Alt r spcas12a nuclease v3

For the CRISPR-Cas9 system, we used syn-crRNA-TS-crRNA (5′-GCUGUCCCCAGUGCAUAUUCguuuuagagcuaugcu-3′), Hipp11-crRNA (5′-GAACACUAGUGCACUUAUCCguuuuagagcuaugcu-3′), Rosa26-crRNA (5′-ACUCCAGUCUUUCUAGAAGAguuuuagagcuaugcu-3′), Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies, IDT#1072532), and Sp HiFi Cas9 nuclease V3 (Integrated DNA Technologies, IDT#1081060) in this study. The crRNA and tracrRNA were annealed (gRNAs) in a thermocycler (95 °C for 5 min, ramped down to 77 °C at 2 °C/s, then ramped down to 25 °C at 0.1 °C/s) and then stocked as 50 µM gRNAs. Upper- and lower-case letters indicate sequences of the target-specific protospacer region and the tracrRNA fusion domain, respectively. For the CRISPR-Cas12a system, we prepared syn-crRNA-TS-crRNA (5′-uaauuucuacucuuguagauGCUGUCCCCAGUGCAUAUUCAGG-3′), Rosa26-crRNA (5′-uaauuucuacucuuguagauUAGAAGAUGGGCGGGAGUCUUCU-3′), and Alt-R SpCas12a nuclease V3 (Integrated DNA Technologies, IDT#1076158). Here, upper- and lower-case letters indicate sequences of the target-specific protospacer region and the loop domain, respectively. The Cas9-crRNA, tracrRNA, Cas12a-crRNA, and Cas nucleases were obtained from Integrated DNA Technologies.

The DNA cleavage activity was assayed using pCriMGET_9-12a_SpmCherry_c plasmid as a substrate. Mixtures of ribonucleoprotein complex (syn-crRNA-TS-crRNA:tracrRNA (Integrated DNA Technologies, IDT#1072532) and Sp HiFi Cas9 nuclease V3 (Integrated DNA Technologies, IDT#1081060), or syn-crRNA-TS-crRNA and Alt-R SpCas12a nuclease V3 (Integrated DNA Technologies, IDT#1076158) were added into reaction tubes together with 10 × NEB buffer 3.1 and pCriMGET_9-12a_SpmCherry_c plasmid. The mixtures were incubated at 37 °C for 1 h, followed by treatment with 20 mg/mL Proteinase K (Nacalai Tesque) for 10 min at 56 °C to release the DNA substrate from the Cas nuclease. The products of each reaction were electrophoresed on 1.0% agarose gel. As a single digestion control, EcoRV-HF (NEB) digested plasmid DNA was used.