Expanded targeting scope of LbCas12a variants allows editing of multiple oncogenic mutations

Eunyoung Choi; Hye-Yeon Hwang; Eunji Kwon; Daesik Kim; Taeyoung Koo

doi:10.1016/j.omtn.2022.09.005

Expanded targeting scope of LbCas12a variants allows editing of multiple oncogenic mutations

Mol Ther Nucleic Acids. 2022 Sep 19:30:131-142. doi: 10.1016/j.omtn.2022.09.005. eCollection 2022 Dec 13.

Authors

Eunyoung Choi¹, Hye-Yeon Hwang², Eunji Kwon³, Daesik Kim², Taeyoung Koo^{1

3}

Affiliations

¹ Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea.
² Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Republic of Korea.
³ Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea.

Abstract

RNA-guided CRISPR-Cas12a endonucleases are promising tools for genome engineering. Here we demonstrate that LbCas12a variants derived from Lachnospiraceae bacterium show a broad PAM preference, recognizing certain non-canonical PAMs with high efficiency. Furthermore, we engineered LbABE8e to carry G532R and/or K595R mutations, altering its original PAM specificities; these variants exhibited superior base editing activity in human cells compared with wild-type LbABE8e at sites with non-canonical PAMs. Based on this finding, we utilized the most effective LbCas12a and LbABE8e variants to demonstrate multiplexed and mutant-allele-specific gene editing in oncogenes, made possible by the variant's recognition of non-canonical PAMs. Importantly, LbCas12a-G532R/K595R and LbABE8e-G532R/K595R with optimized crRNA arrays targeted to triple oncogenic mutations inhibited colon cancer cell proliferation. Taken together, these results demonstrate the potential of engineered LbCas12a and LbABE8e as tools for targeting sites with alternative PAMs for genome engineering and therapeutic editing in cancer cells.

Keywords: CRC; CRISPR; LbABE8e; LbCas12a; MT: RNA/DNA editing; base editing; colorectal cancer; gene therapy; non-canonical PAMs; oncogenic mutation.