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In vivo genome editing using Staphylococcus aureus Cas9
Open accessAbstract
The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.
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“Also, the requirement of a specific PAM Sequence for targeted genome editing is crucial, and even though it reduces off-target activity, it often results in suboptimal DNA cleavage, editing efficiencies (Ran et al., 2015; Edraki et al., 2019) and restricting the scope of targeted edition.”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Ran et al., 2015
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In the citing paper
“Cas9 genomic localization is delimited by the protospacer adjacent motif (PAM), which is NGG for SpCas9 while evolved variants of SpCas9 tolerate a broader range of PAM sequences17–21.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [20]
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In the citing paper
“The SpCas9 homolog derived from Staphylococcus aureus (SaCas9) has been extensively characterized as a highly efficient and significantly smaller alternative to SpCas9.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [20]
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“It recognizes an NNGRRT PAM (where R = A or G)20.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [20]
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In the citing paper
“Recent advancements in gene-editing technologies have led to the development of artificial Cas9 variants with relaxed PAM specificities, which improve efficiency but are associated with an increase in off-target effects and a decrease in target specificity (Ran et al., 2015).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Ran et al., 2015
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In the citing paper
“Recently, a smaller form of Cas9 was developed, and shown capable of packing and delivery with sgRNA by AAV in vivo12.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [12]
In the citing paper
“Adenovirus or adeno-associated virus (AAV)-mediated CRISPR-Cas9 delivery has been successfully applied to knockout genes in the mouse brain and liver12,13.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [12]
In the citing paper
“A smaller form of Cas9 (Staphylococcus aureus Cas9), which fits into AAV vectors, has been recently used to knockout genes by NHEJ in the mouse liver. More than 40% indel formations in Pcsk9 locus was reported12.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [12]
In the citing paper
“For example, less than 10% indels at the Apob locus in the mouse liver was reported in the same study12.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [12]
In the citing paper
“However, the large genetic size of Cas9 (~ 4.5 kb) and the total plasmid size (> 7 kb) significantly increase the difficulty of delivery and expression of CRISPR/Cas9 systems [94].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [94]
In the citing paper
“Although the newly discovered Cas9 form from Staphylococcus aureus strain (SaCas9) is about 1 kb shorter than SpCas9, the overall genetic size is still too large for application [94, 140].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [94]
In the citing paper
“The Cas9 from Staphylococcus aureus (SaCas9) is over 1 kb shorter (3.15 kb) than SpCas9 [69].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [69]
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Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
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Metadata
- Authors
- Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V., Sharp, P. A., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Massachusetts Institute of Technology (US)
- Koch Institute for Integrative Cancer Research At MIT (US)
- National Institutes of Health (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature
- Published
- 2015-04
- Volume
- 520
- Issue
- 7546
- Pages
- 186-191
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4393360
- OpenAlex
- W2070614858
- Cited by
- 2,687
- References
- 51
- Citations by year
- 2026: 126 · 2025: 167 · 2024: 180 · 2023: 199 · 2022: 285 · 2021: 263 · 2020: 274 · 2019: 261 · 2018: 264 · 2017: 288 · 2016: 264 · 2015: 113
- FWCI
- 134.53
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- RePEc: Research Papers in Economics (submittedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- PMC
- Funders
- Howard Hughes Medical Institute
- National Institute of Mental Health (DP1MH100706)
- National Eye Institute (R01EY024259, P30EY012196)
- National Cancer Institute (R01CA133404, P01CA042063, P30CA014051, P30-CA-14051, P01 CA 42063)
- National Institute of General Medical Sciences (R01GM034277, T32GM008313, R01 GM34277, T32GM007753)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- National Center for Chronic Disease Prevention and Health Promotion (5DP1-MH100706)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- SaCas9, SpCas9, CRISPR/Cas9, in vivo gene editing, adeno-associated virus delivery, PCSK9, cholesterol regulation, targeting specificity, single guide RNA
- MeSH terms
- Proprotein Convertase 9, Animals, Base Sequence, Cholesterol, Genetic Engineering, Liver, Male, Mice, Inbred C57BL, Serine Endopeptidases, Staphylococcus aureus, Substrate Specificity, Genome, Gene Targeting, Proprotein Convertases, Mice, CRISPR-Associated Proteins
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