Construction of Inducible CasRx Lentiviral Vector

The pLenti-TRE-CasRx-U6-gRNA vector was prepared by PCR, restriction digestion, and ligation. The U6 promoter and PAC were amplified from pLenti-CRISPR-V2 (addgene 52961). An SV40 early promoter was amplified from plenti4 (invitrogen). TRE was cut from pLenti6-CMV-Tight-DEST (Addgene_26433). rtTA3 was amplified and assembled from pLenti-CMV-rtTA3 Blast (Addgene 26429) and Fuw-M2-rtTA (Addgene 20342).
gRNA sequences were picked from the online source https://cas13design.nygenome.org/ (Wessels et al., 2020 (link)). Forward primers with 5′-AAAC overhang and reverse primer with 3′-AAAA overhang were annealed together and treated with polynucleotide kinase. Afterward, annealed oligo was ligated to pLenti-TRE-CasRx-U6 cut with BsmBI and treated with alkaline phosphatase. Sequences of all insertions were then verified by Sanger sequencing. Guide RNA sequences used are listed in Supplementary Table S1.