State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Department of Clinical Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China.
PLoS Biol. 2024 May 30;22(5):e3002619. doi: 10.1371/journal.pbio.3002619. eCollection 2024 May.
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
CRISPR 相关内切酶 Cas12a 因其易于使用和低脱靶性而成为生物医学研究中强大的基因组编辑工具。然而，Cas12a 的大小严重限制了其临床应用，如腺相关病毒（AAV）为基础的基因治疗。在这里，我们从目前未分类的产甲烷菌的宏基因组组装基因组中鉴定了一种新型紧凑 Cas12a 同源物，称为 EbCas12a。它具有 5'-TTTV-3'（V = A、G、C）的 PAM 序列，是迄今为止报道的 Cas12a 同源物中最小的约 3.47kb。此外，还设计了增强型 EbCas12a（enEbCas12a），使其在多个靶位点在哺乳动物细胞中具有与 AsCas12a 和 LbCas12a 相当的编辑效率和更高的特异性。基于紧凑的 enEbCas12a，开发了一种带有 Cas12a crRNA 的一体式 AAV 递送系统，用于体外和体内应用。总的来说，新型最小的高保真 enEbCas12a，以及首例一体式 AAV 递送 Cas12a 的案例，将极大地推动未来的基因治疗和科学研究。
