Experimental validation of mononucleotide features associated with crRNA efficiency. (A) Establishment of the in vivo plasmid assay. Plasmid substrates containing target sequences for otx2b_AA/AB, pax2a_AJ and sox19a-KO_4 crRNAs were first injected into 1-cell stage zebrafish embryos and corresponding HiFi Cas9 RNP complexes were subsequently injected to ensure in vivo cleavage reaction. Plasmid and genomic DNAs were extracted from isolated nuclei of injected embryos at 24 hpf for the TIDE analysis. Plasmid substrates were cleaved in a similar manner to genome targets, albeit less efficiently. Means of two injection experiments are shown with standard errors. (B−D) In vivo plasmid assays for mutant crRNAs of the otx2b_AA (B), otx2b_AB (C), and pax2a_AJ (D) using HiFi Cas9. In target sequences on the plasmid, nucleotides with high P-values were mutated into nucleotides with the opposite effect, as indicated by red squares. H and L denote mutations with disfavored to favored nucleotides and vice versa, respectively. Means of two injection experiments are shown as values relative to wild-type crRNA (Mutant/Wild-type), which were obtained by dividing indel frequencies of mutant plasmid targets induced by mutant crRNAs by those for corresponding wild-type crRNAs. Standard errors are also shown. (E) In vivo plasmid assays for mutant crRNAs of sox19a-KO_4 using WT Cas9 and HiFi Cas9. Means of two (WT Cas9) or three (HiFi Cas9) injection experiments are shown as values relative to WT crRNA with standard errors.
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