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Characterization of a thermostable Cas12a ortholog

Authors :
Jing Wu
Pan Gao
Yajing Shi
Caixiang Zhang
Xiaohan Tong
Huidi Fan
Xi Zhou
Ying Zhang
Hao Yin
Source :
Cell Insight, Vol 2, Iss 6, Pp 100126- (2023)
Publication Year :
2023
Publisher :
Elsevier, 2023.

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.

Details

Language :
English
ISSN :
27728927
Volume :
2
Issue :
6
Database :
Directory of Open Access Journals
Journal :
Cell Insight
Publication Type :
Academic Journal
Accession number :
edsdoj.91490d19e4144362836042d735bc527f
Document Type :
article
Full Text :
https://doi.org/10.1016/j.cellin.2023.100126