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

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. ​

Literature Information ​

PMID23992846
JournalCell
Impact Factor42.5
JCR QuartileQ1
Publication Year2013
Times Cited1753
KeywordsCRISPR Cas9, genome editing, specificity, double-strand breaks, guide RNA
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.
ISSN0092-8674
Pages1380-9
Issue154(6)
AuthorsF Ann Ran, Patrick D Hsu, Chie-Yu Lin, Jonathan S Gootenberg, Silvana Konermann, Alexandro E Trevino, David A Scott, Azusa Inoue, Shogo Matoba, Yi Zhang, Feng Zhang

TL;DR ​

This study presents a novel genome editing approach utilizing a Cas9 nickase mutant combined with paired guide RNAs to achieve targeted double-strand breaks while significantly reducing off-target effects by 50- to 1,500-fold, thus enhancing the precision of gene editing. This strategy not only maintains on-target cleavage efficiency but also broadens the potential for diverse applications in research and medical fields that demand high specificity in genome editing.

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CRISPR Cas9 · genome editing · specificity · double-strand breaks · guide RNA

Abstract ​

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

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Primary Questions Addressed ​

  1. What are the potential implications of reduced off-target activity in clinical applications of CRISPR technology?
  2. How does the use of paired guide RNAs compare to traditional single-guide RNA approaches in terms of specificity and efficiency?
  3. What challenges might arise when implementing double nicking techniques in various organisms beyond mouse zygotes?
  4. In what ways can the double nicking strategy be optimized for different types of genomic targets or editing purposes?
  5. How does the mechanism of repair for individual nicks influence the overall outcomes of genome editing experiments?

Key Findings ​

Research Background and Objective ​

Targeted genome editing technologies, particularly those utilizing the CRISPR-Cas9 system, have revolutionized both research and medical fields. However, the potential for off-target effects—unintended mutations at non-target sites—remains a significant concern. The objective of this study is to enhance the specificity of CRISPR-Cas9 genome editing by employing a Cas9 nickase mutant combined with paired guide RNAs to reduce off-target mutagenesis while maintaining efficient on-target cleavage.

Main Methods/Materials/Experimental Design ​

The study utilized a novel approach involving the Cas9 nickase mutant and paired guide RNAs to create targeted double-strand breaks in the genome. The experimental design included the following key steps:

  1. Selection of Guide RNAs: Two guide RNAs were designed to target the same genomic locus but were offset to allow for simultaneous nicking.
  2. Nickase Activity: The Cas9 nickase mutant was employed to introduce single-strand nicks in the DNA, which are repaired with high fidelity.
  3. Double-Strand Break Formation: The combination of paired guide RNAs facilitated the formation of double-strand breaks through coordinated nicking.
  4. Evaluation of Off-Target Effects: The reduction in off-target activity was quantified in cell lines and mouse zygotes.
  5. Gene Knockout Efficiency: The efficiency of gene knockout was assessed in mouse zygotes while monitoring on-target cleavage.

The technical workflow can be represented as follows:

Mermaid diagram

Key Results and Findings ​

  • The use of paired guide RNAs with the Cas9 nickase mutant resulted in a significant reduction of off-target activity, achieving a decrease of 50- to 1,500-fold compared to traditional Cas9 methods.
  • The approach successfully facilitated gene knockout in mouse zygotes, demonstrating effective on-target cleavage without compromising specificity.
  • The study provided evidence that simultaneous nicking can enhance the precision of genome editing applications.

Main Conclusions/Significance/Innovativeness ​

This research introduces a versatile strategy that significantly enhances the specificity of genome editing by minimizing off-target effects through the use of paired guide RNAs and a Cas9 nickase mutant. The ability to achieve high efficiency in gene knockout while maintaining on-target fidelity is crucial for advancing therapeutic applications of genome editing technologies. This innovative approach may pave the way for safer and more effective genetic modifications in various fields, including medicine and agriculture.

Research Limitations and Future Directions ​

While the study presents promising results, several limitations and future research directions were identified:

LimitationsFuture Directions
The study primarily focused on cell lines and mouse zygotes, limiting generalizability to other organisms.Explore the application of this method in a broader range of species and cell types.
The long-term effects of the nicking strategy on genome stability were not assessed.Investigate the long-term consequences of using paired nicking on genomic integrity.
The efficiency of the approach in complex genomic environments remains to be fully characterized.Test the method in more complex genomic contexts, such as multicellular organisms or tissues.

This research lays the groundwork for future studies aimed at refining genome editing techniques to enhance their safety and efficacy across diverse applications.

