A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.

Shao-Lin Xie,Wan-Ping Bian,De-Sheng Pei,Muhammad Junaid,Chao Wang,Ji-Xing Zou

doi:10.1038/srep34555

Abstract

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system.

Highlights

Loss-of-function is an important approach for in vivo functional study of a gene of interest in transgenic animals
Nakajima and Yaoita reported that the gene editing activity of transcription activator like effector nucleases (TALENs) was improved by injecting TALEN capped RNAs into the oocytes of Xenopus laevis[48]
Little is known about translational capacity of mature oocytes in zebrafish by using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system

Summary

Introduction

Loss-of-function is an important approach for in vivo functional study of a gene of interest in transgenic animals. We reported a modified in vitro oocyte storage strategy to increase the efficiency of genome editing by CRISPR/Cas[9] system in zebrafish. This novel technique with in vitro oocyte injection prior to their storage improved the efficiencies of gene targeting and germline transmission. Contrary to the conventional genome editing approaches, this method utilized the inherent translation system to impel the oocytes to produce Cas[9] proteins in advance, which greatly increased the efficiency of genome editing It improved the efficiency of germline transmission. Our novel technique will increase the speed of generating heritable mutants of zebrafish using CRISPR/Cas[9] system

Methods

Results

Conclusion