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Curated Papers > Highly Cited Authoritative Literature on "CRISPR Gene Editing"

In vivo genome editing using Staphylococcus aureus Cas9. ​

Literature Information ​

DOI10.1038/nature14299
PMID25830891
JournalNature
Impact Factor48.5
JCR QuartileQ1
Publication Year2015
Times Cited1425
KeywordsGenome editing, Staphylococcus aureus Cas9, Adeno-associated virus, Pcsk9, Specificity
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0028-0836
Pages186-91
Issue520(7546)
AuthorsF Ann Ran, Le Cong, Winston X Yan, David A Scott, Jonathan S Gootenberg, Andrea J Kriz, Bernd Zetsche, Ophir Shalem, Xuebing Wu, Kira S Makarova, Eugene V Koonin, Phillip A Sharp, Feng Zhang

TL;DR ​

This study explores the use of Staphylococcus aureus Cas9 (SaCas9), a smaller alternative to Streptococcus pyogenes Cas9 (SpCas9), demonstrating that SaCas9 can achieve genome editing efficiencies comparable to SpCas9 while being over 1 kilobase shorter. The research highlights the potential of SaCas9 for therapeutic applications, evidenced by successful gene modification and reduced cholesterol levels in mice following AAV-mediated delivery.

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Genome editing · Staphylococcus aureus Cas9 · Adeno-associated virus · Pcsk9 · Specificity

Abstract ​

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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Primary Questions Addressed ​

  1. What are the advantages of using SaCas9 over SpCas9 in terms of delivery efficiency and specificity in gene editing?
  2. How does the efficiency of SaCas9 compare to other smaller Cas9 orthologues in different biological systems?
  3. What potential therapeutic applications could benefit from the use of SaCas9 in genome editing?
  4. How does the genome-wide targeting specificity of SaCas9 affect its safety and efficacy in clinical settings?
  5. What are the implications of achieving over 40% gene modification in vivo for future gene therapy strategies?

Key Findings ​

Research Background and Objectives ​

The study focuses on the development of a smaller Cas9 ortholog from Staphylococcus aureus (SaCas9) for genome editing applications, particularly for in vivo delivery using adeno-associated virus (AAV) vectors. Traditional Cas9 from Streptococcus pyogenes (SpCas9) is too large for efficient AAV packaging, limiting its use in therapeutic settings. The goal is to characterize SaCas9's genome editing efficiency and specificity compared to SpCas9.

Main Methods/Materials/Experimental Design ​

The researchers conducted a comprehensive evaluation of six smaller Cas9 orthologs, with a particular focus on SaCas9. Key methodologies included:

  1. In Vitro Cleavage Assays:

    • Comparison of Cas9 orthologs' DNA cleavage capabilities using human embryonic kidney 293FT cells.
    • Development of single guide RNAs (sgRNAs) tailored for each Cas9.

  2. PAM Discovery:

    • Identification of protospacer adjacent motifs (PAMs) necessary for Cas9 activity through a library of plasmids containing variable PAM sequences.

  3. BLESS Technique:

    • Implementation of the BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing) method to assess genome-wide targeting specificity and identify off-target effects.

  4. In Vivo Experiments:

    • Packaging of SaCas9 and sgRNA into AAV vectors for intravenous injection in mice, targeting the cholesterol regulatory gene Pcsk9.

  5. Statistical Analysis:

    • Use of statistical methods to correlate double-strand break (DSB) scores with indel formation at target sites.
Mermaid diagram

Key Results and Findings ​

  • SaCas9 demonstrated genome editing efficiencies comparable to SpCas9, achieving over 40% gene modification in the Pcsk9 gene within one week post-injection.
  • Significant reductions in serum Pcsk9 and total cholesterol levels were observed.
  • The BLESS technique revealed high specificity of SaCas9 with minimal off-target effects, showing low levels of DSB signals at predicted off-target sites in vivo.
  • A strong correlation was established between DSB scores and indel formation, indicating reliable prediction of on-target and off-target activity.

Main Conclusions/Significance/Innovation ​

The study successfully presents SaCas9 as a promising alternative to SpCas9 for in vivo genome editing, particularly due to its smaller size allowing for efficient AAV delivery. The findings underscore the importance of Cas9 specificity and the potential for therapeutic applications in treating conditions related to cholesterol metabolism. This research expands the CRISPR-Cas toolbox, paving the way for more versatile genome editing technologies.

Research Limitations and Future Directions ​

  • The study notes the need for further evaluation of long-term effects and potential off-target modifications in vivo.
  • Future research could focus on optimizing the SaCas9 system and exploring other Cas9 orthologs for additional therapeutic applications.
  • Additional studies should aim to validate the findings across larger cohorts to ensure the safety and efficacy of SaCas9-mediated genome editing in various contexts.

