Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Peter Cameron*, Mary M. Coons, Sanne E. Klompe, Alexandra M. Lied, Stephen C. Smith, Bastien Vidal, Paul D. Donohoue, Tomer Rotstein, Bryan W. Kohrs, David B. Nyer, Rachel Kennedy, Lynda M. Banh, Carolyn Williams, Mckenzi S. Toh, Matthew J. Irby, Leslie S. Edwards, Chun Han Lin, Arthur L.G. Owen, Tim Künne, John van der Oost & 7 others Stan J.J. Brouns, Euan M. Slorach, Chris K. Fuller, Scott Gradia, Steven B. Kanner, Andrew P. May, Samuel H. Sternberg

*Corresponding author for this work

Research output: Contribution to journalLetterAcademicpeer-review

Abstract

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Original languageEnglish
Pages (from-to)1471-1477
JournalNature Biotechnology
Volume37
DOIs
Publication statusPublished - 18 Dec 2019

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Human Engineering
RNA
CRISPR-Associated Proteins
Guide RNA
Genes
Cells
Genome
Dimerization
Eukaryotic Cells
Immune System
Plasmids
Immune system
Electric fuses
Bacteria
Efficiency
Cell Line
Proteins
Gene Editing

Cite this

Cameron, P., Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., ... Sternberg, S. H. (2019). Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37, 1471-1477. https://doi.org/10.1038/s41587-019-0310-0
Cameron, Peter ; Coons, Mary M. ; Klompe, Sanne E. ; Lied, Alexandra M. ; Smith, Stephen C. ; Vidal, Bastien ; Donohoue, Paul D. ; Rotstein, Tomer ; Kohrs, Bryan W. ; Nyer, David B. ; Kennedy, Rachel ; Banh, Lynda M. ; Williams, Carolyn ; Toh, Mckenzi S. ; Irby, Matthew J. ; Edwards, Leslie S. ; Lin, Chun Han ; Owen, Arthur L.G. ; Künne, Tim ; van der Oost, John ; Brouns, Stan J.J. ; Slorach, Euan M. ; Fuller, Chris K. ; Gradia, Scott ; Kanner, Steven B. ; May, Andrew P. ; Sternberg, Samuel H. / Harnessing type I CRISPR–Cas systems for genome engineering in human cells. In: Nature Biotechnology. 2019 ; Vol. 37. pp. 1471-1477.
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abstract = "Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50{\%}. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.",
author = "Peter Cameron and Coons, {Mary M.} and Klompe, {Sanne E.} and Lied, {Alexandra M.} and Smith, {Stephen C.} and Bastien Vidal and Donohoue, {Paul D.} and Tomer Rotstein and Kohrs, {Bryan W.} and Nyer, {David B.} and Rachel Kennedy and Banh, {Lynda M.} and Carolyn Williams and Toh, {Mckenzi S.} and Irby, {Matthew J.} and Edwards, {Leslie S.} and Lin, {Chun Han} and Owen, {Arthur L.G.} and Tim K{\"u}nne and {van der Oost}, John and Brouns, {Stan J.J.} and Slorach, {Euan M.} and Fuller, {Chris K.} and Scott Gradia and Kanner, {Steven B.} and May, {Andrew P.} and Sternberg, {Samuel H.}",
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Cameron, P, Coons, MM, Klompe, SE, Lied, AM, Smith, SC, Vidal, B, Donohoue, PD, Rotstein, T, Kohrs, BW, Nyer, DB, Kennedy, R, Banh, LM, Williams, C, Toh, MS, Irby, MJ, Edwards, LS, Lin, CH, Owen, ALG, Künne, T, van der Oost, J, Brouns, SJJ, Slorach, EM, Fuller, CK, Gradia, S, Kanner, SB, May, AP & Sternberg, SH 2019, 'Harnessing type I CRISPR–Cas systems for genome engineering in human cells', Nature Biotechnology, vol. 37, pp. 1471-1477. https://doi.org/10.1038/s41587-019-0310-0

Harnessing type I CRISPR–Cas systems for genome engineering in human cells. / Cameron, Peter; Coons, Mary M.; Klompe, Sanne E.; Lied, Alexandra M.; Smith, Stephen C.; Vidal, Bastien; Donohoue, Paul D.; Rotstein, Tomer; Kohrs, Bryan W.; Nyer, David B.; Kennedy, Rachel; Banh, Lynda M.; Williams, Carolyn; Toh, Mckenzi S.; Irby, Matthew J.; Edwards, Leslie S.; Lin, Chun Han; Owen, Arthur L.G.; Künne, Tim; van der Oost, John; Brouns, Stan J.J.; Slorach, Euan M.; Fuller, Chris K.; Gradia, Scott; Kanner, Steven B.; May, Andrew P.; Sternberg, Samuel H.

In: Nature Biotechnology, Vol. 37, 18.12.2019, p. 1471-1477.

Research output: Contribution to journalLetterAcademicpeer-review

TY - JOUR

T1 - Harnessing type I CRISPR–Cas systems for genome engineering in human cells

AU - Cameron, Peter

AU - Coons, Mary M.

AU - Klompe, Sanne E.

AU - Lied, Alexandra M.

AU - Smith, Stephen C.

AU - Vidal, Bastien

AU - Donohoue, Paul D.

AU - Rotstein, Tomer

AU - Kohrs, Bryan W.

AU - Nyer, David B.

AU - Kennedy, Rachel

AU - Banh, Lynda M.

AU - Williams, Carolyn

AU - Toh, Mckenzi S.

AU - Irby, Matthew J.

AU - Edwards, Leslie S.

AU - Lin, Chun Han

AU - Owen, Arthur L.G.

AU - Künne, Tim

AU - van der Oost, John

AU - Brouns, Stan J.J.

AU - Slorach, Euan M.

AU - Fuller, Chris K.

AU - Gradia, Scott

AU - Kanner, Steven B.

AU - May, Andrew P.

AU - Sternberg, Samuel H.

PY - 2019/12/18

Y1 - 2019/12/18

N2 - Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

AB - Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

U2 - 10.1038/s41587-019-0310-0

DO - 10.1038/s41587-019-0310-0

M3 - Letter

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EP - 1477

JO - Nature Biotechnology

JF - Nature Biotechnology

SN - 1087-0156

ER -

Cameron P, Coons MM, Klompe SE, Lied AM, Smith SC, Vidal B et al. Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology. 2019 Dec 18;37:1471-1477. https://doi.org/10.1038/s41587-019-0310-0