, and to predict (and generally maximize) on-target activity as well as avoid off-target activity. Some tools are able to generate guides for other enzymes, such as Cas12a, that may preferentially recognize a different PAM. Different design tools utilize different algorithms prioritizing different parameters, and may return slightly different results.
Regardless of how reliable an in-silico prediction of sgRNA on- and off-target activity is, it is still imperative to verify empirically that the enzyme is cutting where it is expected to cut, and to determine where else in the genome is being affected. In vitro endonuclease cleavage assays can characterize the efficacy of a guide RNAs to form a cleavage-competent complex. A variety of methods are used to detect the results, including mismatch-sensitive enzymes, RFLP analysis, melting curve analysis, and PCR amplification.
Up the game
The use of high-fidelity enzymes allows for less off-target editing.
Open chromatin correlates with cleavage efficiency. It may behoove a researcher to transiently overexpress a targeted transcriptional activator when editing closed chromatin regions. The use of multiple guides per gene helps assure that that gene has been disrupted.
NHEJ is more likely to occur than HDR. To shift the balance to (the desired) insertion, various strategies have been identified that can help improve the efficiency of a CRISPR-mediated gene knock-in. For example, rather than using double-stranded DNA as donor, chemically modified single-stranded oligos have been shown to be more stable templates. Using longer (single-stranded) templates is also associated with an improvement in insertion efficiency.
Multi-trick pony
The CRISPR-Cas system has been adapted to accomplish tasks well beyond double-stranded break-and-repair. These are generally based on a mutant (nuclease-deficient) Cas, often fuses to another functional protein motif, and so most principles discussed above—such as delivery, guide design, and genetic control of the components—carry forward.
CRISPR has been used to target RNA, or to create epigenetic modification. It can allow for purification of any genomic sequence—expanding on ChIP—specified by the sgRNA.
Among the most exciting takes on CRISPR-Cas is its use in individual base editing—changing a single nucleotide of choice without even the need to create a double-stranded break in the DNA.
Whatever the task at hand, the power of CRISPR for basic research and medical advancements seems almost limitless. Use it well and wisely.