MRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay

Jennifer L Anderson,Shao J Du,Meng-Chieh Shen,Hui Wang,Elisabeth M Busch-Nentwich,Frederick J Tan,Steven A Farber,Timothy S Mulligan,Catherine M Scahill,Mary C Mullins

doi:10.1371/journal.pgen.1007105

Abstract

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

Highlights

The recent increased use of reverse genetic approaches has been largely driven by the ease, affordability of construction, and implementation of the CRISPR/Cas9 and TALEN systems
MRNA processing in mutants produces transcripts that escape nonsense-mediated decay
This study reveals that mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function

Summary

Introduction

The recent increased use of reverse genetic approaches has been largely driven by the ease, affordability of construction, and implementation of the CRISPR/Cas and TALEN systems. While genome editing methods, such as the CRISPR/Cas and TALEN systems, have proven to be an efficient and effective way to reduce or eliminate gene function, a frequent lack of a mutant phenotype is observed, often explained by genetic compensation. This is a process wherein related genes or pathway members are differentially regulated in the mutants to compensate for the targeted loss of a specific gene [3]

Methods

Results

Discussion

Conclusion