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

Natasa Savic; Femke Cas Ringnalda; Helen Lindsay; Christian Berk; Katja Bargsten; Yizhou Li; Dario Neri; Mark D Robinson; Constance Ciaudo; Jonathan Hall; Martin Jinek; Gerald Schwank

doi:10.7554/eLife.33761

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

Natasa Savic, Femke Cas Ringnalda, Helen Lindsay, Christian Berk, Katja Bargsten, Yizhou Li, Dario Neri, Mark D Robinson, Constance Ciaudo, Jonathan Hall, Martin Jinek, Gerald Schwank

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Journal	eLife
Volume	7
DOIs	https://doi.org/10.7554/eLife.33761
Publication status	Published - 29 May 2018
Externally published	Yes

Keywords

CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems
Cell Nucleus/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
DNA/chemistry
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA Replication
Gene Editing/methods
Genetic Loci
Guanidines/chemistry
HEK293 Cells
Humans
Oligodeoxyribonucleotides/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics
Recombinant Fusion Proteins/genetics
Recombinational DNA Repair
Staining and Labeling/methods

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10.7554/eLife.33761

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@article{54680e84b3fb451dbba882e898b22ae9,

title = "Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair",

abstract = "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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author = "Natasa Savic and Ringnalda, {Femke Cas} and Helen Lindsay and Christian Berk and Katja Bargsten and Yizhou Li and Dario Neri and Robinson, {Mark D} and Constance Ciaudo and Jonathan Hall and Martin Jinek and Gerald Schwank",

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year = "2018",

month = may,

day = "29",

doi = "10.7554/eLife.33761",

language = "English",

volume = "7",

journal = "eLife",

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T1 - Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

AU - Savic, Natasa

AU - Ringnalda, Femke Cas

AU - Lindsay, Helen

AU - Berk, Christian

AU - Bargsten, Katja

AU - Li, Yizhou

AU - Neri, Dario

AU - Robinson, Mark D

AU - Ciaudo, Constance

AU - Hall, Jonathan

AU - Jinek, Martin

AU - Schwank, Gerald

PY - 2018/5/29

Y1 - 2018/5/29

N2 - 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.

AB - 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.

KW - CRISPR-Associated Protein 9/genetics

KW - CRISPR-Cas Systems

KW - Cell Nucleus/genetics

KW - Clustered Regularly Interspaced Short Palindromic Repeats

KW - DNA/chemistry

KW - DNA Breaks, Double-Stranded

KW - DNA End-Joining Repair

KW - DNA Replication

KW - Gene Editing/methods

KW - Genetic Loci

KW - Guanidines/chemistry

KW - HEK293 Cells

KW - Humans

KW - Oligodeoxyribonucleotides/chemistry

KW - RNA, Guide, CRISPR-Cas Systems/genetics

KW - Recombinant Fusion Proteins/genetics

KW - Recombinational DNA Repair

KW - Staining and Labeling/methods

U2 - 10.7554/eLife.33761

DO - 10.7554/eLife.33761

M3 - Article

C2 - 29809142

SN - 2050-084X

VL - 7

JO - eLife

JF - eLife

ER -

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

Abstract

Keywords

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