Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.

Polly Gravells,Timothy C Humphrey,Julia M Hong,Rajani K Vangala,Kazunori Tomita,Sara Ahrabi,James T Brash,Andrew C.G Porter,Lena A Brustle,Aikaterini Kaloudi,Christopher Chung

doi:10.1093/hmg/ddv409

Abstract

Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Highlights

Inappropriate repair of chromosomal DNA double-strand breaks (DSBs) can lead to mutagenesis, gross chromosomal instability and genetic disease [1]
Using an I-SceI-sensitive hypoxanthine phosphoribosyltransferase (HPRT) minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides
We have developed a system for measuring the repair of I-SceI- or Cas9-induced DSBs by three different mechanisms, all at the same site in the endogenous HPRT gene

Summary

Introduction

Inappropriate repair of chromosomal DNA double-strand breaks (DSBs) can lead to mutagenesis, gross chromosomal instability and genetic disease [1]. Knowledge of DSB repair pathways, and their defects in particular cancers, has helped to define oncogenic mechanisms and to develop new therapeutic strategies [2]. Such knowledge underpins powerful genome engineering methods that use customized endonucleases, including clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNAguided nucleases, to make targeted chromosomal DSBs [3]. We use the term accNHEJ to describe the joining of any two DNA ends that have undergone no gain or loss of nucleotides In some circumstances, such as the absence of Ku heterodimer, NHEJ is more likely to involve limited endresection, resulting in insertions and/or deletions (indels) at the site of the DSB. In an analogous pathway termed single-strand annealing (SSA), that can be considered a form of non-conservative RAD51-independent HR, deletions result from extensive end-resection followed by annealing of longer (e.g. >100 nt) repeats [6]

Methods

Results

Conclusion