Department of Gastroenterology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China.
Center of Infectious Diseases, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China.
Biotechnol Bioeng. 2019 Jun;116(6):1463-1474. doi: 10.1002/bit.26938. Epub 2019 Feb 11.
As the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (previously known as Cpf1) system cleaves double-stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a-EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR-Cas12a-assisted one-pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72-100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one-pot DNA editing tool.
作为簇状规律间隔短回文重复 (CRISPR)-Cas12a（以前称为 Cpf1）系统切割双链 DNA 并产生粘性末端，它可以作为 DNA 组装/编辑的有用工具。为了拓宽其应用范围，生成了各种具有扩展原间隔基序 (PAM) 要求的工程化 FnCas12a 蛋白。两种变体（FnCas12a-EP15 和 EP16）将 FnCas12a 的靶向范围扩大了约四倍。它们可以有效地识别广泛的 PAM 序列，包括 YN（Y= C 或 T）、TAC 和 CAA。同时，基于我们证明 FnCas12a 在 16 至 60°C 之间具有活性，我们开发了一种“改进的 CRISPR-Cas12a 辅助一锅 DNA 编辑”（iCOPE）方法，通过在一个锅中结合 crRNA 转录、消化和连接来促进 DNA 编辑。通过应用 iCOPE，两个 DNA 片段组装的编辑效率达到 72-100%，对于 21kb 大的 DNA 构建修饰，编辑效率可达 100%。由于 Cas12a 的优势，iCOPE 仅用一种消化酶即可替代当前的多种限制酶，几乎没有序列限制地在一个锅中进行克隆。总之，本研究扩大了 CRISPR 系统的 DNA 靶向范围，并可作为一种高效无缝的一锅 DNA 编辑工具。
