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

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

Literature Information

DOI	10.1038/nature17946
PMID	27096365
Journal	Nature
Impact Factor	48.5
JCR Quartile	Q1
Publication Year	2016
Times Cited	2522
Keywords	Base Editing, Genome Editing, Point Mutation, CRISPR/Cas9, DNA Repair
Literature Type	Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN	0028-0836
Pages	420-4
Issue	533(7603)
Authors	Alexis C Komor, Yongjoo B Kim, Michael S Packer, John A Zuris, David R Liu

TL;DR

This research introduces 'base editing,' a novel genome-editing technique that allows for the precise conversion of one DNA base into another without inducing double-stranded DNA breaks, thereby overcoming the inefficiencies and random mutations associated with traditional methods. By engineering CRISPR/Cas9 fusions with a cytidine deaminase, the study demonstrates effective correction of various point mutations in human and murine cells, achieving a significant reduction in undesired indel formation and expanding the potential for treating genetic diseases.

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Base Editing · Genome Editing · Point Mutation · CRISPR/Cas9 · DNA Repair

Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

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Primary Questions Addressed

1. How does the efficiency of base editing compare to traditional genome-editing methods for correcting point mutations?
2. What are the potential applications of base editing in treating specific genetic diseases beyond those mentioned in the study?
3. Can base editing techniques be adapted for use in organisms other than human and murine cell lines, and what challenges might arise?
4. What are the implications of minimal indel formation in base editing for long-term genetic stability and safety in therapeutic applications?
5. How do the engineered fusions of CRISPR/Cas9 and cytidine deaminase enhance the specificity of base editing compared to other genome-editing technologies?

Key Findings

Research Background and Purpose

Current genome-editing technologies primarily rely on inducing double-stranded DNA breaks (DSBs) to facilitate gene correction. However, these methods often lead to unintended insertions and deletions (indels) due to cellular repair responses, which are especially problematic for correcting point mutations responsible for many genetic diseases. This study introduces a novel technique called base editing, which allows for the direct conversion of one DNA base to another without requiring DSBs or donor templates.

Main Methods/Materials/Experimental Design

The authors developed base editors by fusing a catalytically inactive Cas9 (dCas9) with cytidine deaminase enzymes. The primary experimental components included:

• Base Editor Constructs: Different constructs (BE1, BE2, BE3) were created by varying the fusions of dCas9 with the deaminase rAPOBEC1 and the uracil DNA glycosylase inhibitor (UGI).
• Activity Window: The editing efficiency was assessed by determining the "activity window," which was optimized to approximately five nucleotides.
• Cell Line Testing: The efficiency of base editing was tested in human and murine cell lines, focusing on correcting specific T→C mutations related to diseases.

The technical workflow can be represented in the following Mermaid code:

Key Results and Findings

• Editing Efficiency: The base editors showed high efficiency in converting cytidine to uridine, achieving correction rates of 15-75% in cellular DNA with minimal indel formation (≤ 1%).
• Generation of BE2 and BE3: The addition of UGI (BE2) increased editing efficiency by threefold compared to BE1. Further modification to nick the non-edited strand (BE3) enhanced efficiency up to 37%.
• Disease Models: Successful correction of disease-relevant mutations was demonstrated in mammalian cells, including the conversion of the Alzheimer's-associated APOE4 allele and the cancer-associated p53 mutation.

Main Conclusions/Significance/Innovation

The development of base editing represents a significant advancement in genome editing technology, providing a more efficient and precise method for correcting point mutations without the drawbacks associated with DSBs. This method holds potential for treating a wide range of genetic disorders, expanding the therapeutic possibilities of genome editing.

Research Limitations and Future Directions

• Off-Target Effects: While off-target base editing was detected, it was limited to known Cas9 off-target sites. Further studies are needed to comprehensively evaluate off-target activity.
• Editing Context: The efficiency of base editing can be influenced by the sequence context surrounding the target base. Understanding these effects will be crucial for optimizing base editing strategies.
• Future Applications: The study suggests potential for developing additional base editors targeting other types of modifications, such as methylation, which could broaden the scope of genome and epigenome editing.

In summary, this research highlights the innovative approach of base editing, showcasing its efficacy in correcting genetic mutations while minimizing unwanted genomic alterations, paving the way for future therapeutic applications.

References

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Literatures Citing This Work

1. Cre-dependent DNA recombination activates a STING-dependent innate immune response. - Geneviève Pépin;Jonathan Ferrand;Klara Höning;W Samantha N Jayasekara;Jason E Cain;Mark A Behlke;Daniel J Gough;Bryan R G Williams;Veit Hornung;Michael P Gantier - Nucleic acids research (2016)
2. Development of autologous blood cell therapies. - Ah Ram Kim;Vijay G Sankaran - Experimental hematology (2016)
3. Genome-editing technologies for gene correction of hemophilia. - Chul-Yong Park;Dongjin R Lee;Jin Jea Sung;Dong-Wook Kim - Human genetics (2016)
4. Genetically modified (GM) crops: milestones and new advances in crop improvement. - Ayushi Kamthan;Abira Chaudhuri;Mohan Kamthan;Asis Datta - TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik (2016)
5. The present and future of genome editing in cancer research. - Xiaoyi Li;Raymond Wu;Andrea Ventura - Human genetics (2016)
6. Genome editing comes of age. - Jin-Soo Kim - Nature protocols (2016)
7. Beyond CRISPR: A guide to the many other ways to edit a genome. - Heidi Ledford - Nature (2016)
8. The new editor-targeted genome engineering in the absence of homology-directed repair. - A J Kueh;M J Herold - Cell death discovery (2016)
9. Opportunities and challenges in modeling human brain disorders in transgenic primates. - Charles G Jennings;Rogier Landman;Yang Zhou;Jitendra Sharma;Julia Hyman;J Anthony Movshon;Zilong Qiu;Angela C Roberts;Anna Wang Roe;Xiaoqin Wang;Huihui Zhou;Liping Wang;Feng Zhang;Robert Desimone;Guoping Feng - Nature neuroscience (2016)
10. Applications of CRISPR Genome Engineering in Cell Biology. - Fangyuan Wang;Lei S Qi - Trends in cell biology (2016)

... (2512 more literatures)

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