THE PROMISE AND CHALLENGE OF THERAPEUTIC GENOME EDITING

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Abstract

Genome editing, involving precise manipulation of cellular DNA sequences to alter cell fates and organism traits, offers the potential to both understand human genetics and cure genetic disease as never before. Scientific, technical and ethical aspects of employing CRISPR technology for therapeutic applications in humans are discussed, focusing on specific examples that highlight both opportunities and challenges. Genome editing is or will soon be in the clinic for several diseases, with more applications in the pipeline. The rapid pace of the field demands active efforts to ensure responsible use of this breakthrough technology to treat, cure and prevent genetic disease.

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Most cited references 115

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A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

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Multiplex genome engineering using CRISPR/Cas systems.

Le Cong, F. Ran, David T. Cox … (2013)

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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RNA-guided human genome engineering via Cas9.

P. Mali, L. YANG, K. Esvelt … (2013)

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

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Author and article information

Journal

Journal ID (nlm-journal-id): 0410462

Journal ID (pubmed-jr-id): 6011

Journal ID (nlm-ta): Nature

Journal ID (iso-abbrev): Nature

Title: Nature

ISSN (Print): 0028-0836

ISSN (Electronic): 1476-4687

Publication date Nihms-submitted: 29 March 2022

Publication date (Print): February 2020

Publication date (Electronic): 12 February 2020

Publication date PMC-release: 08 April 2022

Volume: 578

Issue: 7794

Pages: 229-236

Affiliations

[1 ]Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

[2 ]Department of Chemistry, University of California, Berkeley, California 94720, USA.

[3 ]California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720, USA.

[4 ]Innovative Genomics Institute, University of California, Berkeley, California 94720, USA.

[5 ]Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.

[6 ]MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

[7 ]Gladstone Institutes, University of California, San Francisco, California 94114, USA.

Author notes

[* ]Correspondence: doudna@ 123456berkeley.edu

Article

Manuscript ID: NIHMS1776701

DOI: 10.1038/s41586-020-1978-5

PMC ID: 8992613

PubMed ID: 32051598

SO-VID: ada12f69-9a37-49a9-8bcf-e5233f6063dd

License:

This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.

THE PROMISE AND CHALLENGE OF THERAPEUTIC GENOME EDITING

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Abstract

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CRISPR/Cas9 editing in human blood

Most cited references 115

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

Multiplex genome engineering using CRISPR/Cas systems.

RNA-guided human genome engineering via Cas9.

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