Sp hifi cas9 nuclease v3

Alt-R CRISPR-Cas9 reagents (S.p. HiFi Cas9 Nuclease V3, Electroporation enhancer, tracrRNA ATTO550, and crRNAs) and ssODN (HDR Donor Oligos) were purchased from Integrated DNA Technologies. PCR primers were obtained from GeneriBiotech. Nano-Glo HiBiT Lytic Detection System and Nano-Glo HiBiT Extracellular Detection System were purchased from Promega. Antibodies: mouse anti-human CFTR antibodies (569, TJA9; CF Foundation), donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor 647; Invitrogen). siRNA: CFTR Human siRNA Oligo Duplex (Locus ID 1080; Origene), RAB5 (RAB5A) Human siRNA Oligo Duplex (Locus ID 5868), and RAB11 Human siRNA Oligo Duplex (Locus ID 8766).

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.