Curated Papers > High-impact authoritative literature on "CRISPR gene editing"

Genome engineering using the CRISPR-Cas9 system.

Literature Information

DOI	10.1038/nprot.2013.143
PMID	24157548
Journal	Nature protocols
Impact Factor	16.0
JCR Quartile	Q1
Publication Year	2013
Times Cited	6330
Keywords	Genome Engineering, CRISPR-Cas9, RNA-guided, Gene Editing, Cell Lines
Literature Type	Journal Article, Research Support, N.I.H., Extramural
ISSN	1750-2799
Pages	2281-2308
Issue	8(11)
Authors	F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott, Feng Zhang

TL;DR

This study presents a comprehensive toolkit for using the Cas9 nuclease for precise genome editing in mammalian cells through nonhomologous end joining (NHEJ) and homology-directed repair (HDR), while also introducing a double-nicking strategy to reduce off-target effects. The findings provide practical guidelines for target selection and cleavage efficiency, enabling rapid gene modifications and the generation of modified cell lines for functional studies within a few weeks, highlighting the potential of CRISPR technology in genetic research.

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Genome Engineering · CRISPR-Cas9 · RNA-guided · Gene Editing · Cell Lines

Abstract

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

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Primary Questions Addressed

1. What are the potential applications of CRISPR-Cas9 technology in gene therapy and disease treatment?
2. How does the choice between nonhomologous end joining (NHEJ) and homology-directed repair (HDR) affect the outcomes of genome editing?
3. What are the latest advancements in minimizing off-target effects in CRISPR-Cas9 genome editing?
4. How can modified cell lines generated through CRISPR-Cas9 be utilized in functional studies and drug discovery?
5. What are the ethical considerations surrounding the use of CRISPR-Cas9 in human genome editing?

Key Findings

Research Background and Objectives

The CRISPR-Cas9 system is a revolutionary tool for genome engineering, enabling precise alterations in DNA across various organisms. This protocol aims to provide comprehensive guidelines for utilizing the CRISPR-Cas9 system in mammalian cells, focusing on methods for targeted genome editing via nonhomologous end joining (NHEJ) and homology-directed repair (HDR). The goal is to streamline the design, execution, and validation of CRISPR experiments.

Main Methods/Materials/Experimental Design

The protocol encompasses several key steps for effective genome editing using the CRISPR-Cas9 system, including target site selection, sgRNA design, plasmid construction, and cell transfection.

Experimental Design Overview

1. Target Design: Identify a 20-bp genomic sequence adjacent to a PAM site (5'-NGG) for Cas9 targeting.
2. sgRNA Design: Use an online tool to generate suitable sgRNA sequences and evaluate potential off-target sites.
3. Construct sgRNA Plasmid: Clone the sgRNA into a Cas9 expression vector (pSpCas9) to create a functional plasmid.
4. Transfect Cells: Introduce the plasmid into mammalian cells (e.g., HEK 293FT) using Lipofectamine or nucleofection techniques.
5. Assess Editing Efficiency: Evaluate the efficiency of genome editing through assays such as SURVEYOR or sequencing.
6. Isolate Clonal Lines: Use FACS or serial dilution methods to isolate individual clones with desired modifications.
7. Functional Validation: Perform additional assays to confirm successful editing and assess phenotypic changes.

Key Results and Findings

• The CRISPR-Cas9 system allows for efficient genome editing in various cell types, achieving significant modification rates (e.g., ~65-68% for targeted loci).
• The use of double nicking with the Cas9 nickase mutant can reduce off-target effects while maintaining editing efficiency.
• The protocol demonstrates the generation of stable cell lines with specific genetic alterations, which can be used for functional studies.

Main Conclusions/Significance/Innovation

The outlined protocol provides a robust framework for implementing CRISPR-Cas9 genome editing in mammalian cells. Its strengths include:

• High Efficiency: The method allows for rapid generation of genetically modified cell lines.
• Versatility: Applicable to various mammalian cell types and organisms, enhancing the scope of genetic research.
• Innovation in Design: The incorporation of a web-based CRISPR design tool and double-nicking strategies exemplifies cutting-edge approaches to minimize off-target effects.

Limitations and Future Directions

• Off-Target Effects: Despite improvements, the potential for unintended mutations remains a concern, necessitating ongoing refinement of targeting strategies.
• Cell Type Variability: Editing efficiency can vary significantly between different cell types, highlighting the need for tailored approaches in diverse biological contexts.
• Future Research: Continued exploration of alternative Cas9 variants, enhanced delivery methods, and more refined HDR templates could further improve the precision and applicability of CRISPR technologies.

Summary Table of Key Steps

Step	Description	Timeframe
1	Target Design	1 day
2	sgRNA Design	1 day
3	sgRNA Plasmid Construction	2-3 days
4	Cell Transfection	3-4 days
5	Editing Efficiency Assessment	5-6 hours
6	Clonal Isolation	2-3 hours hands-on; 2-3 weeks
7	Functional Validation	3-4 hours

This structured approach to CRISPR-Cas9 genome editing not only facilitates the practical application of this technology but also sets the stage for future innovations in genetic research and therapeutic development.

References

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10. 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)

Literatures Citing This Work

1. Human genome editing as a tool to establish causality. - Fyodor D Urnov - Proceedings of the National Academy of Sciences of the United States of America (2014)
2. Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation. - Laurel Oldach;Jin Zhang - Chemistry & biology (2014)
3. IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances. - Monica Gostissa;Bjoern Schwer;Amelia Chang;Junchao Dong;Robin M Meyers;Gregory T Marecki;Vivian W Choi;Roberto Chiarle;Ali A Zarrin;Frederick W Alt - Proceedings of the National Academy of Sciences of the United States of America (2014)
4. CRISPR-Cas systems for editing, regulating and targeting genomes. - Jeffry D Sander;J Keith Joung - Nature biotechnology (2014)
5. Enterococcus infection biology: lessons from invertebrate host models. - Grace J Yuen;Frederick M Ausubel - Journal of microbiology (Seoul, Korea) (2014)
6. CRISPR-Cas system: a powerful tool for genome engineering. - Liang Liu;Xiu-Duo Fan - Plant molecular biology (2014)
7. Designer microbes for biosynthesis. - Maureen B Quin;Claudia Schmidt-Dannert - Current opinion in biotechnology (2014)
8. iPS cell technologies: significance and applications to CNS regeneration and disease. - Hideyuki Okano;Shinya Yamanaka - Molecular brain (2014)
9. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids. - Ning Wang;Hongyu Zhang;Bing-Qiang Zhang;Wei Liu;Zhonglin Zhang;Min Qiao;Hongmei Zhang;Fang Deng;Ningning Wu;Xian Chen;Sheng Wen;Junhui Zhang;Zhan Liao;Qian Zhang;Zhengjian Yan;Liangjun Yin;Jixing Ye;Youlin Deng;Hue H Luu;Rex C Haydon;Houjie Liang;Tong-Chuan He - PloS one (2014)
10. Targeted mutagenesis using CRISPR/Cas system in medaka. - Satoshi Ansai;Masato Kinoshita - Biology open (2014)

... (6320 more literatures)

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