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

CRISPR-Cas systems for editing, regulating and targeting genomes. ​

Literature Information ​

DOI10.1038/nbt.2842
PMID24584096
JournalNature biotechnology
Impact Factor41.7
JCR QuartileQ1
Publication Year2014
Times Cited1402
KeywordsCRISPR technology, gene editing, RNA-guided nucleases, gene expression regulation, genome targeting
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., Review
ISSN1087-0156
Pages347-55
Issue32(4)
AuthorsJeffry D Sander, J Keith Joung

TL;DR ​

The rapid adoption of CRISPR technology for targeted genome editing has revolutionized biological research by enabling efficient and customizable modifications of genes in various organisms, including those previously difficult to manipulate. This advancement not only enhances our understanding of gene functions but also holds significant potential for developing innovative therapeutic strategies for treating human diseases.

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CRISPR technology · gene editing · RNA-guided nucleases · gene expression regulation · genome targeting

Abstract ​

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

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Primary Questions Addressed ​

  1. How do different Cas proteins compare in terms of efficiency and specificity for genome editing?
  2. What are the potential off-target effects of CRISPR-Cas systems, and how can they be minimized?
  3. In what ways can CRISPR technology be adapted for therapeutic applications beyond gene editing?
  4. What are the ethical considerations surrounding the use of CRISPR-Cas systems in human germline editing?
  5. How does the development of CRISPR-based tools influence the future of synthetic biology and biotechnology?

Key Findings ​

Research Background and Purpose ​

The advent of CRISPR-Cas systems has revolutionized genome editing, providing a powerful and efficient method for targeted modifications across a wide range of organisms. This review discusses the mechanisms of CRISPR technology, its applications in biological research and potential therapeutic uses, as well as the challenges associated with specificity and off-target effects.

Main Methods/Materials/Experimental Design ​

The CRISPR-Cas9 system operates by creating double-stranded breaks (DSBs) in DNA, which can be repaired through two primary pathways: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). The method is characterized by the following steps:

Mermaid diagram
  • Components: The system consists of the Cas9 nuclease and a guide RNA (gRNA) that directs Cas9 to the target DNA sequence.
  • Targeting: The gRNA must bind to a protospacer adjacent motif (PAM) for effective cleavage.
  • Applications: This system has been applied in various cell types and organisms, including plants and animals, to induce gene knockouts, insertions, and modifications.

Key Results and Findings ​

  • Efficiency: CRISPR-Cas9 demonstrates high efficiency in generating targeted mutations, with alteration frequencies often exceeding 50%.
  • Versatility: The system has been successfully utilized in a diverse range of organisms, from bacteria to mammals, facilitating genetic modifications across species.
  • Off-Target Effects: While the technology is powerful, there are concerns regarding off-target cleavage, with studies showing unintended mutations at sites with slight sequence variations.

Main Conclusions/Significance/Innovation ​

The CRISPR-Cas9 system represents a significant advancement in genetic engineering, enabling precise and efficient genome editing. Its applications extend beyond simple gene editing to include gene regulation and epigenome modification, thus opening new avenues for research and therapeutic strategies in treating genetic diseases. The ability to multiplex gRNAs allows for complex genetic manipulations, enhancing the potential for comprehensive genetic screening.

Research Limitations and Future Directions ​

  • Specificity: There is an urgent need for methods to improve the specificity of CRISPR-Cas9 to minimize off-target effects. Current strategies include using paired nickases and modified gRNAs.
  • Delivery Mechanisms: Optimizing delivery methods for different cell types and organisms remains a challenge. Developing tissue-specific expression systems could enhance therapeutic applications.
  • Long-Term Effects: Future research should focus on understanding the long-term implications of CRISPR modifications, particularly in therapeutic contexts.

Overall, while the CRISPR-Cas9 technology has transformed genetic research, ongoing improvements in specificity and delivery are crucial for its successful application in clinical settings.

