ArticleMolecular therapy. Nucleic acids2020
A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells.
Article in Molecular therapy. Nucleic acids, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 26 citations in OpenAlex.
- Article
- Enrichment strategies to enhance genome editing.Journal of biomedical science · 2023Review
- A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.International journal of molecular sciences · 2023Article
- Selecting for CRISPR-Edited Knock-In Cells.International journal of molecular sciences · 2022Review
- Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing.The Journal of biological chemistry · 2022Article
- Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle.International journal of molecular sciences · 2022Review
- Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.Current issues in molecular biology · 2022Article
- DNA in extracellular vesicles: from evolution to its current application in health and disease.Cell & bioscience · 2022Review
- Article
- A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system.Journal of Zhejiang University. Science. B · 2022Article
- chi-miR-487b-3p Inhibits Goat Myoblast Proliferation and Differentiation by Targeting IRS1 through the IRS1/PI3K/Akt Signaling Pathway.International journal of molecular sciences · 2021Article
- Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2021Review
- CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing.International journal of molecular sciences · 2021Article
- Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.Cell structure and function · 2021Article
- A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.Methods in molecular biology (Clifton, N.J.) · 2021Article
- CRISPR-based strategies for targeted transgene knock-in and gene correction.Faculty reviews · 2020Review
Corrections and comments
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Authors and funding
8 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the "HDR-USR" system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.
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