Characterization of a thermostable Cas12a ortholog

Jing Wu; Pan Gao; Yajing Shi; Caixiang Zhang; Xiaohan Tong; Huidi Fan; Xi Zhou; Ying Zhang; Hao Yin

doi:10.1016/j.cellin.2023.100126

Characterization of a thermostable Cas12a ortholog

Cell Insight. 2023 Oct 11;2(6):100126. doi: 10.1016/j.cellin.2023.100126. eCollection 2023 Dec.

Authors

Jing Wu^{1

2}, Pan Gao^{1

3}, Yajing Shi^{1

3}, Caixiang Zhang^{1

3}, Xiaohan Tong^{1

2}, Huidi Fan⁴, Xi Zhou⁴, Ying Zhang^{1

3}, Hao Yin^{1

2

5

6}

Affiliations

¹ Department of Clinical Laboratory, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430071, China.
² State Key Laboratory of Virology, TaiKang Centre for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, 430071, China.
³ Department of Rheumatology and Immunology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430071, China.
⁴ State Key Laboratory of Virology, Wuhan Institute of Virology, Wuhan, 430071, China.
⁵ Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, 430071, China.
⁶ Department of Urology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430071, China.

Abstract

CRISPR-Cas12a has been used for genome editing and molecular diagnosis. The well-studied Cas12a orthologs have a T-rich PAM and are usually categorized as non-thermally stable enzymes. Here, we identified a new Cas12a ortholog from Clostridium thermobutyricum, which survives at 60 °C. This Cas12a ortholog is named as CtCas12a and exhibits low sequence similarity to the known Cas12a family members. CtCas12a is active in a wide temperature range from 17 to 77 °C. Moreover, this ortholog has a relaxed PAM of YYV (Y=C or T, V = A or C or G). We optimized the conditions for trans-cleavage and enabled its detection of nucleic acids. CtCas12a executed genome editing in human cells and generated up to 26% indel formation in the EGFP locus. With the ability to be active at high temperatures as well as having a relaxed PAM sequence, CtCas12a holds potential to be further engineered for pathogen detection and editing a wide range of genomic sequences.