T4 dna ligase

The protein-coding sequence of AsCas12a (RRID:Addgene_84739) was subcloned into a lentiviral expression vector, EFS-FLAG-P2A-Puro (RRID:Addgene_108100). NLS sequences were incorporated into overlapping DNA oligonucleotides homologous to the Cas-containing backbone by PCR. AsCas12a (E174R, S542R) was cloned in two rounds of mutagenesis PCR on the completed WT AsCas12a-6xNLS vector to generate a final lentiviral vector, opAsCas12a (pRG232, Addgene_149723). All vector cloning was performed using the In-Fusion HD Cloning system (TBUSA).
The AsCas12a crRNA expression vector (pRG212, Addgene_149722) was built by replacing the sgRNA region in the CROPseq-Guide-Puro plasmid (RRID:Addgene_86708) with AsCas12a DRs flanking a short filler that contained BsmbI restriction sites. In addition, the puromycin resistance cassette was replaced with EGFP-P2A-Neomycin, subcloned from LRG2.1-Neomycin (RRID:Addgene_125593).
The resulting pRG212 (EFS-GFP-P2A-Neo-U6-crRNA) vector was BsmbI-digested, and crRNAs were ligated in with T4 DNA ligase (NEB). Single-crRNA and dual-crRNA were cloned by annealing and phosphorylating complementary DNA oligonucleotides with T4 polynucleotide kinase (NEB).

ABEmax‐NG and BE4max were kind gifts from Dr Xingxu Huang's laboratory from ShanghaiTech University. Oligos used for sgRNA construction were synthesized, annealed and cloned into BsaI site of the pGL3‐U6‐sgRNA‐PGK‐puromycin (Addgene, 51133) expression vector with T4 DNA ligase (NEB). The sequences for synthesis are listed in Table S1.