CRISPR-Cas9 genome editing has revolutionized genomics research and will continue to do so for years to come. The Alt-R® CRISPR-Cas9 System from IDT has all the reagents needed for successful genome editing, and offers numerous advantages over alternative methods. But even with an optimized system like Alt-R, effective genome editing is difficult to achieve. To ensure the best results, consider these points on target selection as you design your experiments.
1. Does your target gene express multiple transcripts?
Due to alternative exon splicing, a single gene can express multiple transcripts, or splice variants. If your target gene expresses splice variants which do not contain your target exon, your gene may retain activity in knockout experiments. Use Ensembl (or other genome databases) to determine whether multiple transcripts exist for your target gene. If so, design the crRNA such that the exon you are targeting is present in every transcript. See Figure 1 for examples of alternatively spliced transcripts to consider.
Figure 1. Constitutive and alternative exon splicing. In constitutive splicing, each exon is joined together in the order it occurs in nuclear RNA (pre-mRNA). Alternative splicing of these exons can produce a variety of mRNA isoforms. The 5 possible alternative splicing events that produce them are: exon skipping, mutually exclusive exons, alternative splice-site selection (5′ and 3′), and intron retention. For an individual pre-mRNA, individual exons can exhibit different types of alternative splicing patterns .
2. Are there SNPs present in your target site?
When single nucleotide polymorphisms (SNPs) are present in your target sequence, the protospacer element of your crRNA may have difficulties binding to it (Figure 2). This can ultimately lead to inefficiencies in genome editing. Check reliable databases, such as the NCBI dbSNP or UCSC Genome Browser, to ensure your target sequence does not contain SNPs.
Figure 2. Components of the Alt-R CRISPR-Cas9 System for directing Cas9 endonuclease to genomic targets. The cleavage site is specified by the protospacer element of the crRNA (thick green bar). The crRNA protospacer element recognizes 19 or 20 nt on the opposite strand of the NGG PAM site.
3. What is the ploidy of your cells?
The existence of multiple alleles will inevitably lead to an increase in the number of possible editing events. For this reason, it is important to take your cells’ ploidy into account, especially when working with triploid or tetraploid cell lines or organisms. While T7EI mismatch endonuclease assays can be effective in estimating editing efficiency , DNA sequencing is the recommended method for identifying the type of editing event(s) that took place, whether in diploid or multiploid cells.
4. Is there a known phenotype associated with the targeted gene?
If you are unaware of your target gene’s associated phenotype, you may wrongly attribute your results to incorrect causal factors. For example, successful genome editing experiments that result in lethality, proliferation, or differentiation can be easily misinterpreted as errors in methodology. Being familiar with expected phenotypic effects will eliminate this confusion and lead to more meaningful results.
5. Are you selecting for monoclonal populations?
If you plan to select clonal populations that contain specific editing events, we recommend checking the editing efficiency of the entire cell population first. By doing this, you can assess the chances of picking individual clones. Also, if you are making limited dilutions, do so as soon as possible after your editing experiment, as non-edited cells could potentially outgrow edited cells.