Analysis of Single Nucleotide Variants in CRISPR-Cas9 Edited Zebrafish Exomes Shows No Evidence of Off-Target Inflation.

Marie R Mooney,Erica E Davis,Nicholas Katsanis

doi:10.3389/fgene.2019.00949

Marie R Mooney, Erica E Davis + Show 1 more

Open Access

https://doi.org/10.3389/fgene.2019.00949

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Abstract

Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design (mother, father, offspring) to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

Highlights

CRISPR-Cas9 gene editing technology has offered powerful investigative tools and opened new potential avenues for the treatment of genetic disorders
We focused on three different genes for which a) we have substantial experience in this model organism and b) give reproducible, quantitative defects in kidney morphogenesis (Hall et al, 2018), mandibular and neuronal development (Tsai et al, 2018), and craniofacial morphogenesis (Shaw et al, 2017)
We required that each single guide RNA (sgRNA) was predicted to generate few off-target effects

Summary

Introduction

CRISPR-Cas gene editing technology has offered powerful investigative tools and opened new potential avenues for the treatment of genetic disorders. The alteration of off-target regions decreases precipitously with the use of stringently designed sgRNA sequences and Cas enzymes engineered for high specificity (Fu et al, 2013; Doench et al, 2016; Hu et al, 2018), though recent work demonstrates that precise control over the nature of editing even at on-target sites remains challenging (Kosicki et al, 2018). In rodents, these same factors influence the efficiency and specificity of CRISPR-Cas editing (Anderson et al, 2018). Examination of atypical CRISPR-Cas influence on organisms remains limited; it is often focused primarily on predicted off-target assessment and is not always agnostic (Varshney et al, 2015)

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