Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor.

Deshani C Ranawakage,Yusuke Kamachi,Takuya Takada,Yuya Kawaguchi,Yuki Kuninobu-Bonkohara,Kota Sugio,Keita Okada

doi:10.3389/fcell.2020.598634

Abstract

Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently “knocked-in” into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3′ homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5′ and 3′ junctions indicated that knock-in allele frequencies were higher at the 3′ side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

Highlights

The ability to achieve precise and sequence-specific genome editing using the prokaryotic CRISPR-Cas9 system has revolutionized genomic engineering, enabling an unprecedented potential to modify the genomes of almost any organism (Mali et al, 2013; Ran et al, 2013; Barman et al, 2020)
We first selected the SoxB1 transcription factor gene sox3 as a knock-in target, as we had previously demonstrated through mRNA injection that the Sox3 protein that was C-terminally tagged with the FLAG composite tag was stably expressed in zebrafish embryos (Ranawakage et al, 2019)
We chose a CRISPR-Cas9 system that utilizes an in vitro assembled RNP complex that consists of three components: two synthetic RNA oligonucleotides, and a high-fidelity recombinant Streptococcus pyogenes Cas9 protein that was developed by Integrated DNA Technologies, Inc. (IDT) (Figure 2A)

Summary

Introduction

The ability to achieve precise and sequence-specific genome editing using the prokaryotic CRISPR-Cas system has revolutionized genomic engineering, enabling an unprecedented potential to modify the genomes of almost any organism (Mali et al, 2013; Ran et al, 2013; Barman et al, 2020). Upon recognition of the target DNA sequence, Cas mediates the cleavage of target DNA upstream of PAM to create a double-strand break (DSB). This DSB is repaired by cellular machinery through either the non-homologous end joining (NHEJ) or the homology-directed repair (HDR) pathways (Jasin and Haber, 2016; Salsman et al, 2017; Gallagher and Haber, 2018; Barman et al, 2020). This results in a low success rate when attempting to generate knock-in alleles in multicellular model organisms, such as the zebrafish (Danio rerio) (Boel et al, 2018; Cornet et al, 2018; Prykhozhij and Berman, 2018; Liu et al, 2019)

Objectives

Methods

Results

Conclusion