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Curated Papers > High-Impact Authority Literature on "CRISPR Gene Editing"

Resources for the design of CRISPR gene editing experiments. ​

Literature Information ​

DOI10.1186/s13059-015-0823-x
PMID26612492
JournalGenome biology
Impact Factor9.4
JCR QuartileQ1
Publication Year2015
Times Cited50
KeywordsCRISPR gene editing, gene function, experimental design
Literature TypeJournal Article, Research Support, Non-U.S. Gov't, Review
ISSN1474-7596
Pages260
Issue16()
AuthorsDaniel B Graham, David E Root

TL;DR ​

This paper reviews the rapid adoption of CRISPR-based methods for gene perturbation and their role in functional genomics, emphasizing important design considerations for genome editing experiments. It also surveys the available tools and resources that facilitate the effective use of this powerful technology, highlighting its significance in advancing genetic research.

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CRISPR gene editing · gene function · experimental design

Abstract ​

CRISPR-based approaches have quickly become a favored method to perturb genes to uncover their functions. Here, we review the key considerations in the design of genome editing experiments, and survey the tools and resources currently available to assist users of this technology.

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Primary Questions Addressed ​

  1. What are the common pitfalls in the design of CRISPR gene editing experiments that researchers should be aware of?
  2. How do different CRISPR tools compare in terms of efficiency and specificity for gene editing?
  3. What ethical considerations should be taken into account when designing CRISPR experiments?
  4. How can researchers optimize the delivery methods for CRISPR components in different cell types?
  5. What are the best practices for validating the outcomes of CRISPR gene editing experiments?

Key Findings ​

Research Background and Objectives ​

CRISPR technology has revolutionized the field of genetics by enabling precise gene editing. This review by Graham and Root focuses on the design considerations for CRISPR gene editing experiments, providing a comprehensive overview of the tools and resources available for researchers to effectively utilize this technology.

Main Methods/Materials/Experimental Design ​

The review outlines the critical steps involved in CRISPR experiments, which include:

  1. Delivery of CRISPR Components: The Cas9 protein and single guide RNAs (sgRNAs) must be introduced into target cells. This can be done via transfection or viral transduction, with the latter being necessary for pooled screens.

  2. Designing sgRNAs: Selecting optimal sgRNAs is essential for maximizing on-target activity and minimizing off-target effects. Researchers are encouraged to use multiple sgRNAs per target gene to increase the chances of successful gene editing.

  3. Validation of Edits: It is crucial to confirm the on-target efficacy and evaluate any off-target effects. Various assays, including sequencing, are employed to assess the modifications.

The following flowchart illustrates the overall experimental design process for CRISPR:

Mermaid diagram

Key Results and Findings ​

  1. Efficiency of CRISPR: The authors highlight that the editing efficiency typically ranges from 30% to 60%, and achieving a uniform population of edited cells often requires single-cell cloning.

  2. Off-target Activity: The review emphasizes the variability in off-target effects across different sgRNAs and the need for empirical validation of sgRNA specificity.

  3. Technological Tools: Several software tools are recommended for designing sgRNAs and predicting off-target sites, aiding researchers in optimizing their experiments.

Main Conclusions/Significance/Innovativeness ​

The review provides valuable insights into the practical aspects of designing CRISPR experiments. It emphasizes the importance of careful planning in selecting target genes, designing sgRNAs, and validating results to ensure the success of gene editing efforts. The authors advocate for the continued development of tools and methodologies to enhance the efficiency and specificity of CRISPR technology.

Research Limitations and Future Directions ​

The authors acknowledge several limitations, including the inherent variability in CRISPR efficiency and the challenges in predicting off-target effects accurately. Future research should focus on:

  • Improving the efficiency of homology-directed repair (HDR) to enhance the precision of gene edits.
  • Developing new Cas9 variants and alternative CRISPR systems to broaden the applicability and specificity of gene editing.
  • Standardizing validation methods to confirm on-target and off-target effects across different experimental contexts.

Overall, the review underscores the transformative potential of CRISPR technology while highlighting the need for ongoing research to address its limitations.

References ​

  1. Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. - Jeffry D Sander;Peter Zaback;J Keith Joung;Daniel F Voytas;Drena Dobbs - Nucleic acids research (2007)
  2. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. - Michael R Lieber - Annual review of biochemistry (2010)
  3. 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)
  4. Regulation of homologous recombination in eukaryotes. - Wolf-Dietrich Heyer;Kirk T Ehmsen;Jie Liu - Annual review of genetics (2010)
  5. 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)
  6. Multiplex genome engineering using CRISPR/Cas systems. - Le Cong;F Ann Ran;David Cox;Shuailiang Lin;Robert Barretto;Naomi Habib;Patrick D Hsu;Xuebing Wu;Wenyan Jiang;Luciano A Marraffini;Feng Zhang - Science (New York, N.Y.) (2013)
  7. RNA-guided human genome engineering via Cas9. - Prashant Mali;Luhan Yang;Kevin M Esvelt;John Aach;Marc Guell;James E DiCarlo;Julie E Norville;George M Church - Science (New York, N.Y.) (2013)
  8. RNA-programmed genome editing in human cells. - Martin Jinek;Alexandra East;Aaron Cheng;Steven Lin;Enbo Ma;Jennifer Doudna - eLife (2013)
  9. 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)
  10. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. - Haoyi Wang;Hui Yang;Chikdu S Shivalila;Meelad M Dawlaty;Albert W Cheng;Feng Zhang;Rudolf Jaenisch - Cell (2013)

Literatures Citing This Work ​

  1. Genome editing: the end of the beginning. - Jennifer A Doudna;Charles A Gersbach - Genome biology (2015)
  2. A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research. - Joseph M Miano;Qiuyu Martin Zhu;Charles J Lowenstein - Arteriosclerosis, thrombosis, and vascular biology (2016)
  3. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes. - Juan C Oliveros;Mònica Franch;Daniel Tabas-Madrid;David San-León;Lluis Montoliu;Pilar Cubas;Florencio Pazos - Nucleic acids research (2016)
  4. An Overview of CRISPR-Based Tools and Their Improvements: New Opportunities in Understanding Plant-Pathogen Interactions for Better Crop Protection. - Abdellah Barakate;Jennifer Stephens - Frontiers in plant science (2016)
  5. A Broad Overview and Review of CRISPR-Cas Technology and Stem Cells. - Simon N Waddington;Riccardo Privolizzi;Rajvinder Karda;Helen C O'Neill - Current stem cell reports (2016)
  6. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control. - Lindsey C Perkin;Sherry L Adrianos;Brenda Oppert - Insects (2016)
  7. Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line. - Van Trung Chu;Robin Graf;Tristan Wirtz;Timm Weber;Jeremy Favret;Xun Li;Kerstin Petsch;Ngoc Tung Tran;Michael H Sieweke;Claudia Berek;Ralf Kühn;Klaus Rajewsky - Proceedings of the National Academy of Sciences of the United States of America (2016)
  8. CRISPR/Cas9 in zebrafish: an efficient combination for human genetic diseases modeling. - Jiaqi Liu;Yangzhong Zhou;Xiaolong Qi;Jia Chen;Weisheng Chen;Guixing Qiu;Zhihong Wu;Nan Wu - Human genetics (2017)
  9. Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing. - Levi Lowder;Aimee Malzahn;Yiping Qi - Frontiers in plant science (2016)
  10. Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies. - Gang Wang;Luhan Yang;Dennis Grishin;Xavier Rios;Lillian Y Ye;Yong Hu;Kai Li;Donghui Zhang;George M Church;William T Pu - Nature protocols (2017)

... (40 more literatures)

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