a Department of Rutgers University, Rutgers The State University of New Jersey, Waksman Institute of Microbiology , Piscataway , NJ , USA.
b Skolkovo Institute of Science and Technology , Center of Life Sciences , Skolkovo , Russia.
RNA Biol. 2019 Apr;16(4):413-422. doi: 10.1080/15476286.2018.1495492. Epub 2018 Aug 17.
Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.
CRISPR-Cas 核糖核蛋白效应物的靶标结合是由 Cas 蛋白部分识别双链 PAM 基序引发的，随后是靶标的不稳定、局部融化以及 CRISPR RNA 部分的引导部分对靶标的询问。后一过程取决于种子序列，即靶标中必须与 CRISPR RNA 引导严格互补的部分。靶标和 CRISPR RNA 引导序列之外的种子序列中的错配仅对靶标结合产生较小的影响，从而导致 CRISPR-Cas 效应物的脱靶活性。在这里，我们定义了来自嗜热解淀粉芽孢杆菌的 V 型 Cas12b 效应物的种子序列。虽然 Cas12b 的种子只有五个碱基长，但与迄今为止表征的所有其他效应物不同，靶标切割位点的核苷酸碱基对靶标结合有很强的贡献。在分析来自嗜酸耐热杆菌的 Cas12b 效应物的靶标识别时，确认了这种额外要求的普遍性。因此，虽然短种子可能导致 Cas12b 的混杂性，但在切割位点的额外特异性决定因素可能具有补偿作用，使 Cas12b 适合专门的基因组编辑应用。
