User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9.

Liangliang Chen,Xue Zhang,Heyao Zhang,Ying Ye,Rapolas Spalinskas,Qiang Wu,Zhexin Zhu,Wensheng Zhang,Hongxia Dai,Wenyan Ren

doi:10.1155/2018/9576959

Abstract

Loss-of-function studies are critically important in gene functional analysis of model organisms and cells. However, conditional gene inactivation in diploid cells is difficult to achieve, as it involves laborious vector construction, multifold electroporation, and complicated genotyping. Here, a strategy is presented for generating biallelic conditional gene and DNA regulatory region knockouts in mouse embryonic stem cells by codelivery of CRISPR-Cas9 and short-homology-arm targeting vectors sequentially or simultaneously. Collectively, a simple and rapid method was presented to knock out any DNA element conditionally. This approach will facilitate the functional studies of essential genes and regulatory regions during development.

Highlights

Gene function analysis is of crucial importance to understanding normal physiology and disease pathogenesis
RNA-guided nuclease Cas9 efficiently induces doublestrand breaks (DSB) at the targeted locus, which could be repaired by two mechanisms: nonhomologous end joining (NHEJ) and homology-directed repair (HDR)
The conventional knockout technology requires complicated targeting vector construction that normally involves the use of bacterial artificial chromosomes (BAC) and recombineering technology [16, 17]

Summary

Introduction

Gene function analysis is of crucial importance to understanding normal physiology and disease pathogenesis. Andersson-Rolf et al employed the CRISPR/Cas technique together with invertible elements to conditionally knock out coding genes [6]. Stem Cells International targeting any genomic region is desirable and important for dissecting the function of coding genes as well as numerous DNA regulatory elements. The easy-made targeting vectors are aided by the CRISPR/Cas technique to insert LoxP elements on the flanking regions of the targeted loci sequentially or simultaneously, by which conditional gene knockouts are generated, such as Eed (embryonic ectoderm development) and SRCAP (Snf2-related CREBBP activator protein) in mouse embryonic stem cells (mESCs). A simple method to conditionally target any DNA element was established, which could be used for gene editing in human cells

Materials and Methods

FNFL targeting

Simultaneous LNL and FNFL targeting

Discussion