Cited by 145CRISPR and Genetic EngineeringRNA regulation and diseaseCAR-T cell therapy research

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High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells

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Kim, Hui Kwon, Lee, Sungtae, Kim, Younggwang, Park, Jinman, Min, Seonwoo, Choi, Jae Woo, Huang, Tony P., Yoon, Sungroh, Liu, David R., Kim, Hyongbum Henry·2020·Nature Biomedical Engineering

DOI 10.1038/s41551-019-0505-1

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Abstract

The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.

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Authors: Kim, H. K., Lee, S., Kim, Y., Park, J., Min, S., Choi, J. W., Huang, T. P., Yoon, S., Liu, D. R., & Kim, H. H.
Affiliations: Yonsei University (KR)
Seoul National University (KR)
Broad Institute (US)
ORCID: Kim, Hui Kwon
Kim, Younggwang
Park, Jinman
Min, Seonwoo
Choi, Jae Woo
Huang, Tony P.
Yoon, Sungroh
Liu, David R.
Kim, Hyongbum Henry
Venue: Nature Biomedical Engineering
Publisher: Springer Science and Business Media LLC
Published: 2020-01-14
Volume: 4
Issue: 1
Pages: 111-124
ISSN: 2157-846X (ISSN-L)
DOI: 10.1038/s41551-019-0505-1
OpenAlex: W2999201570
Cited by: 145
References: 66
Citations by year: 2026: 19 · 2025: 18 · 2024: 23 · 2023: 22 · 2022: 20 · 2021: 26 · 2020: 17
FWCI: 6.89
Type: journal article
Language: en
Open access: green
ir.ymlib.yonsei.ac.kr
Licence: cc-by-nc-nd
www.springernature.com/gp/researchers/text-and-data-mining (tdm)
www.springernature.com/gp/researchers/text-and-data-mining (vor)
Locations: Nature Biomedical Engineering (publishedVersion)
PubMed (publishedVersion)
Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
YUHSpace (Yonsei University Medical Library) (submittedVersion, cc-by-nc-nd)
Funders: Foundation for the National Institutes of Health (RM1 HG009490, R01 EB022376, R35 GM118062)
Howard Hughes Medical Institute (David Liu)
National Research Foundation of Korea (2017R1A2B3004198, 2017M3A9B4062403, 2018R1A5A2025079)
Ministry of Health and Welfare (HI17C0676, HI16C1012)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Health Sciences › Medicine › Oncology › CAR-T cell therapy research
Keywords: SpCas9, protospacer adjacent motif, on-target activity, off-target effects, CRISPR gene editing
MeSH terms: Gene Editing, CRISPR-Associated Protein 9, Deep Learning, Genetic Vectors, Humans, Streptococcus pyogenes, Lentivirus, HEK293 Cells, CRISPR-Cas Systems

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High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells

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