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Efficient genome editing in zebrafish using a CRISPR-Cas system. ​

Literature Information ​

DOI10.1038/nbt.2501
PMID23360964
JournalNature biotechnology
Impact Factor41.7
JCR QuartileQ1
Publication Year2013
Times Cited1475
KeywordsGenome Editing, CRISPR-Cas System, Zebrafish
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
ISSN1087-0156
Pages227-9
Issue31(3)
AuthorsWoong 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

TL;DR ​

This study demonstrates that the CRISPR-Cas system can effectively induce targeted genetic modifications in zebrafish embryos, achieving efficiencies comparable to traditional methods like zinc finger nucleases and transcription activator-like effector nucleases. The findings highlight the potential of CRISPR-Cas technology for precise genetic engineering in live organisms, expanding its applications in developmental biology and genetic research.

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Genome Editing · CRISPR-Cas System · Zebrafish

Abstract ​

In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.

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Primary Questions Addressed ​

  1. What are the potential advantages of using CRISPR-Cas systems over traditional methods like zinc finger nucleases in zebrafish genome editing?
  2. How does the efficiency of CRISPR-Cas system in zebrafish compare to its application in other model organisms?
  3. What specific challenges might researchers face when implementing CRISPR-Cas technology in zebrafish embryos?
  4. In what ways could the findings from zebrafish genome editing using CRISPR-Cas systems be applied to other fields, such as medicine or agriculture?
  5. What future developments in CRISPR technology could enhance its effectiveness in zebrafish or other species?

Key Findings ​

Research Background and Objective ​

The study explores the potential of RNA-guided nucleases, specifically the CRISPR/Cas9 system, for efficient in vivo genome editing. CRISPR/Cas9, originally a bacterial immune mechanism, has been adapted for targeted genetic modifications. The primary aim is to demonstrate that this system can effectively induce targeted genetic changes in zebrafish embryos, achieving efficiencies comparable to established methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

Main Methods/Materials/Experimental Design ​

The authors utilized a combination of CRISPR/Cas9 technology and zebrafish embryos to test the efficacy of genome editing. The methodology included:

  1. Construction of Expression Vectors: Vectors were created for the expression of Cas9 nuclease and guide RNAs (gRNAs) using T7 RNA polymerase.
  2. Microinjection: Zebrafish embryos were injected with varying concentrations of gRNA and Cas9 mRNA to determine optimal conditions for inducing mutations.
  3. Mutation Detection: The T7 Endonuclease I (T7EI) assay was employed to assess the frequency of targeted insertions/deletions (indels).
  4. Sequencing: Mutated alleles were sequenced to confirm the presence and nature of the modifications.

The following flowchart illustrates the technical route:

Mermaid diagram

Key Results and Findings ​

  • The CRISPR/Cas9 system demonstrated high efficiency, with successful mutagenesis at 9 out of 11 targeted sites in zebrafish.
  • Indel frequencies ranged from 10.0% to 59.4%, depending on the concentration of injected RNAs.
  • The system achieved significant genetic modifications at sites previously unalterable by TALENs.
  • The nature of mutations induced by CRISPR/Cas9 was consistent with those observed using ZFNs and TALENs, confirming its robustness.
Target GeneIndel Frequency (%)Previous Method Efficacy
fh (site #1)52.7TALENs: Failed
gsk3b24.1ZFNs: Comparable
drd359.4ZFNs: Comparable

Main Conclusions/Significance/Innovation ​

The study concludes that the CRISPR/Cas9 system is a powerful and efficient tool for genome editing in whole organisms, particularly zebrafish. Its advantages include:

  • Simplicity in design and construction of gRNAs.
  • Higher mutagenesis rates at difficult target sites compared to ZFNs and TALENs.
  • Potential for multiplex genome editing due to the use of a single Cas9 protein and short gRNA sequences.

This research underscores the potential of CRISPR/Cas9 as a widely applicable technology for genetic studies and therapeutic applications.

Research Limitations and Future Directions ​

  • The study acknowledges the need to investigate the genome-wide specificity of RNA-guided Cas9 nucleases to prevent off-target effects.
  • Future research should focus on understanding why certain gRNAs fail to induce efficient mutations and optimizing gRNA design for improved efficacy.
  • Larger-scale tests of the targeting range of the gRNA/Cas9 system are warranted to further establish its utility across various organisms.

In summary, this research represents a significant advancement in the field of genome editing, highlighting the CRISPR/Cas9 system's robustness and efficiency, while also pointing towards future avenues for enhancing its application in biomedical research.