References ​

  1. 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)
  2. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. - Thomas Gaj;Jing Guo;Yoshio Kato;Shannon J Sirk;Carlos F Barbas - Nature methods (2012)
  3. Locus-specific editing of histone modifications at endogenous enhancers. - Eric M Mendenhall;Kaylyn E Williamson;Deepak Reyon;James Y Zou;Oren Ram;J Keith Joung;Bradley E Bernstein - Nature biotechnology (2013)
  4. 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)
  5. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. - Yonatan B Tzur;Ari E Friedland;Saravanapriah Nadarajan;George M Church;John A Calarco;Monica P Colaiácovo - Genetics (2013)
  6. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. - Morgan L Maeder;James F Angstman;Marcy E Richardson;Samantha J Linder;Vincent M Cascio;Shengdar Q Tsai;Quan H Ho;Jeffry D Sander;Deepak Reyon;Bradley E Bernstein;Joseph F Costello;Miles F Wilkinson;J Keith Joung - Nature biotechnology (2013)
  7. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. - Vikram Pattanayak;Steven Lin;John P Guilinger;Enbo Ma;Jennifer A Doudna;David R Liu - Nature biotechnology (2013)
  8. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. - David A Wright;Stacey Thibodeau-Beganny;Jeffry D Sander;Ronnie J Winfrey;Andrew S Hirsh;Magdalena Eichtinger;Fengli Fu;Matthew H Porteus;Drena Dobbs;Daniel F Voytas;J Keith Joung - Nature protocols (2006)
  9. Genome engineering with zinc-finger nucleases. - Dana Carroll - Genetics (2011)
  10. Efficient genome editing in zebrafish using a CRISPR-Cas system. - Woong Y Hwang;Yanfang Fu;Deepak Reyon;Morgan L Maeder;Shengdar Q Tsai;Jeffry D Sander;Randall T Peterson;J-R Joanna Yeh;J Keith Joung - Nature biotechnology (2013)

Literatures Citing This Work ​

  1. Gene editing at CRISPR speed. - Monya Baker - Nature biotechnology (2014)
  2. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. - Shengdar Q Tsai;Nicolas Wyvekens;Cyd Khayter;Jennifer A Foden;Vishal Thapar;Deepak Reyon;Mathew J Goodwin;Martin J Aryee;J Keith Joung - Nature biotechnology (2014)
  3. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. - Lior Nissim;Samuel D Perli;Alexandra Fridkin;Pablo Perez-Pinera;Timothy K Lu - Molecular cell (2014)
  4. Targeted genome editing in human repopulating haematopoietic stem cells. - Pietro Genovese;Giulia Schiroli;Giulia Escobar;Tiziano Di Tomaso;Claudia Firrito;Andrea Calabria;Davide Moi;Roberta Mazzieri;Chiara Bonini;Michael C Holmes;Philip D Gregory;Mirjam van der Burg;Bernhard Gentner;Eugenio Montini;Angelo Lombardo;Luigi Naldini - Nature (2014)
  5. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. - James A Gagnon;Eivind Valen;Summer B Thyme;Peng Huang;Laila Akhmetova;Laila Ahkmetova;Andrea Pauli;Tessa G Montague;Steven Zimmerman;Constance Richter;Alexander F Schier - PloS one (2014)
  6. Computational and experimental approaches to reveal the effects of single nucleotide polymorphisms with respect to disease diagnostics. - Tugba G Kucukkal;Ye Yang;Susan C Chapman;Weiguo Cao;Emil Alexov - International journal of molecular sciences (2014)
  7. Development and applications of CRISPR-Cas9 for genome engineering. - Patrick D Hsu;Eric S Lander;Feng Zhang - Cell (2014)
  8. Key applications of plant metabolic engineering. - Warren Lau;Michael A Fischbach;Anne Osbourn;Elizabeth S Sattely - PLoS biology (2014)
  9. The prospect of molecular therapy for Angelman syndrome and other monogenic neurologic disorders. - Barbara J Bailus;David J Segal - BMC neuroscience (2014)
  10. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. - Tetsushi Sakuma;Ayami Nishikawa;Satoshi Kume;Kazuaki Chayama;Takashi Yamamoto - Scientific reports (2014)

... (1392 more literatures)

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