A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

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A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. / Vakulskas, Christopher A; Dever, Daniel P; Rettig, Garrett R; Turk, Rolf; Jacobi, Ashley M; Collingwood, Michael A; Bode, Nicole M; McNeill, Matthew S; Yan, Shuqi; Camarena, Joab; Lee, Ciaran M; Park, So Hyun; Wiebking, Volker; Bak, Rasmus O; Gomez-Ospina, Natalia; Pavel-Dinu, Mara; Sun, Wenchao; Bao, Gang; Porteus, Matthew H; Behlke, Mark A.

I: Nature Medicine, Bind 24, Nr. 8, 2018, s. 1216-1224.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Vakulskas, CA, Dever, DP, Rettig, GR, Turk, R, Jacobi, AM, Collingwood, MA, Bode, NM, McNeill, MS, Yan, S, Camarena, J, Lee, CM, Park, SH, Wiebking, V, Bak, RO, Gomez-Ospina, N, Pavel-Dinu, M, Sun, W, Bao, G, Porteus, MH & Behlke, MA 2018, 'A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells', Nature Medicine, bind 24, nr. 8, s. 1216-1224. https://doi.org/10.1038/s41591-018-0137-0

APA

Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., Lee, C. M., Park, S. H., Wiebking, V., Bak, R. O., Gomez-Ospina, N., Pavel-Dinu, M., Sun, W., Bao, G., Porteus, M. H., & Behlke, M. A. (2018). A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine, 24(8), 1216-1224. https://doi.org/10.1038/s41591-018-0137-0

CBE

Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, Bode NM, McNeill MS, Yan S, Camarena J, Lee CM, Park SH, Wiebking V, Bak RO, Gomez-Ospina N, Pavel-Dinu M, Sun W, Bao G, Porteus MH, Behlke MA. 2018. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine. 24(8):1216-1224. https://doi.org/10.1038/s41591-018-0137-0

MLA

Vancouver

Author

Vakulskas, Christopher A ; Dever, Daniel P ; Rettig, Garrett R ; Turk, Rolf ; Jacobi, Ashley M ; Collingwood, Michael A ; Bode, Nicole M ; McNeill, Matthew S ; Yan, Shuqi ; Camarena, Joab ; Lee, Ciaran M ; Park, So Hyun ; Wiebking, Volker ; Bak, Rasmus O ; Gomez-Ospina, Natalia ; Pavel-Dinu, Mara ; Sun, Wenchao ; Bao, Gang ; Porteus, Matthew H ; Behlke, Mark A. / A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. I: Nature Medicine. 2018 ; Bind 24, Nr. 8. s. 1216-1224.

Bibtex

@article{4b4d40653da74e2799e89fddf109e827,
title = "A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells",
abstract = "Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.",
keywords = "BONE-MARROW, CRISPR-CAS9 NUCLEASES, GENOME, GUIDE RNA, HUMAN T-CELLS, IN-VIVO, MAMMALIAN-CELLS, MARROW TRANSPLANTATION, MESSENGER-RNA, OFF-TARGET",
author = "Vakulskas, {Christopher A} and Dever, {Daniel P} and Rettig, {Garrett R} and Rolf Turk and Jacobi, {Ashley M} and Collingwood, {Michael A} and Bode, {Nicole M} and McNeill, {Matthew S} and Shuqi Yan and Joab Camarena and Lee, {Ciaran M} and Park, {So Hyun} and Volker Wiebking and Bak, {Rasmus O} and Natalia Gomez-Ospina and Mara Pavel-Dinu and Wenchao Sun and Gang Bao and Porteus, {Matthew H} and Behlke, {Mark A}",
year = "2018",
doi = "10.1038/s41591-018-0137-0",
language = "English",
volume = "24",
pages = "1216--1224",
journal = "Nature Medicine",
issn = "1078-8956",
publisher = "Nature Publishing Group",
number = "8",

}

RIS

TY - JOUR

T1 - A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

AU - Vakulskas, Christopher A

AU - Dever, Daniel P

AU - Rettig, Garrett R

AU - Turk, Rolf

AU - Jacobi, Ashley M

AU - Collingwood, Michael A

AU - Bode, Nicole M

AU - McNeill, Matthew S

AU - Yan, Shuqi

AU - Camarena, Joab

AU - Lee, Ciaran M

AU - Park, So Hyun

AU - Wiebking, Volker

AU - Bak, Rasmus O

AU - Gomez-Ospina, Natalia

AU - Pavel-Dinu, Mara

AU - Sun, Wenchao

AU - Bao, Gang

AU - Porteus, Matthew H

AU - Behlke, Mark A

PY - 2018

Y1 - 2018

N2 - Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

AB - Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

KW - BONE-MARROW

KW - CRISPR-CAS9 NUCLEASES

KW - GENOME

KW - GUIDE RNA

KW - HUMAN T-CELLS

KW - IN-VIVO

KW - MAMMALIAN-CELLS

KW - MARROW TRANSPLANTATION

KW - MESSENGER-RNA

KW - OFF-TARGET

UR - http://www.scopus.com/inward/record.url?scp=85051279904&partnerID=8YFLogxK

U2 - 10.1038/s41591-018-0137-0

DO - 10.1038/s41591-018-0137-0

M3 - Journal article

C2 - 30082871

VL - 24

SP - 1216

EP - 1224

JO - Nature Medicine

JF - Nature Medicine

SN - 1078-8956

IS - 8

ER -