References ​

  1. CRISPR-based adaptive immune systems. - Michael P Terns;Rebecca M Terns - Current opinion in microbiology (2011)
  2. Heritable gene targeting in zebrafish using customized TALENs. - Peng Huang;An Xiao;Mingguo Zhou;Zuoyan Zhu;Shuo Lin;Bo Zhang - Nature biotechnology (2011)
  3. Genome editing with engineered zinc finger nucleases. - Fyodor D Urnov;Edward J Rebar;Michael C Holmes;H Steve Zhang;Philip D Gregory - Nature reviews. Genetics (2010)
  4. FLASH assembly of TALENs for high-throughput genome editing. - Deepak Reyon;Shengdar Q Tsai;Cyd Khayter;Jennifer A Foden;Jeffry D Sander;J Keith Joung - Nature biotechnology (2012)
  5. CRISPR provides acquired resistance against viruses in prokaryotes. - Rodolphe Barrangou;Christophe Fremaux;Hélène Deveau;Melissa Richards;Patrick Boyaval;Sylvain Moineau;Dennis A Romero;Philippe Horvath - Science (New York, N.Y.) (2007)
  6. Mfold web server for nucleic acid folding and hybridization prediction. - Michael Zuker - Nucleic acids research (2003)
  7. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. - Jeffry D Sander;Morgan L Maeder;Deepak Reyon;Daniel F Voytas;J Keith Joung;Drena Dobbs - Nucleic acids research (2010)
  8. Small CRISPR RNAs guide antiviral defense in prokaryotes. - Stan J J Brouns;Matthijs M Jore;Magnus Lundgren;Edze R Westra;Rik J H Slijkhuis;Ambrosius P L Snijders;Mark J Dickman;Kira S Makarova;Eugene V Koonin;John van der Oost - Science (New York, N.Y.) (2008)
  9. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). - Jeffry D Sander;Elizabeth J Dahlborg;Mathew J Goodwin;Lindsay Cade;Feng Zhang;Daniel Cifuentes;Shaun J Curtin;Jessica S Blackburn;Stacey Thibodeau-Beganny;Yiping Qi;Christopher J Pierick;Ellen Hoffman;Morgan L Maeder;Cyd Khayter;Deepak Reyon;Drena Dobbs;David M Langenau;Robert M Stupar;Antonio J Giraldez;Daniel F Voytas;Randall T Peterson;Jing-Ruey J Yeh;J Keith Joung - Nature methods (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. 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)
  2. A library of TAL effector nucleases spanning the human genome. - Yongsub Kim;Jiyeon Kweon;Annie Kim;Jae Kyung Chon;Ji Yeon Yoo;Hye Joo Kim;Sojung Kim;Choongil Lee;Euihwan Jeong;Eugene Chung;Doyoung Kim;Mi Seon Lee;Eun Mi Go;Hye Jung Song;Hwangbeom Kim;Namjin Cho;Duhee Bang;Seokjoong Kim;Jin-Soo Kim - Nature biotechnology (2013)
  3. A CRISPR way to engineer the human genome. - Sivaprakash Ramalingam;Narayana Annaluru;Srinivasan Chandrasegaran - Genome biology (2013)
  4. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. - Lei S Qi;Matthew H Larson;Luke A Gilbert;Jennifer A Doudna;Jonathan S Weissman;Adam P Arkin;Wendell A Lim - Cell (2013)
  5. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. - James E DiCarlo;Julie E Norville;Prashant Mali;Xavier Rios;John Aach;George M Church - Nucleic acids research (2013)
  6. Biotechnology: Rewriting a genome. - Emmanuelle Charpentier;Jennifer A Doudna - Nature (2013)
  7. RNA guides genome engineering. - Claudio Mussolino;Toni Cathomen - Nature biotechnology (2013)
  8. A CRISPR view of genome sequences. - Amy K Cain;Christine J Boinett - Nature reviews. Microbiology (2013)
  9. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. - Nannan Chang;Changhong Sun;Lu Gao;Dan Zhu;Xiufei Xu;Xiaojun Zhu;Jing-Wei Xiong;Jianzhong Jeff Xi - Cell research (2013)
  10. The CRISPR system--keeping zebrafish gene targeting fresh. - Patrick R Blackburn;Jarryd M Campbell;Karl J Clark;Stephen C Ekker - Zebrafish (2013)

... (1465 more literatures)

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