Pu6 lb crrna

pET-dLbCpf1-BE, a plasmid encoding a human codon-optimized dLbCpf1-BE with a His purification tag at the N terminus (His₆-NLS-APOBEC1-XTEN-dLbCpf1(D832A + E925A + D1148A)-NLS-UGI-NLS), was generated using NEBuilder^® HiFi DNA Assembly Master Mix (New England Biolabs) to insert dLbCpf1-BE from pCMV-dLbCpf1-BE (Addgene, #107685) into the pET28a vector (Novagen).
pCMV-dLbCpf1-BE (Addgene, #107685) was modified to incorporate mutations in the LbCpf1 domain (N256A, N260A, S348A, K514A, K881A, or K897A; these mutations respectively correspond to N278A, N282A, S376A, K523A, K949A, or K965A in AsCpf1) and combinations of mutations in the APOBEC1 domain (YE1: W90Y + R126E; YE2: W90Y + R132E; EE: R126E + R132E; YEE: W90Y + R126E + R132E) using site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs). crRNA-encoding plasmids were constructed by ligation (Quick Ligation Kit, New England Biolabs) of annealed oligonucleotides to pU6-Lb-crRNA (Addgene, #78957) digested with BsmBI.

We used human codon-optimized spCas9 [p3s-Cas9HC (Addgene plasmids #43945)], LbCpf1 [pY016 (Addgene plasmids #69988)], and AsCpf1 [pY010 (Addgene plasmids #69982)]. Plasmid DNAs for orthogonal gene manipulation, lenti dCAS-VP64_Blast (Addgene plasmid #61425) and lenti MS2-p65-HSF1_Hygro (Addgene plasmid #61426) were gifts from Feng Zhang. pU6-As-crRNA (Addgene plasmid #78956) and pU6-Lb-crRNA (Addgene plasmid #78957) were used for cloning the gRNA of LbCpf1 and AsCpf1. To construct fgRNA-cloning vector, 5′-scaffold sequences of LbCpf1 (5′-AATTTCTACTAAGTGTAGAT-3′) and AsCpf1 (5′-TAATTTCTACTCTTGTAGAT-3′) were inserted behind the U6 promoter sequences of the Cas9 gRNA-cloning vector. The target sequences of each gRNA are listed in Supplementary Table 1.