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Characterization of a thermostable Cas12a ortholog

Cell Insight. 2023-10; 
Jing Wu, Pan Gao, Yajing Shi, Caixiang Zhang, Xiaohan Tong, Huidi Fan, Xi Zhou, Ying Zhang, Hao Yin
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Molecular Biology Reagents … gene were synthesized and cloned into the vectors (GenScript Biotech, Nanjing, China). The … the mammalian codon optimized CtCas12a sequence (GenScript) into the modified PY016 … Get A Quote

摘要

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 , 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 -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... More

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