Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency

Marella D Canny; Nathalie Moatti; Leo C K Wan; Amélie Fradet-Turcotte; Danielle Krasner; Pedro A Mateos-Gomez; Michal Zimmermann; Alexandre Orthwein; Yu-Chi Juang; Wei Zhang; Sylvie M Noordermeer; Eduardo Seclen; Marcus D Wilson; Andrew Vorobyov; Meagan Munro; Andreas Ernst; Timothy F Ng; Tiffany Cho; Paula M Cannon; Sachdev S Sidhu; Frank Sicheri; Daniel Durocher

doi:10.1038/nbt.4021

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency

Marella D Canny, Nathalie Moatti, Leo C K Wan, Amélie Fradet-Turcotte, Danielle Krasner, Pedro A Mateos-Gomez, Michal Zimmermann, Alexandre Orthwein, Yu-Chi Juang, Wei Zhang, Sylvie M Noordermeer, Eduardo Seclen, Marcus D Wilson, Andrew Vorobyov, Meagan Munro, Andreas Ernst, Timothy F Ng, Tiffany Cho, Paula M Cannon, Sachdev S SidhuFrank Sicheri, Daniel Durocher

School of Biological Sciences

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

Abstract

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

Original language	English
Pages (from-to)	95-102
Number of pages	8
Journal	Nature Biotechnology
Volume	36
Issue number	1
DOIs	https://doi.org/10.1038/nbt.4021
Publication status	Published - 27 Nov 2017

Keywords / Materials (for Non-textual outputs)

Animals
CRISPR-Cas Systems/genetics
DNA Damage/genetics
DNA End-Joining Repair/genetics
DNA Repair/genetics
Gene Editing
Gene Expression Regulation/genetics
Humans
Mice
Recombinational DNA Repair/genetics
Tumor Suppressor p53-Binding Protein 1/antagonists & inhibitors

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10.1038/nbt.4021

Marcus Wilson
- School of Biological Sciences - Sir Henry Dale Fellow
Person: Academic: Research Active

Cite this

Canny, M. D., Moatti, N., Wan, L. C. K., Fradet-Turcotte, A., Krasner, D., Mateos-Gomez, P. A., Zimmermann, M., Orthwein, A., Juang, Y.-C., Zhang, W., Noordermeer, S. M., Seclen, E., Wilson, M. D., Vorobyov, A., Munro, M., Ernst, A., Ng, T. F., Cho, T., Cannon, P. M., ... Durocher, D. (2017). Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nature Biotechnology, 36(1), 95-102. https://doi.org/10.1038/nbt.4021

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title = "Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency",

abstract = "Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.",

keywords = "Animals, CRISPR-Cas Systems/genetics, DNA Damage/genetics, DNA End-Joining Repair/genetics, DNA Repair/genetics, Gene Editing, Gene Expression Regulation/genetics, Humans, Mice, Recombinational DNA Repair/genetics, Tumor Suppressor p53-Binding Protein 1/antagonists & inhibitors",

author = "Canny, {Marella D} and Nathalie Moatti and Wan, {Leo C K} and Am{\'e}lie Fradet-Turcotte and Danielle Krasner and Mateos-Gomez, {Pedro A} and Michal Zimmermann and Alexandre Orthwein and Yu-Chi Juang and Wei Zhang and Noordermeer, {Sylvie M} and Eduardo Seclen and Wilson, {Marcus D} and Andrew Vorobyov and Meagan Munro and Andreas Ernst and Ng, {Timothy F} and Tiffany Cho and Cannon, {Paula M} and Sidhu, {Sachdev S} and Frank Sicheri and Daniel Durocher",

note = "AAM needs to be provided by new PI - he was in Canada before so exception can be used, although AAM is required. 29/03/2019 EN",

year = "2017",

month = nov,

day = "27",

doi = "10.1038/nbt.4021",

language = "English",

volume = "36",

pages = "95--102",

journal = "Nature Biotechnology",

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Canny, MD, Moatti, N, Wan, LCK, Fradet-Turcotte, A, Krasner, D, Mateos-Gomez, PA, Zimmermann, M, Orthwein, A, Juang, Y-C, Zhang, W, Noordermeer, SM, Seclen, E, Wilson, MD, Vorobyov, A, Munro, M, Ernst, A, Ng, TF, Cho, T, Cannon, PM, Sidhu, SS, Sicheri, F & Durocher, D 2017, 'Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency', Nature Biotechnology, vol. 36, no. 1, pp. 95-102. https://doi.org/10.1038/nbt.4021

TY - JOUR

T1 - Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency

AU - Canny, Marella D

AU - Moatti, Nathalie

AU - Wan, Leo C K

AU - Fradet-Turcotte, Amélie

AU - Krasner, Danielle

AU - Mateos-Gomez, Pedro A

AU - Zimmermann, Michal

AU - Orthwein, Alexandre

AU - Juang, Yu-Chi

AU - Zhang, Wei

AU - Noordermeer, Sylvie M

AU - Seclen, Eduardo

AU - Wilson, Marcus D

AU - Vorobyov, Andrew

AU - Munro, Meagan

AU - Ernst, Andreas

AU - Ng, Timothy F

AU - Cho, Tiffany

AU - Cannon, Paula M

AU - Sidhu, Sachdev S

AU - Sicheri, Frank

AU - Durocher, Daniel

N1 - AAM needs to be provided by new PI - he was in Canada before so exception can be used, although AAM is required. 29/03/2019 EN

PY - 2017/11/27

Y1 - 2017/11/27

N2 - Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

AB - Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

KW - Animals

KW - CRISPR-Cas Systems/genetics

KW - DNA Damage/genetics

KW - DNA End-Joining Repair/genetics

KW - DNA Repair/genetics

KW - Gene Editing

KW - Gene Expression Regulation/genetics

KW - Humans

KW - Mice

KW - Recombinational DNA Repair/genetics

KW - Tumor Suppressor p53-Binding Protein 1/antagonists & inhibitors

U2 - 10.1038/nbt.4021

DO - 10.1038/nbt.4021

M3 - Letter

C2 - 29176614

SN - 1087-0156

VL - 36

SP - 95

EP - 102

JO - Nature Biotechnology

JF - Nature Biotechnology

IS - 1

ER -

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency

Abstract

Keywords / Materials (for Non-textual outputs)

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Marcus Wilson

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