Figure 2.

LAHR utilizes both the homologous arm and the sticky end of the repair template. (A) Schematic of the EGFP^Y66Sreporter sequence and the repair template. The dsDNA region between T180 and G207 indicates the AsCas12a editing region, in which the silent mutations, C180T and C181T (blue uppercases) were introduced to generate an AsCas12 PAM sequence (underlined by a green arrow, and the arrow orientated the direction of the PAM sequence); the cleavage site is indicated by green arrowheads; the missense mutation A200C (red uppercase) causes a tyrosine-to-serine substitution (Y66S) that eliminated the EGFP fluorescence. The repair template contained a sticky end, which was compatible to the AsCas12a-generated distal sticky end on the reporter gene, and a homologous arm (green box). Adjacent to the sticky end of the repair template a repairing A/T base pair (green uppercases) was introduced to restore the codon of tyrosine. (B) Correction of the A200C mutation using repair templates with a same sticky end, but varying lengths of the homologous arms (from 20 bp to 200 bp). Indicated percentage of EGFP-positive cells was determined by flow cytometry (See also supplementary figure S12). Error bars indicate the standard deviation of the average of n = 3 parallel samples. The experiment was repeated three times and a representative dataset is presented here. Statistical test: two-tailed unpaired t-test, ns P > 0.05, * P < 0.05, ** P < 0.01. (C) Correction of the A200C mutation using repair templates sharing a same 80-bp homologous arm, but with indicated sticky ends. Indicated percentage of EGFP-positive cells was determined by flow cytometry (See also supplementary figure S12). Error bars indicate the standard deviation of the average of n = 3 parallel samples. The experiment was repeated three times and a representative dataset is presented here. Statistical test: two-tailed unpaired t-test, ns P > 0.05, * P < 0.05, *** P < 0.001.