Fig. 2: In cellulo characterization of zCRISPR-Cas12a.

a Overall workflow of zCRISPR-Cas12a in cellulo investigation. Created with BioRender.com. b Genome editing performance affected by the number of Z substitution in the spacer region. The crRNAs with the incremental number of A or Z base (from 0 to 12) in the spacer region were used for genome editing. c Editing efficiency of A-crRNA and Z-crRNA mediated AsCas12a at two different endogenous sites with crRNA bearing variable length 3’ end truncations or extensions. d Characterization of the effect of Z substitution in PAM proximal region with seven endogenous sites (left), and the impact shown by the subtracted average indel frequency (the average indel frequency of Z-crRNA minus the average indel frequency of A-crRNA, right). e Characterization of the effect of Z substitution in PAM distal region with seven endogenous sites (left), and the impact shown by the subtracted average indel frequency (right). f Validation of zCRISPR-Cas12a on reported low-editing-efficiency sites. 24 previously characterized low-editing-efficiency sites²⁴ with all types of PAMs were selected to verify the efficacy of zCRISPR-Cas12a in HCT116 cells. A, C, G, and T represent four kinds of PAM sequences: TTTA, TTTC, TTTG, and TTTT. g Performance of coupling Z-crRNA and engineered AsCas12a variant. AsCas12a Ultra was used to further improve the on-target efficiency of ten inefficient enhanced sites in (f). h Relationship between the amount of RNPs and the editing efficiency. Different amounts of RNPs were transfected into HCT116 cells. The indel frequency was measured by NGS after 72 h cell culture. Error bars, s.e.m.; n = 3; nt, nucleotide. Statistical analysis was performed using one-tailed Welch’s t-tests, ns = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Exact p-values are provided in the Source Data.