Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks

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Abstract

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.

Abstract

Here the authors fuse hRad51 and variants thereof to Cas9 nickase to facilitate homology-directed repair without generating double strand breaks, minimizing indel formation and off-target editing. This tool represents progress towards the goal of performing HDR without an excess of undesired side products.

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Most cited references 31

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CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.

Alexis Komor, Ahmed H Badran, David R. Liu (2017)

The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.

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Reprogramming human T cell function and specificity with non-viral genome targeting

Theodore L Roth, Cristina Puig-Saus, Ruby Yu … (2018)

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes 1 using recombinant viral vectors, which do not target transgenes to specific genomic sites 2,3 . In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR) 4,5 . Here, we developed a CRISPR-Cas9 genome targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (> 1kb) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we apply this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, demonstrating improved signaling function. Second, we replace the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognize tumour antigen and mount productive anti-tumour cell responses in vitro and in vivo. Taken together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

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Accurate classification of BRCA1 variants with saturation genome editing

Gregory M. Findlay, Riza M Daza, Beth Martin … (2018)

Variants of uncertain significance (VUS) fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumor suppressor in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here, we employ saturation genome editing to assay 96.5% of all possible single nucleotide variants (SNVs) in 13 exons encoding functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as ~300 SNVs that disrupt expression. We predict that these results will be immediately useful for clinical interpretation of BRCA1 variants, and that this paradigm can be extended to overcome the challenge of VUS in additional clinically actionable genes.

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Author and article information

Contributors

David R. Liu:

ORCID: http://orcid.org/0000-0002-9943-7557

drliu@fas.harvard.edu

Journal

Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature Communications

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2041-1723

Publication date (Electronic): 17 May 2019

Publication date PMC-release: 17 May 2019

Publication date Collection: 2019

Volume: 10

Electronic Location Identifier: 2212

Affiliations

[1 ]GRID grid.66859.34, Merkin Institute of Transformative Technologies in Healthcare, , Broad Institute of Harvard and MIT, ; Cambridge, MA 02142 USA

[2 ]ISNI 000000041936754X, GRID grid.38142.3c, Howard Hughes Medical Institute, , Harvard University, ; Cambridge, MA 02142 USA

[3 ]ISNI 000000041936754X, GRID grid.38142.3c, Department of Chemistry and Chemical Biology, , Harvard University, ; Cambridge, MA 02138 USA

[4 ]ISNI 000000041936754X, GRID grid.38142.3c, Program in Speech and Hearing Bioscience and Technology, , Harvard Medical School, ; Boston, MA 02115 USA

Author information

David R. Liu http://orcid.org/0000-0002-9943-7557

Article

Publisher ID: 9983

DOI: 10.1038/s41467-019-09983-4

PMC ID: 6525190

PubMed ID: 31101808

SO-VID: 37a91769-45e9-412d-8188-1bc0f38ed6ed

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 13 February 2019

Date accepted : 4 April 2019

Funding

Funded by: FundRef https://doi.org/10.13039/100000060, U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID);

Award ID: U01 AI142756