Alt r cas12a crrnas

Cas9 gRNAs were prepared by mixing equimolar amounts of Alt-R crRNA and Alt-R tracrRNA (Integrated DNA Technologies, Coralville, IA, USA) in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heating to 95°C and slowly cooling to room temperature or using Alt-R sgRNA (Integrated DNA Technologies) hydrated in IDTE (pH 7.5) (10 mM Tris, pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). Cas12a gRNAs consisted of Alt-R Cas12a crRNAs (Integrated DNA Technologies) hydrated in IDTE (pH 7.5). RNP complexes were assembled by combining the CRISPR-Cas nuclease (Alt-R S.p. Cas9 Nuclease V3 or Alt-R A.s. Cas12a Ultra V3; Integrated DNA Technologies) and the Alt-R gRNA at a 1.2:1 molar ratio of gRNA:protein and incubating at room temperature for 10 min. The target-specific sequences of the gRNAs used in this study are listed in Tables S1 for Cas9 and S2 for Cas12a. The guides chosen were either within the same general genetic context (same amplicon sequencing space; enzyme dependent) or identical between the two cell lines (cell-line dependent) used in this study.

Cas9 gRNAs were prepared by mixing equimolar amounts of Alt-R™ crRNA and Alt-R tracrRNA (Integrated DNA Technologies, Coralville, IA, USA) in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heating to 95 °C and slowly cooling to room temperature or using Alt-R sgRNA (Integrated DNA Technologies) hydrated in IDTE pH 7.5 (10 mM Tris, pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). Cas12a gRNAs consisted of Alt-R Cas12a crRNAs (Integrated DNA Technologies) hydrated in IDTE pH 7.5. RNP complexes were assembled by combining the CRISPR–Cas nuclease (Alt-R S.p. Cas9 Nuclease V3, Alt-R S.p. HiFi Cas9 Nuclease V3, Alt-R S.p. Cas9 D10A V3, Alt-R S.p. Cas9 H840A V3, Alt-R A.s. Cas12a V3, or Alt-R A.s. Cas12a Ultra; Integrated DNA Technologies) and the Alt-R gRNA at a 1:1 to 1.2:1 molar ratio of gRNA:protein and incubating at room temperature for 30 min. For paired nicking experiments, each RNP was formed separately, and two RNPs were mixed together at an equal molar ratio prior to adding to the cells at the time of transfection. The 20-nt target specific sequences of the gRNAs used in this study are listed in Supplementary Table 1.