Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
- ETH Zurich, Switzerland
- University of Zurich, Switzerland
Abstract
The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.
Data availability
The data that support the findings of this study are available within the paper and its Supplementary files. Source data files have been provided for Figure 4, Figure 5, Figure 6, Figure Supplement 1, Figure Supplement 2, and Figure Supplement 3.Scripts for mapping sequencing data, counting mutations and generating plots are available at https://github.com/HLindsay/Savic_CRISPR_HDR. Fastq files have been uploaded to ArrayExpress, and accession number is E-MTAB-6808.
Article and author information
Author details
Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (PMPDP3_171388)
- Natasa Savic
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_160230)
- Gerald Schwank
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_149393)
- Martin Jinek
Vallee Foundation
- Martin Jinek
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Bernard de Massy, Institute of Human Genetics, CNRS UPR 1142, France
Publication history
- Received: November 23, 2017
- Accepted: May 26, 2018
- Accepted Manuscript published: May 29, 2018 (version 1)
- Version of Record published: June 28, 2018 (version 2)
- Version of Record updated: June 29, 2018 (version 3)
- Version of Record updated: July 4, 2018 (version 4)
- Version of Record updated: March 6, 2019 (version 5)
Copyright
© 2018, Savic et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
eLife 7:e33761.
https://doi.org/10.7554/eLife.33761
Further reading
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- Chromosomes and Gene Expression
- Neuroscience
Sensory feedback is required for the stable execution of learned motor skills, and its loss can severely disrupt motor performance. The neural mechanisms that mediate sensorimotor stability have been extensively studied at systems and physiological levels, yet relatively little is known about how disruptions to sensory input alter the molecular properties of associated motor systems. Songbird courtship song, a model for skilled behavior, is a learned and highly structured vocalization that is destabilized following deafening. Here, we sought to determine how the loss of auditory feedback modifies gene expression and its coordination across the birdsong sensorimotor circuit. To facilitate this system-wide analysis of transcriptional responses, we developed a gene expression profiling approach that enables the construction of hundreds of spatially-defined RNA-sequencing libraries. Using this method, we found that deafening preferentially alters gene expression across birdsong neural circuitry relative to surrounding areas, particularly in premotor and striatal regions. Genes with altered expression are associated with synaptic transmission, neuronal spines, and neuromodulation and show a bias toward expression in glutamatergic neurons and Pvalb/Sst-class GABAergic interneurons. We also found that connected song regions exhibit correlations in gene expression that were reduced in deafened birds relative to hearing birds, suggesting that song destabilization alters the inter-region coordination of transcriptional states. Finally, lesioning LMAN, a forebrain afferent of RA required for deafening-induced song plasticity, had the largest effect on groups of genes that were also most affected by deafening. Combined, this integrated transcriptomics analysis demonstrates that the loss of peripheral sensory input drives a distributed gene expression response throughout associated sensorimotor neural circuitry and identifies specific candidate molecular and cellular mechanisms that support the stability and plasticity of learned motor skills.
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- Chromosomes and Gene Expression
In songbirds, deafening leads to changes in gene expression which have now been mapped at the single-cell level across the neural circuit involved in song production.
