Jiang Yuewen, Pan Qihua, Wang Zhi, Lu Ke, Xia Bilin, Chen Tiansheng
Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China.
State Key Laboratory of Mariculture Breeding, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Fisheries College of Jimei University, Xiamen 361021, China.
J Zhejiang Univ Sci B. 2024 Dec 15;25(12):1083-1096. doi: 10.1631/jzus.B2300899.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Cas9 (SaCas9) system to edit the tyrosinase (, oculocutaneous albinism II (), and paired box 6.1 () genes in the fish model medaka(), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）系统属于II型CRISPR/Cas系统，是一种广泛应用于不同生物体的有效基因编辑工具，但Cas9（SpCas9）的大小相当大（4.3 kb），不利于载体递送。在本研究中，我们使用密码子优化的Cas9（SaCas9）系统在鱼类模型青鳉中编辑酪氨酸酶（tyrosinase）、眼皮肤白化病II型（oculocutaneous albinism II）和配对盒6.1（paired box 6.1）基因，其中SaCas9（3.3 kb）的大小要小得多，并且所需的原间隔序列临近基序（protospacer-adjacent motif，PAM）序列为5'-NNGRRT-3'。我们还使用了转运RNA（tRNA）-单向导RNA（sgRNA）系统在体内或体外通过转录来表达功能性sgRNA，并将SaCas9和tRNA-sgRNA组合用于编辑青鳉基因组中的相关基因。结果表明，SaCas9/sgRNA和SaCas9/tRNA-sgRNA系统能够有效编辑青鳉基因组，而PAM序列是编辑效率的重要组成部分。此外，tRNA可通过使sgRNA受诸如巨细胞病毒等常见启动子的控制来提高系统的灵活性。而且，一体化盒式巨细胞病毒（CMV）-SaCas9-tRNA-sgRNA-tRNA在青鳉基因编辑中具有功能。综上所述，密码子优化的SaCas9系统为编辑青鳉基因组以及潜在地编辑其他鱼类基因组提供了一种更小的替代工具。
