Guide sequences were chosen by locating the PAM motif 5′-NNAGAAG-3′ in promoter, 5′-UTR or coding regions and selecting the 20 nucleotides immediately downstream as the protospacer. Forward oligonucleotides included a 5′-GGGAG extension, while reverse oligonucleotides were reverse complements bearing a 5′-AAAC prefix and a terminal 3′C; all oligos were synthesized by Tsingke Biotechnology. Annealed duplexes with compatible overhangs were ligated into the BsmBI-digested backbone and multiplex assemblies followed established protocols; primer pairs are listed in Supplementary Table S1.
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