FNLS-BE3-NG was generated using the NEBuilder HIFI DNA Assembly kit (New England Biolabs(NEB) cat# E2621L) according to manufacturer’s instructions, by combining a PCR-amplified FNLS-APOBEC1 DNA from pLenti-TRE3G-FNLS-PGK-Puro (Addgene#110847), PCR-amplified Cas9n-NG DNA from pX330-SpCas9-NG (Addgene#117919), PCR-amplified UGI DNA from pLenti-TRE3G-FNLS-PGK-Puro (Addgene#110847) and an NheI/PmeI-digested piggyBac dox-inducible expression vector PB-TRE-Cas955 (link) including a puromycin selection marker. The evoAPOBEC1-BE4max-NG DNA from pBT375 (addgene#125616) were cloned between the NotI and PmeI sites of the PB-TRE-Cas9 with NotI restriction enzyme site insertion. NEB Stable Competent E. coli (NEB cat# C3040I) was used following the manufacturer’s instructions. Q5 High-Fidelity 2X Master Mix (NEB cat# M0494S) was used for all PCRs. All enzymes and buffers were obtained from New England Biolabs unless otherwise noted. Nuclease-free water (Life Technologies cat# 10977-015) was used for cloning and PCR reactions. All primers and oligos were synthesized by IDT.
Chen Y., Hysolli E., Chen A., Casper S., Liu S., Yang K., Liu C, & Church G. (2022). Multiplex base editing to convert TAG into TAA codons in the human genome. Nature Communications, 13, 4482.
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