School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China; School of Marine Sciences, Ningbo University, Ningbo 315832, Zhejiang, China.
School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Mol Cell. 2023 Aug 3;83(15):2768-2780.e6. doi: 10.1016/j.molcel.2023.06.014. Epub 2023 Jul 3.
Type V CRISPR-associated systems (Cas)12 family nucleases are considered to have evolved from transposon-associated TnpB, and several of these nucleases have been engineered as versatile genome editors. Despite the conserved RNA-guided DNA-cleaving functionality, these Cas12 nucleases differ markedly from the currently identified ancestor TnpB in aspects such as guide RNA origination, effector complex composition, and protospacer adjacent motif (PAM) specificity, suggesting the presence of earlier evolutionary intermediates that could be mined to develop advanced genome manipulation biotechnologies. Using evolutionary and biochemical analyses, we identify that the miniature type V-U4 nuclease (referred to as Cas12n, 400-700 amino acids) is likely the earliest evolutionary intermediate between TnpB and large type V CRISPR systems. We demonstrate that with the exception of CRISPR array emergence, CRISPR-Cas12n shares several similar characteristics with TnpB-ωRNA, including a miniature and likely monomeric nuclease for DNA targeting, origination of guide RNA from nuclease coding sequence, and generation of a small sticky end following DNA cleavage. Cas12n nucleases recognize a unique 5'-AAN PAM sequence, of which the A nucleotide at the -2 position is also required for TnpB. Moreover, we demonstrate the robust genome-editing capacity of Cas12n in bacteria and engineer a highly efficient CRISPR-Cas12n (termed Cas12Pro) with up to 80% indel efficiency in human cells. The engineered Cas12Pro enables base editing in human cells. Our results further expand the understanding regarding type V CRISPR evolutionary mechanisms and enrich the miniature CRISPR toolbox for therapeutic applications.
V 型 CRISPR 相关系统（Cas）12 家族核酸酶被认为是从转座子相关的 TnpB 进化而来的，其中一些核酸酶已被工程化为多功能基因组编辑工具。尽管具有保守的 RNA 指导的 DNA 切割功能，但这些 Cas12 核酸酶在引导 RNA 起源、效应复合物组成和原间隔序列邻近基序（PAM）特异性等方面与目前鉴定的祖先 TnpB 明显不同，这表明存在早期进化中间体，可以从中挖掘开发先进的基因组操作生物技术。通过进化和生化分析，我们确定微型 V 型-U4 核酸酶（称为 Cas12n，400-700 个氨基酸）可能是 TnpB 和大型 V 型 CRISPR 系统之间最早的进化中间体。我们证明，除了 CRISPR 阵列的出现外，CRISPR-Cas12n 与 TnpB-ωRNA 具有几个相似的特征，包括用于 DNA 靶向的微型和可能的单体核酸酶、从核酸酶编码序列起源的引导 RNA 以及 DNA 切割后产生小粘性末端。Cas12n 核酸酶识别独特的 5'-AAN PAM 序列，其中-2 位的 A 核苷酸也是 TnpB 所必需的。此外，我们证明了 Cas12n 在细菌中的强大基因组编辑能力，并通过在人类细胞中高达 80%的插入缺失效率工程化了一种高效的 CRISPR-Cas12n（称为 Cas12Pro）。工程化的 Cas12Pro 能够在人类细胞中进行碱基编辑。我们的研究结果进一步扩展了对 V 型 CRISPR 进化机制的理解，并丰富了用于治疗应用的微型 CRISPR 工具包。
