Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, s...

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Main Authors:	Zhang, Jian-Ping, Li, Xiao-Lan, Neises, Amanda, Chen, Wanqiu, Hu, Lin-Ping, Ji, Guang-Zhen, Yu, Jun-Yao, Xu, Jing, Yuan, Wei-Ping, Cheng, Tao, Zhang, Xiao-Bing
Format:	Online
Language:	English
Published:	Nature Publishing Group 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919781/

id	pubmed-4919781
recordtype	oai_dc
spelling	pubmed-49197812016-06-28 Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency Zhang, Jian-Ping Li, Xiao-Lan Neises, Amanda Chen, Wanqiu Hu, Lin-Ping Ji, Guang-Zhen Yu, Jun-Yao Xu, Jing Yuan, Wei-Ping Cheng, Tao Zhang, Xiao-Bing Article CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable. Nature Publishing Group 2016-06-24 /pmc/articles/PMC4919781/ /pubmed/27338021 http://dx.doi.org/10.1038/srep28566 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Zhang, Jian-Ping Li, Xiao-Lan Neises, Amanda Chen, Wanqiu Hu, Lin-Ping Ji, Guang-Zhen Yu, Jun-Yao Xu, Jing Yuan, Wei-Ping Cheng, Tao Zhang, Xiao-Bing
spellingShingle	Zhang, Jian-Ping Li, Xiao-Lan Neises, Amanda Chen, Wanqiu Hu, Lin-Ping Ji, Guang-Zhen Yu, Jun-Yao Xu, Jing Yuan, Wei-Ping Cheng, Tao Zhang, Xiao-Bing Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
author_facet	Zhang, Jian-Ping Li, Xiao-Lan Neises, Amanda Chen, Wanqiu Hu, Lin-Ping Ji, Guang-Zhen Yu, Jun-Yao Xu, Jing Yuan, Wei-Ping Cheng, Tao Zhang, Xiao-Bing
author_sort	Zhang, Jian-Ping
title	Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
title_short	Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
title_full	Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
title_fullStr	Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
title_full_unstemmed	Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
title_sort	different effects of sgrna length on crispr-mediated gene knockout efficiency
description	CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.
publisher	Nature Publishing Group
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919781/
_version_	1613599276633423872

Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

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