Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency.

Ignacio García-Tuñón,Manuel Sánchez-Martín,Lucía Méndez,Verónica Alonso-Pérez,Sandra Pérez- Ramos,Julián Sevilla,Elena Vuelta,Raquel Saldaña,Jesús María Hernández-Sánchez,Marta Martín-Izquierdo,Fermín Sánchez- Guijo,Jesús María Hernández-Rivas,María Herrero,Stefan Maas

doi:10.1371/journal.pone.0216674

Abstract

CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

Highlights

With the recent diversification of genome editing tools, including those involving clustered, regularly interspaced short palindromic repeats and their nuclease-associated protein Cas9 (CRISPR/Cas9), the landscape of suppression techniques has dramatically changed
Two groups of sgRNAs were created to study the efficiency of splice donor exon (SDE)-sgRNAs and IE-sgRNAs at generating null alleles in mouse and human cells (Fig 1)
If there is an acceptable degree of efficiency of delivery of CRISPR/Cas9 reagents to the target cell, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene

Summary

Introduction

With the recent diversification of genome editing tools, including those involving clustered, regularly interspaced short palindromic repeats and their nuclease-associated protein Cas (CRISPR/Cas9), the landscape of suppression techniques has dramatically changed. CRISPR/Cas is similar in action and efficacy to protein-based targeted nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)[1], the ease with which these reagents can be designed and tested through the construction of single-. JM HernandezSanchez was supported by a research grant from Fundacion Española de Hematologıa y Hemoterapia (FEHH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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Discussion

Conclusion