References ​

  1. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. - Christian Wolfrum;Shuanping Shi;K Narayanannair Jayaprakash;Muthusamy Jayaraman;Gang Wang;Rajendra K Pandey;Kallanthottathil G Rajeev;Tomoko Nakayama;Klaus Charrise;Esther M Ndungo;Tracy Zimmermann;Victor Koteliansky;Muthiah Manoharan;Markus Stoffel - Nature biotechnology (2007)
  2. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. - Carmela Zincarelli;Stephen Soltys;Giuseppe Rengo;Joseph E Rabinowitz - Molecular therapy : the journal of the American Society of Gene Therapy (2008)
  3. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. - Shengdar Q Tsai;Zongli Zheng;Nhu T Nguyen;Matthew Liebers;Ved V Topkar;Vishal Thapar;Nicolas Wyvekens;Cyd Khayter;A John Iafrate;Long P Le;Martin J Aryee;J Keith Joung - Nature biotechnology (2015)
  4. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. - Josiane E Garneau;Marie-Ève Dupuis;Manuela Villion;Dennis A Romero;Rodolphe Barrangou;Patrick Boyaval;Christophe Fremaux;Philippe Horvath;Alfonso H Magadán;Sylvain Moineau - Nature (2010)
  5. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. - Elitza Deltcheva;Krzysztof Chylinski;Cynthia M Sharma;Karine Gonzales;Yanjie Chao;Zaid A Pirzada;Maria R Eckert;Jörg Vogel;Emmanuelle Charpentier - Nature (2011)
  6. State-of-the-art gene-based therapies: the road ahead. - Mark A Kay - Nature reviews. Genetics (2011)
  7. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. - Federico Mingozzi;Katherine A High - Nature reviews. Genetics (2011)
  8. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. - Ekaterina Semenova;Matthijs M Jore;Kirill A Datsenko;Anna Semenova;Edze R Westra;Barry Wanner;John van der Oost;Stan J J Brouns;Konstantin Severinov - Proceedings of the National Academy of Sciences of the United States of America (2011)
  9. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. - Rimantas Sapranauskas;Giedrius Gasiunas;Christophe Fremaux;Rodolphe Barrangou;Philippe Horvath;Virginijus Siksnys - Nucleic acids research (2011)
  10. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. - Martin Jinek;Krzysztof Chylinski;Ines Fonfara;Michael Hauer;Jennifer A Doudna;Emmanuelle Charpentier - Science (New York, N.Y.) (2012)

Literatures Citing This Work ​

  1. High-throughput functional genomics using CRISPR-Cas9. - Ophir Shalem;Neville E Sanjana;Feng Zhang - Nature reviews. Genetics (2015)
  2. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9. - Taeyoung Koo;Jungjoon Lee;Jin-Soo Kim - Molecules and cells (2015)
  3. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. - Vyas Ramanan;Amir Shlomai;David B T Cox;Robert E Schwartz;Eleftherios Michailidis;Ankit Bhatta;David A Scott;Feng Zhang;Charles M Rice;Sangeeta N Bhatia - Scientific reports (2015)
  4. Precision cancer mouse models through genome editing with CRISPR-Cas9. - Haiwei Mou;Zachary Kennedy;Daniel G Anderson;Hao Yin;Wen Xue - Genome medicine (2015)
  5. Pathophysiology, diagnosis and management of nephrogenic diabetes insipidus. - Detlef Bockenhauer;Daniel G Bichet - Nature reviews. Nephrology (2015)
  6. Development of an intein-mediated split-Cas9 system for gene therapy. - Dong-Jiunn Jeffery Truong;Karin Kühner;Ralf Kühn;Stanislas Werfel;Stefan Engelhardt;Wolfgang Wurst;Oskar Ortiz - Nucleic acids research (2015)
  7. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. - Dan Wang;Haiwei Mou;Shaoyong Li;Yingxiang Li;Soren Hough;Karen Tran;Jia Li;Hao Yin;Daniel G Anderson;Erik J Sontheimer;Zhiping Weng;Guangping Gao;Wen Xue - Human gene therapy (2015)
  8. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. - Benjamin P Kleinstiver;Michelle S Prew;Shengdar Q Tsai;Ved V Topkar;Nhu T Nguyen;Zongli Zheng;Andrew P W Gonzales;Zhuyun Li;Randall T Peterson;Jing-Ruey Joanna Yeh;Martin J Aryee;J Keith Joung - Nature (2015)
  9. Application of CRISPR/Cas9 for biomedical discoveries. - Sean M Riordan;Daniel P Heruth;Li Q Zhang;Shui Qing Ye - Cell & bioscience (2015)
  10. Expression of CRISPR/Cas single guide RNAs using small tRNA promoters. - Adam L Mefferd;Anand V R Kornepati;Hal P Bogerd;Edward M Kennedy;Bryan R Cullen - RNA (New York, N.Y.) (2015)

... (1415 more literatures)

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