Department of Bioengineering, University of Illinois at Urbana-Champaign, Champaign, IL, 61801, USA.
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, IL, 61801, USA.
Nat Commun. 2024 May 7;15(1):3823. doi: 10.1038/s41467-024-48012-x.
The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.
CRISPR-Cas12a 系统在特异性和多重性方面比广泛使用的 CRISPR-Cas9 系统更具优势。然而，其靶标编辑效率通常远低于 CRISPR-Cas9 系统。在这里，我们通过简单地将 2-氨基腺嘌呤（碱基 Z，可改变典型的沃森-克里克碱基配对）整合到 crRNA 中，提高了其与互补 DNA 靶标的结合亲和力，从而提高了其靶标编辑效率。由此产生的 CRISPR-Cas12a（此后称为 zCRISPR-Cas12a）显示出与 CRISPR-Cas9 系统相当的靶标编辑效率，但在哺乳动物细胞中的脱靶效应远低于 CRISPR-Cas9 系统。此外，zCRISPR-Cas12a 可用于精确的基因敲入和高效的多重基因组编辑。总体而言，zCRISPR-Cas12a 系统优于 CRISPR-Cas9 系统，我们的简单 crRNA 工程策略也可能扩展到其他 CRISPR-Cas 家族成员及其衍生物。
