Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Background: While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. Results: We systematically c...

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Main Authors: Wang, Yuanming, Liu, Ivy Kaiwen, Sutrisnoh, Norfala-Aliah, Srinivasan, Harini, Zhang, Junyi, Li, Jia, Zhang, Fan, Lalith, Charles Richard John, Xing, Heyun, Shanmugam, Raghuvaran, Foo, Jia Nee, Yeo, Hwee Ting, Ooi, Kean Hean, Bleckwehl, Tore, Par, Rachel Yi Yun, Lee, Shi Mun, Ismail, Nur Nadiah, Sanwari, Nur Aidah, Lee, Vanessa Si Ting, Lew, Jan, Tan, Meng How
Other Authors: School of Chemical and Biomedical Engineering
Format: Article
Language:English
Published: 2018
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Online Access:https://hdl.handle.net/10356/87501
http://hdl.handle.net/10220/45417
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Institution: Nanyang Technological University
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spelling sg-ntu-dr.10356-875012023-12-29T06:46:50Z Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells Wang, Yuanming Liu, Ivy Kaiwen Sutrisnoh, Norfala-Aliah Srinivasan, Harini Zhang, Junyi Li, Jia Zhang, Fan Lalith, Charles Richard John Xing, Heyun Shanmugam, Raghuvaran Foo, Jia Nee Yeo, Hwee Ting Ooi, Kean Hean Bleckwehl, Tore Par, Rachel Yi Yun Lee, Shi Mun Ismail, Nur Nadiah Sanwari, Nur Aidah Lee, Vanessa Si Ting Lew, Jan Tan, Meng How School of Chemical and Biomedical Engineering School of Biological Sciences Lee Kong Chian School of Medicine (LKCMedicine) CRISPR Genome Editing Background: While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. Results: We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes. Conclusions: Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version 2018-07-31T06:48:41Z 2019-12-06T16:43:14Z 2018-07-31T06:48:41Z 2019-12-06T16:43:14Z 2018 Journal Article Wang, Y., Liu, I. K., Sutrisnoh, N.-A., Srinivasan, H., Zhang, J., Li, J., et al. (2018). Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells. Genome Biology, 19(1), 62-. 1474-760X https://hdl.handle.net/10356/87501 http://hdl.handle.net/10220/45417 10.1186/s13059-018-1445-x en Genome Biology © 2018 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 16 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic CRISPR
Genome Editing
spellingShingle CRISPR
Genome Editing
Wang, Yuanming
Liu, Ivy Kaiwen
Sutrisnoh, Norfala-Aliah
Srinivasan, Harini
Zhang, Junyi
Li, Jia
Zhang, Fan
Lalith, Charles Richard John
Xing, Heyun
Shanmugam, Raghuvaran
Foo, Jia Nee
Yeo, Hwee Ting
Ooi, Kean Hean
Bleckwehl, Tore
Par, Rachel Yi Yun
Lee, Shi Mun
Ismail, Nur Nadiah
Sanwari, Nur Aidah
Lee, Vanessa Si Ting
Lew, Jan
Tan, Meng How
Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
description Background: While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. Results: We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes. Conclusions: Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.
author2 School of Chemical and Biomedical Engineering
author_facet School of Chemical and Biomedical Engineering
Wang, Yuanming
Liu, Ivy Kaiwen
Sutrisnoh, Norfala-Aliah
Srinivasan, Harini
Zhang, Junyi
Li, Jia
Zhang, Fan
Lalith, Charles Richard John
Xing, Heyun
Shanmugam, Raghuvaran
Foo, Jia Nee
Yeo, Hwee Ting
Ooi, Kean Hean
Bleckwehl, Tore
Par, Rachel Yi Yun
Lee, Shi Mun
Ismail, Nur Nadiah
Sanwari, Nur Aidah
Lee, Vanessa Si Ting
Lew, Jan
Tan, Meng How
format Article
author Wang, Yuanming
Liu, Ivy Kaiwen
Sutrisnoh, Norfala-Aliah
Srinivasan, Harini
Zhang, Junyi
Li, Jia
Zhang, Fan
Lalith, Charles Richard John
Xing, Heyun
Shanmugam, Raghuvaran
Foo, Jia Nee
Yeo, Hwee Ting
Ooi, Kean Hean
Bleckwehl, Tore
Par, Rachel Yi Yun
Lee, Shi Mun
Ismail, Nur Nadiah
Sanwari, Nur Aidah
Lee, Vanessa Si Ting
Lew, Jan
Tan, Meng How
author_sort Wang, Yuanming
title Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
title_short Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
title_full Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
title_fullStr Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
title_full_unstemmed Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
title_sort systematic evaluation of crispr-cas systems reveals design principles for genome editing in human cells
publishDate 2018
url https://hdl.handle.net/10356/87501
http://hdl.handle.net/10220/45417
_version_ 1787136495191064576