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

Bingzhou Han,Biao Zhang,Yage Zhang,Yang Zhou,Bo Zhang,Xiangjun Tong,Christopher J Krueger,Zuoyan Zhu,Xuetong Bi

doi:10.3390/biology11030411

Abstract

Simple SummaryCRISPR/Cas9 enables efficient mutagenesis and generation of various knockout and knockin alleles in many species including zebrafish. However, the application of the Cas12a nuclease in zebrafish is far from ideal due to demanding experimental conditions, especially the requirements for delivery such as a purified protein and the heatshock of embryos. Here we show that ErCas12a, the only Cas12a reported to be effective when injected as mRNA in zebrafish, is highly efficient for large fragment knockin via either microhomology-mediated or non-homologous end joining pathways with mild heatshock conditions. Moreover, we fused T5 exonuclease to ErCas12a and found that the fusion protein could efficiently induce gene knockout and knockin without heatshock. Therefore, we demonstrated the efficacy of multiple genome-editing applications using ErCas12a and its variant with simplified conditions in zebrafish.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.

Highlights

The CRISPR/Cas system, including the type II effector Cas9 and the type V effectorCas12a, has been widely adopted for genome editing both ex vivo and in vivo
While previous reports suggested that ErCas12a could function in the mRNA form in zebrafish embryos with a 4 h heatshock [11], we found that only one-hour heatshock was sufficient for high efficiency ErCas12a editing, reaching about 80% indel efficiency with tyr or alb pre-crRNA
ErCas12a likely has better application potential, as it can function in the mRNA form [11], and through modification with the T5 exonuclease can function without heatshock, and we have demonstrated its efficacy in microhomology-mediated end joining (MMEJ)- and non-homologous end joining (NHEJ)-based large fragment gene knockin

Summary

Introduction

The CRISPR/Cas system, including the type II effector Cas and the type V effectorCas12a, has been widely adopted for genome editing both ex vivo and in vivo. Given the fact that Cas recognizes a guanine-rich PAM and generates blunt DSB ends [2–4], the application of Cas12a may provide benefits, including expanded target site choice, such as T-rich introns, non-coding RNAs, and UTRs [1]; facile multiplexed genome editing enabled by the pre-crRNA processing ability of Cas12a [5–9]; and increased efficiency of homology-based knockin due to the sticky ends generated by Cas12a cutting [10,11]. Cas12a identified from Eubacterium rectale (ErCas12a, known as MAD7 nuclease), was reported to induce efficient mutagenesis when injected as mRNA in zebrafish embryos, and requires heatshock treatment [11]. We hypothesized that the mRNA-active ErCas12a could be an ideal tool for large fragment knockin in zebrafish, for homology-based approaches, which might be facilitated by the staggered DSBs. In addition, its different target site selection criteria compared with Cas should greatly increase the targetable range as well as flexibility for the experimental design to achieve large fragment integration, especially for T-rich sequences such as introns

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Discussion

Conclusion