ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

• Type of Material: Article
• Authors: Bingzhou Han, Peking University; Yage Zhang, Peking University; Yang Zhou, Peking University; Biao Zhang, Peking University; Christopher J Krueger, Emory University; Xuetong Bi, Peking University; Zuoyan Zhu, Peking University; Xiangjun Tong, Peking University; Bo Zhang, Peking University
• Language: English
• Date: 2022-03-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2022 by the authors.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/biology11030411
• Title of Journal or Parent Work: Biology
• Volume: 11
• Issue: 3
• Grant/Funding Information: This research was partially funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China (NSFC) [2019YFA0802800, 2018YFA0801000, 31871458, 32070824, 2016YFA0100500, and 31671500].
• Keywords: knockin; NHEJ; zebrafish; genome editing; MMEJ; ErCas12a
• Research Categories: Engineering, Biomedical

Abstract

In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement of delivery as purified protein and the necessity of heatshock of injected embryos. To explore the potential of CRISPR/Cas12a-mediated genome editing and simplify its application in zebrafish, we took advantage of the recently reported mRNA-active ErCas12a and investigated its efficacy for the knockin of large DNA fragments, such as fluorescent reporter genes. For knockin via either microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, ErCas12a-injected embryos with a brief heatshock displayed comparable knockin efficiency with Cas9 injection. Through the fusion of T5 exonuclease (T5exo) to the N-terminus of ErCas12a (T5exo-ErCas12a), we further demonstrated high efficiency gene knockout and knockin at a normal incubation temperature, eliminating the embryo-damaging heatshock step. In summary, our results demonstrate the feasibility of ErCas12a-and T5exo-ErCas12a-mediated genome manipulation under simplified conditions, and further expand the genome editing toolbox for various applications in zebrafish.