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The Alt-R® CRISPR-Cas9 crRNA ordering tool (accessible here) accommodates 19 and 20 nucleotide (nt) protospacer sequences; however, we recommend 20 nt sequences for most experiments. Other formats can be ordered as custom RNAs.
There are reports in the literature suggesting that CRISPR-Cas9 nuclease specificity can be improved through use of truncated guide RNAs. For example, 17 nt protospacer elements have been reported to reduce off-target effects.
In contrast, our research investigating the effect of shorter protospacer element length on CRISPR-Cas9 nuclease specificity demonstrated that 20 nt protospacer elements were optimal, with 19 nt protospacers providing similar strong editing efficacy in most cases (see figure). When using Alt-R S.p. HiFi Cas9 Nuclease, 20 nt protospacer sequences provide the greatest amount of genomic editing (data not shown).
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