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.
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
Further reading
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- Cell Biology
- Chromosomes and Gene Expression
Meiotic crossover recombination is essential for both accurate chromosome segregation and the generation of new haplotypes for natural selection to act upon. This requirement is known as crossover assurance and is one example of crossover control. While the conserved role of the ATPase, PCH-2, during meiotic prophase has been enigmatic, a universal phenotype when pch-2 or its orthologs are mutated is a change in the number and distribution of meiotic crossovers. Here, we show that PCH-2 controls the number and distribution of crossovers by antagonizing their formation. This antagonism produces different effects at different stages of meiotic prophase: early in meiotic prophase, PCH-2 prevents double-strand breaks from becoming crossover-eligible intermediates, limiting crossover formation at sites of initial double-strand break formation and homolog interactions. Later in meiotic prophase, PCH-2 winnows the number of crossover-eligible intermediates, contributing to the designation of crossovers and ultimately, crossover assurance. We also demonstrate that PCH-2 accomplishes this regulation through the meiotic HORMAD, HIM-3. Our data strongly support a model in which PCH-2’s conserved role is to remodel meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors and coordinate meiotic recombination with synapsis, ensuring the progressive implementation of meiotic recombination and explaining its function in the pachytene checkpoint and crossover control.
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- Cell Biology
In animals, mitosis involves the breakdown of the nucleus. The reassembly of a nucleus after mitosis requires the reformation of the nuclear envelope around a single mass of chromosomes. This process requires Ankle2 (also known as LEM4 in humans) which interacts with PP2A and promotes the function of the Barrier-to-Autointegration Factor (BAF). Upon dephosphorylation, BAF dimers cross-bridge chromosomes and bind lamins and transmembrane proteins of the reassembling nuclear envelope. How Ankle2 functions in mitosis is incompletely understood. Using a combination of approaches in Drosophila, along with structural modeling, we provide several lines of evidence that suggest that Ankle2 is a regulatory subunit of PP2A, explaining how it promotes BAF dephosphorylation. In addition, we discovered that Ankle2 interacts with the endoplasmic reticulum protein Vap33, which is required for Ankle2 localization at the reassembling nuclear envelope during telophase. We identified the interaction sites of PP2A and Vap33 on Ankle2. Through genetic rescue experiments, we show that the Ankle2/PP2A interaction is essential for the function of Ankle2 in nuclear reassembly and that the Ankle2/Vap33 interaction also promotes this process. Our study sheds light on the molecular mechanisms of post-mitotic nuclear reassembly and suggests that the endoplasmic reticulum is not merely a source of membranes in the process, but also provides localized enzymatic activity.
