Characterization of a thermostable Cas12a ortholog

Cell insight Pub Date : 2023-10-11 DOI:10.1016/j.cellin.2023.100126

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

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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 (YC 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.

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耐热Cas12a同源物的表征

CRISPR-Cas12a已被用于基因组编辑和分子诊断。研究充分的Cas12a同源物具有富含t的PAM，通常被归类为非热稳定酶。在这里，我们从热丁酸梭菌中鉴定了一个新的Cas12a同源物，它可以在60°C下存活。该Cas12a同源基因被命名为CtCas12a，与已知的Cas12a家族成员序列相似性较低。CtCas12a在17 ~ 77℃的宽温度范围内具有活性。此外，该同源物具有YYV的松弛PAM (YC或T, V = a或C或G)。我们优化了反式切割的条件，使其能够检测核酸。CtCas12a在人类细胞中进行基因组编辑，并在EGFP位点产生高达26%的indel形成。由于CtCas12a在高温下具有活性，并且具有宽松的PAM序列，因此CtCas12a具有进一步设计用于病原体检测和编辑广泛基因组序列的潜力。

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