Faculty Opinions recommendation of Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

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5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

10.1101/354480 ◽

2018 ◽

Cited By ~ 7

Author(s):

Krishna S. Ghanta ◽

Gregoriy A. Dokshin ◽

Aamir Mir ◽

Pranathi Meda Krishnamurthy ◽

Hassan Gneid ◽

...

Keyword(s):

Dna Repair ◽

Genome Editing ◽

Genetic Basis ◽

Cell Types ◽

Great Promise ◽

Precise Form ◽

Homology Directed Repair ◽

Genomic Location ◽

Repair Template ◽

Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.

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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

eLife ◽

10.7554/elife.33761 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 49

Author(s):

Natasa Savic ◽

Femke CAS Ringnalda ◽

Helen Lindsay ◽

Christian Berk ◽

Katja Bargsten ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Types ◽

Dna Double Strand Breaks ◽

End Joining ◽

Covalent Linkage ◽

Homology Directed Repair ◽

Cas9 Nuclease ◽

Repair Mechanisms ◽

Multiple Cell ◽

Donor Template

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

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Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

10.1101/218149 ◽

2017 ◽

Cited By ~ 4

Author(s):

Natasa Savic ◽

Femke Ringnalda ◽

Katja Bargsten ◽

Yizhou Li ◽

Christian Berk ◽

...

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Homology Directed Repair ◽

Genetic Modifications ◽

Repair Template ◽

Single Base Pair ◽

Non Homologous End Joining

AbstractThe CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.

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

Communications Biology ◽

10.1038/s42003-018-0054-2 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 77

Author(s):

Eric J. Aird ◽

Klaus N. Lovendahl ◽

Amber St. Martin ◽

Reuben S. Harris ◽

Wendy R. Gordon

Keyword(s):

Dna Repair ◽

Homology Directed Repair ◽

Repair Efficiency ◽

Repair Template

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A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity

International Journal of Molecular Sciences ◽

10.3390/ijms22083834 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3834

Author(s):

Kevin Bloh ◽

Natalia Rivera-Torres

Keyword(s):

Dna Repair ◽

Mechanism Of Action ◽

Gene Editing ◽

Strand Break ◽

Consensus Model ◽

Homology Directed Repair ◽

Dna Repair Mechanisms ◽

Repair Mechanisms ◽

Repair Template ◽

The Many

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.

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Impact of hypoxia on DNA repair and genome integrity

Mutagenesis ◽

10.1093/mutage/gez019 ◽

2019 ◽

Vol 35 (1) ◽

pp. 61-68 ◽

Cited By ~ 5

Author(s):

Alanna R Kaplan ◽

Peter M Glazer

Keyword(s):

Dna Repair ◽

Cancer Progression ◽

Base Excision Repair ◽

Mutation Frequency ◽

Excision Repair ◽

Tumour Microenvironment ◽

Genome Integrity ◽

Future Directions ◽

Homology Directed Repair ◽

Base Excision

Abstract Hypoxia is a hallmark of the tumour microenvironment with profound effects on tumour biology, influencing cancer progression, the development of metastasis and patient outcome. Hypoxia also contributes to genomic instability and mutation frequency by inhibiting DNA repair pathways. This review summarises the diverse mechanisms by which hypoxia affects DNA repair, including suppression of homology-directed repair, mismatch repair and base excision repair. We also discuss the effects of hypoxia mimetics and agents that induce hypoxia on DNA repair, and we highlight areas of potential clinical relevance as well as future directions.

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H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

Wellcome Open Research ◽

10.12688/wellcomeopenres.11589.3 ◽

2021 ◽

Vol 2 ◽

pp. 83

Author(s):

Jayakumar Sundarraj ◽

Gillian C.A. Taylor ◽

Alex von Kriegsheim ◽

Madapura M Pradeepa

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Embryonic Stem ◽

Adaptor Proteins ◽

Transcriptional Elongation ◽

Pwwp Domain ◽

Stable Isotope Labelling ◽

Homology Directed Repair ◽

Dna Repair Proteins

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

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