Rapid genome editing by CRISPR-Cas9-POLD3 fusion
- University of Oslo, Norway
- University of Bergen, Norway
- University of Helsinki, Finland
- University of Oslo, Finland
- Oslo University Hospital, Norway
- Akershus University Hospital, Norway
- Karolinska Institute, Sweden
- University of Cambridge, United Kingdom
Abstract
Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
Data availability
The following data sets were generated Reint et. al., (2021), Sequence Read Archive (SRA), BioProject ID: PRJNA782085. The following previously published data sets were used: Tsai et al., (2015) Sequence Read Archive (SRA), SRP050338. Custom scripts used to extract UMI from the demultiplexed fastq reads for the GUIDE-Seq analysis is publicly available at: https://bitbucket.org/valenlab/guide-seq-pold3. Sequences of Cas9 nuclease and GFP-BFP reporter cassette used in this study are available in Supplementary file 8.
Article and author information
Author details
Bernhard Schmierer
Funding
Barncancerfonden
- Kornel Labun
Norwegian Research Council
- Emma Haapaniemi
South-Eastern Norway Regional Health Authority ((Grant no. 279922 to Hilde Nilsen))
- Emma Haapaniemi
Knut och Alice Wallenbergs Stiftelse
- Jussi Taipale
Cancerfonden
- Emma Haapaniemi
Barncancerfonden
- Emma Haapaniemi
Instrumentariumin Tiedesäätiö
- Emma Haapaniemi
Science for Life Laboratory
- Bernhard Schmierer
Academy of Finland
- Markku Varjosalo
- Jussi Taipale
- Emma Haapaniemi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jessica K Tyler, Weill Cornell Medicine, United States
Version history
- Received: November 13, 2021
- Accepted: November 15, 2021
- Accepted Manuscript published: December 13, 2021 (version 1)
- Version of Record published: January 10, 2022 (version 2)
- Version of Record updated: May 27, 2022 (version 3)
Copyright
© 2021, Reint 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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Rapid genome editing by CRISPR-Cas9-POLD3 fusion
eLife 10:e75415.
https://doi.org/10.7554/eLife.75415
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