Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells

Philip J.R. Roche; Heidi Gytz; Faiz Hussain; Christopher J.F. Cameron; Denis Paquette; Mathieu Blanchette; Josée Dostie; Bhushan Nagar; Uri David Akavia

doi:10.1089/crispr.2018.0045

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells

Philip J.R. Roche, Heidi Gytz, Faiz Hussain, Christopher J.F. Cameron, Denis Paquette, Mathieu Blanchette, Josée Dostie, Bhushan Nagar, Uri David Akavia

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

Abstract Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9?donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9?donor conjugation in addition to delivery influence HDR efficiency.

Original language	English
Journal	The CRISPR Journal
Volume	1
Issue	6
Pages (from-to)	414-430
Number of pages	17
ISSN	2573-1599
DOIs	https://doi.org/10.1089/crispr.2018.0045
Publication status	Published - 20 Dec 2018

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10.1089/crispr.2018.0045

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@article{208f70ced66b4d999af07ad09b909686,

title = "Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells",

abstract = "Abstract Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9?donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9?donor conjugation in addition to delivery influence HDR efficiency.",

author = "Roche, {Philip J.R.} and Heidi Gytz and Faiz Hussain and Cameron, {Christopher J.F.} and Denis Paquette and Mathieu Blanchette and Jos{\'e}e Dostie and Bhushan Nagar and Akavia, {Uri David}",

note = "doi: 10.1089/crispr.2018.0045",

year = "2018",

month = dec,

day = "20",

doi = "10.1089/crispr.2018.0045",

language = "English",

volume = "1",

pages = "414--430",

journal = "The CRISPR Journal",

issn = "2573-1599",

publisher = "Mary Ann Liebert Inc.",

number = "6",

}

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells. / Roche, Philip J.R.; Gytz, Heidi; Hussain, Faiz et al.
In: The CRISPR Journal, Vol. 1, No. 6, 20.12.2018, p. 414-430.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells

AU - Roche, Philip J.R.

AU - Gytz, Heidi

AU - Hussain, Faiz

AU - Cameron, Christopher J.F.

AU - Paquette, Denis

AU - Blanchette, Mathieu

AU - Dostie, Josée

AU - Nagar, Bhushan

AU - Akavia, Uri David

N1 - doi: 10.1089/crispr.2018.0045

PY - 2018/12/20

Y1 - 2018/12/20

N2 - Abstract Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9?donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9?donor conjugation in addition to delivery influence HDR efficiency.

AB - Abstract Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9?donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9?donor conjugation in addition to delivery influence HDR efficiency.

U2 - 10.1089/crispr.2018.0045

DO - 10.1089/crispr.2018.0045

M3 - Journal article

C2 - 31021244

SN - 2573-1599

VL - 1

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EP - 430

JO - The CRISPR Journal

JF - The CRISPR Journal

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