Allen Aleique, Cooper Brendon H, Singh Jaideep, Rohs Remo, Qin Peter Z
Department of Chemistry, University of Southern California, 3430 S Vermont Ave., Los Angeles, CA, 90089, USA.
Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA, 90089, USA.
Sci Rep. 2025 Feb 10;15(1):4930. doi: 10.1038/s41598-025-87565-9.
Cas12a is a class 2 type V CRISPR-associated nuclease that uses an effector complex comprised of a single protein activated by a CRISPR-encoded small RNA to cleave double-stranded DNA at specific sites. Cas12a processes unique features as compared to other CRISPR effector nucleases such as Cas9, and has been demonstrated as an effective tool for manipulating complex genomes. Prior studies have indicated that DNA flexibility at the region adjacent to the protospacer-adjacent-motif (PAM) contributes to Cas12a target recognition. Here, we adapted a SELEX-seq approach to further examine the connection between PAM-adjacent DNA flexibility and off-target binding by Cas12a. A DNA library containing DNA-DNA mismatches at PAM + 1 to + 6 positions was generated and subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The bound and unbound populations were sequenced to determine the propensity for off-target binding for each of the individual sequences. Analyzing the position and nucleotide dependency of the DNA-DNA mismatches showed that PAM-dependent Cas12a off-target binding requires unpairing of the protospacer at PAM + 1 and increases with unpairing at PAM + 2 and + 3. This revealed that PAM-adjacent DNA flexibility can tune Cas12a off-target binding. The work adds support to the notion that physical properties of the DNA modulate Cas12a target discrimination, and has implications on Cas12a-based applications.
Cas12a是一种2类V型CRISPR相关核酸酶，它利用由CRISPR编码的小RNA激活的单一蛋白质组成的效应复合物在特定位点切割双链DNA。与其他CRISPR效应核酸酶如Cas9相比，Cas12a具有独特的特征，并已被证明是一种用于操纵复杂基因组的有效工具。先前的研究表明，原间隔相邻基序（PAM）附近区域的DNA灵活性有助于Cas12a的靶标识别。在这里，我们采用了一种SELEX-seq方法来进一步研究PAM相邻DNA灵活性与Cas12a脱靶结合之间的联系。生成了一个在PAM +1至+6位置含有DNA-DNA错配的DNA文库，并在RNA引导物与DNA靶标之间不存在配对的情况下，使其与FnCas12a进行体外结合。对结合和未结合的群体进行测序，以确定每个单独序列的脱靶结合倾向。分析DNA-DNA错配的位置和核苷酸依赖性表明，PAM依赖性Cas12a脱靶结合需要PAM +1处原间隔的解配对，并随着PAM +2和+3处的解配对而增加。这表明PAM相邻DNA灵活性可以调节Cas12a的脱靶结合。这项工作为DNA的物理性质调节Cas12a靶标识别这一观点提供了支持，并对基于Cas12a的应用具有启示意义。
