Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

Aron Ferenczi,Charlotte Von Koppenfels,Andrew Hudson,Yen Peng Chew,Erika Kroll,Attila Molnar

doi:10.1038/s41467-021-27004-1

Aron Ferenczi, Charlotte Von Koppenfels + Show 4 more

Open Access

https://doi.org/10.1038/s41467-021-27004-1

Copy DOI

Journal: Nature Communications	Publication Date: Nov 19, 2021
Citations: 17	License type: open-access

Affiliation: University of Edinburgh, Rothamsted Research

Abstract

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

Highlights

Single-stranded oligodeoxynucleotides are widely used as DNA repair templates in CRISPR/Cas precision genome editing
We elucidate the DNA repair events underpinning single-strand templated DNA repair (SSTR) at CRISPR/Cas12a-induced doublestrand breaks (DSBs) in C. reinhardtii. Using both previously used and novel techniques, we find that Singlestranded oligodeoxynucleotides (ssODNs) physically incorporate into the genome during Cas12a-induced DSB repair via the single-strand DNA incorporation (ssDI) form of SSTR
To survey SSTR, we employed our previously developed experimental system whereby DNA DSBs are induced in vivo by delivering CRISPR/Cas12a ribonucleoproteins (RNPs) targeted at the FK506-binding protein 12 (FKB12) locus[3,27]

Summary

Introduction

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. Short ssODNs with homology arms on the order of tens of nucleotides enable precision editing where homologous recombination (HR) occurs at prohibitively low levels[3] The process governing this form of editing is called single-strand templated repair (SSTR), is poorly understood, and has only been studied using Cas[9] in human cells[5,6,7,8,9,10] and yeast[11]. During ssDI ( called ‘bridge model’12), ssODNs physically incorporate into the genome, while during SDSA, ssODNs serve as templates for genomic DNA synthesis without their physical genomic incorporation[5,6,7,8] In human cells, both forms are present at DNA single-strand nicks depending on the orientation of the ssODN relative to the nick[5,6], while SDSA prevails at double-strand breaks (DSBs)[6,7]. Relatively little is known about DNA repair genes in C. reinhardtii[23,24,25,26]

Methods

Results

Conclusion