Fig. 3: Multiplexed base editing by Cas12a variant-mediated base editors in HEK293T cells.

a Schematic illustrating a single CRISPR array processed by Cas12a per se to generate multiple mature sgRNAs for simultaneous multiple loci editing. A tRNA precursor sequence appended to downstream of the array is transcribed along with pre-crRNA and cut by RNase P as previously reported⁵¹. Rhombuses with distinctive colors indicate individual spacers. Orange and gray triangles denote the cleavage sites of Cas12a and RNase P, respectively. U6/T7p U6 or T7 promoter; DR direct repeat sequence; tRNA transfer RNA. b Constructs of CRISPR arrays of hcr3 with three tandem sgRNAs and hcr5 with five tandem sgRNAs targeting to human genome. The sequences of individual targets are listed below, and PAMs are marked in purple. Simultaneous base editing of three loci (CFTR, KLF4, and TET1) in HEK293T cells by Cas12a variant-mediated CBEs (c) and ABEs (d) with multiplex-sgRNA (CRISPR array hcr3) or pooled single sgRNAs, with a single sgRNA for a single locus as the control (n = 3). Simultaneous base editing of five loci (DNMT3B, KLF4, TET1, PRR5L, and CFTR) in HEK293T cells by Cas12a variant-mediated CBEs (e) and ABEs (f) with multiplex-sgRNA (CRISPR array hcr5) or pooled single sgRNAs, with a single sgRNA for a single locus as the control (n = 3). Values and error bars for c–f represent the mean and SEM, respectively. Statistical significance was calculated by Welch’s ANOVA test, and Tamhane’s T2 multiple comparisons test was performed. ns (not significant, p ≥ 0.05) is not shown, and only p < 0.05 is shown (*p < 0.05, **p < 0.01).