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Mol. Ther.-Nucl. Acids, Volume 30, 13 Dec 2022, Pages 131-142 | https://doi.org/10.1016/j.omtn.2022.09.005 Expanded targeting scope of LbCas12a variants allows editing of multiple oncogenic mutations

Eunyoung Choi^1,4, Hye-Yeon Hwang^3,4, Eunji Kwon², Daesik Kim³, Taeyoung Koo^1,2

¹Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea
²Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
³Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Republic of Korea
⁴These authors contributed equally

Corresponding author: Daesik Kim, Taeyoung Koo

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.

논문정보

형식Research article
게재일2022년 12월 (BRIC 등록일 2022-12-16)
연구진국내 연구진
분야 생명과학 > 유전자발현

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