Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

Abstract

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations.

Clinical applications of CRISPR‐based genome editing and diagnostics

“…At this stage, genetic knockouts are facile, but replacing sequences by HDR remains inefficient and technically challenging, although great strides are being made to improve the technique. [65][66][67][68][69][70][71][72] CRISPR systems have generated an extraordinary level of excitement for the clinical potential for curing human disease. In this nascent stage of CRISPR in the clinic, initial applications are treating diseases with the most accessible cells (immune cells and HSPCs edited ex vivo), which avoids the ongoing challenge of tissue-specific delivery in vivo.…”

“…65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies. 17 CURRENT AND NEAR-TERM THERAPEUTIC APPLICATIONS Genetic therapies offer hope for treating and effectively curing monogenic diseases by reversing the underlying genetic cause.…”

A polyclonal allelic expression assay for detecting regulatory effects of transcript variants

“…Since NHEJ often dominates HDR in efficiency, this can result in low numbers of HDR reads. Research in improving the HDR rates in editing is ongoing (Aird et al, 2018;Chu et al, 2015;Maruyama et al, 2015), and likely HDR efficiency will be greatly improved in the future.…”

Interrogating mechanisms of ssDNA binding to a viral HUH-endonuclease by alanine scanning of an electrostatic patch

“…In contrast, this reaction forms a robust phosphotyrosine adduct in vitro , and this protein-DNA conjugate is stable even in denaturing conditions (Chandler et al 2013;Lovendahl, Hayward, and Gordon 2017;Heyraud-Nitschke et al 1995) . Thus, HUH endonucleases pose an attractive option for creating stable protein-DNA linkages in a variety of biotechnology applications, for example improving CRISPR-Cas9 precise gene editing (Aird et al 2018) and tethering proteins of interest in DNA origami applications (Sagredo et al 2016) . Moreover, each HUH endonuclease recognizes a distinct sequence at its native origin of replication, resulting in a sequence-specific covalent adduct.…”