Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways

Abstract

Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFNmediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications.

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

“…Zinc finger nuclease (ZFN) and, more recently, transcription activator-like effector nuclease (TALEN) have been tested as tools for targeted plant gene editing. Several publications have reported successful modifications in native plant genes, but both methods are tedious, expensive, and with only a small number of genes modified so far (18)(19)(20)(21)(22)(23)(24)(25). In sharp contrast, we report specific modifications of seven genes in this study in the span of just a few months, demonstrating the extremely easy-to-use feature of the CRISPR/Cas system.…”

Gene targeting in plants: 25 years later

“…The authors reported GT frequencies of more than 10% in most experiments (Shukla et al, 2009). Very recently, ZFN-mediated GT was documented in Arabidopsis (de Pater et al, 2012;Qi et al, 2013).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…In plants, the same type of results was obtained when using ZFNs targeting the endogenous ADH1 locus in Arabidopsis thaliana. Knock-in was increased up to 16 times in the Atku70 mutant and 3 to 4 times in the Atlig4 and Atsmc6b mutants [100]. Higher rates of homology-driven knock-in were also observed in mammalian cells when using hCas9 fused to proteins involved in cell-cycle dynamics [101,102], allowing a low expression of the nuclease in G1 and a high expression during S/G2/M phases where HDR mediated repair is favored [103,104].…”