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There are reports in the literature suggesting that CRISPR-Cas9 nuclease specificity can be improved by using truncated guide RNAs [Fu Y, Sander JD, et al. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotech, 32(3):279–284.]. For example, 17mer protospacer elements have been reported to reduce off-target effects. However, our research investigating how shortening protospacer element length would affect CRISPR-Cas9 nuclease specificity demonstrated that 20mer protospacer elements were optimal, with 19mers providing similar strong editing efficacy in most cases (See Figure 6 on the Performance tab of the Alt-R CRISPR-Cas9 System product page). Our Alt-R CRISPR configurator accommodates 19 and 20 mer protospacer sequences only. Other formats can be ordered as custom RNAs.
Figure: 19–20 nt protospacer element provides optimal genome editing.
crRNAs with varying protospacer element lengths (17–20 nt) were designed to 12 distinct HPRT target sites. crRNA:tracrRNA complexes were reverse transfected using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) into a HEK293 cell line stably expressing S. pyogenes Cas9. Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7EI mismatch endonuclease (New England Biolabs) and analysis using the Fragment Analyzer™ (Advanced Analytical). At all but one of the 12 target sites, crRNAs with 19 and 20 base protospacer elements provided the greatest amount of genomic editing.