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. Author manuscript; available in PMC: 2023 Nov 5.
Published in final edited form as: Nat Chem Biol. 2023 Jul 3;19(11):1384–1393. doi: 10.1038/s41589-023-01380-9

Fig. 1 |. Engineering AsCas12f for increased genome-editing efficiency.

Fig. 1 |

a, Domain organization of AsCas12f compared with SpCas9, AsCas12a, DpbCasX, and UnCas12f. HNH, REC, and RuvC domains are indicated. Protein lengths are drawn to scale. aa: amino acid. b, Sequence alignment of AsCas12f and its homologous proteins. Representative regions are shown, with candidates for mutagenesis highlighted in red boxes. c, Workflow to determine the cellular activity of AsCas12f and its variants. d, e, Indel levels at TP53-1 (d) and HEXA (e) loci generated by AsCas12f variants that bear one, two, three, four, or five single-point mutations. A list of mutations included in each AsCas12f variant is provided in Supplementary Fig. 4. Two independent replicates were carried out in HEK293T cells. f, g, Time-course in vitro DNA cleavage by wild-type AsCas12f and enAsCas12f at 37 °C (f) and 50 °C (g). Data points were fitted to one-phase exponential association curves. Two independent replicates were carried out. Gel images are provided in Supplementary Fig. 5.