References ​

  1. CRISPR/Cas system and its role in phage-bacteria interactions. - Hélène Deveau;Josiane E Garneau;Sylvain Moineau - Annual review of microbiology (2010)
  2. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. - Scott J Gratz;Alexander M Cummings;Jennifer N Nguyen;Danielle C Hamm;Laura K Donohue;Melissa M Harrison;Jill Wildonger;Kate M O'Connor-Giles - Genetics (2013)
  3. Efficient TALEN-mediated gene knockout in livestock. - Daniel F Carlson;Wenfang Tan;Simon G Lillico;Dana Stverakova;Chris Proudfoot;Michelle Christian;Daniel F Voytas;Charles R Long;C Bruce A Whitelaw;Scott C Fahrenkrug - Proceedings of the National Academy of Sciences of the United States of America (2012)
  4. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. - Giedrius Gasiunas;Rodolphe Barrangou;Philippe Horvath;Virginijus Siksnys - Proceedings of the National Academy of Sciences of the United States of America (2012)
  5. A simple cipher governs DNA recognition by TAL effectors. - Matthew J Moscou;Adam J Bogdanove - Science (New York, N.Y.) (2009)
  6. CRISPR/Cas, the immune system of bacteria and archaea. - Philippe Horvath;Rodolphe Barrangou - Science (New York, N.Y.) (2010)
  7. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. - Feng Zhang;Le Cong;Simona Lodato;Sriram Kosuri;George M Church;Paola Arlotta - Nature biotechnology (2011)
  8. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. - Frank Soldner;Josée Laganière;Albert W Cheng;Dirk Hockemeyer;Qing Gao;Raaji Alagappan;Vikram Khurana;Lawrence I Golbe;Richard H Myers;Susan Lindquist;Lei Zhang;Dmitry Guschin;Lauren K Fong;B Joseph Vu;Xiangdong Meng;Fyodor D Urnov;Edward J Rebar;Philip D Gregory;H Steve Zhang;Rudolf Jaenisch - Cell (2011)
  9. Targeted genome editing across species using ZFNs and TALENs. - Andrew J Wood;Te-Wen Lo;Bryan Zeitler;Catherine S Pickle;Edward J Ralston;Andrew H Lee;Rainier Amora;Jeffrey C Miller;Elo Leung;Xiangdong Meng;Lei Zhang;Edward J Rebar;Philip D Gregory;Fyodor D Urnov;Barbara J Meyer - Science (New York, N.Y.) (2011)
  10. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. - Seung Woo Cho;Sojung Kim;Jong Min Kim;Jin-Soo Kim - Nature biotechnology (2013)

Literatures Citing This Work ​

  1. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. - Zachary L Sebo;Han B Lee;Ying Peng;Yi Guo - Fly (2014)
  2. Genome engineering using the CRISPR-Cas9 system. - F Ann Ran;Patrick D Hsu;Jason Wright;Vineeta Agarwala;David A Scott;Feng Zhang - Nature protocols (2013)
  3. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. - Seung Woo Cho;Sojung Kim;Yongsub Kim;Jiyeon Kweon;Heon Seok Kim;Sangsu Bae;Jin-Soo Kim - Genome research (2014)
  4. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. - Ines Fonfara;Anaïs Le Rhun;Krzysztof Chylinski;Kira S Makarova;Anne-Laure Lécrivain;Janek Bzdrenga;Eugene V Koonin;Emmanuelle Charpentier - Nucleic acids research (2014)
  5. Effect of genetic variation in a Drosophila model of diabetes-associated misfolded human proinsulin. - Bin Z He;Michael Z Ludwig;Desiree A Dickerson;Levi Barse;Bharath Arun;Bjarni J Vilhjálmsson;Pengyao Jiang;Soo-Young Park;Natalia A Tamarina;Scott B Selleck;Patricia J Wittkopp;Graeme I Bell;Martin Kreitman - Genetics (2014)
  6. Newer gene editing technologies toward HIV gene therapy. - N Manjunath;Guohua Yi;Ying Dang;Premlata Shankar - Viruses (2013)
  7. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. - Sandrine Boissel;Jordan Jarjour;Alexander Astrakhan;Andrew Adey;Agnès Gouble;Philippe Duchateau;Jay Shendure;Barry L Stoddard;Michael T Certo;David Baker;Andrew M Scharenberg - Nucleic acids research (2014)
  8. Exploiting CRISPR/Cas systems for biotechnology. - Timothy R Sampson;David S Weiss - BioEssays : news and reviews in molecular, cellular and developmental biology (2014)
  9. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. - Andrew R Bassett;Charlotte Tibbit;Chris P Ponting;Ji-Long Liu - Biology open (2014)
  10. Genetic screens in human cells using the CRISPR-Cas9 system. - Tim Wang;Jenny J Wei;David M Sabatini;Eric S Lander - Science (New York, N.Y.) (2014)

... (1743 more literatures)

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