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Efficient targeted integration directed by short homology in zebrafish and mammalian cells
- Source :
- eLife, Cambridge : eLife Sciences Publications, Ltd, 2020, vol. 9, art. no. e53968, p. [1-25], eLife, Vol 9 (2020), eLife
- Publication Year :
- 2020
-
Abstract
- Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
- Subjects :
- 0301 basic medicine
CRISPR-Associated Proteins
Sus scrofa
Animals, Genetically Modified
end joining
0302 clinical medicine
Genes, Reporter
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knock-In Techniques
Biology (General)
Zebrafish
Transcription activator-like effector nuclease
biology
General Neuroscience
Gene targeting
General Medicine
Medicine
RNA, Guide, Kinetoplastida
Research Article
pig fibroblasts
QH301-705.5
Science
Green Fluorescent Proteins
Computational biology
knock-in
General Biochemistry, Genetics and Molecular Biology
Genome engineering
03 medical and health sciences
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Sequence Homology, Nucleic Acid
Transcription Activator-Like Effector Nucleases
Gene knockin
Animals
Humans
human
Gene
CRISPR/Cas9
Reporter gene
General Immunology and Microbiology
Recombinational DNA Repair
Genetics and Genomics
Fibroblasts
biology.organism_classification
030104 developmental biology
Gene Expression Regulation
targeted integration
CRISPR-Cas Systems
K562 Cells
030217 neurology & neurosurgery
Developmental Biology
Subjects
Details
- Language :
- English
- ISSN :
- 2050084X
- Database :
- OpenAIRE
- Journal :
- eLife, Cambridge : eLife Sciences Publications, Ltd, 2020, vol. 9, art. no. e53968, p. [1-25], eLife, Vol 9 (2020), eLife
- Accession number :
- edsair.doi.dedup.....bfdaa4c2ba4444b21a6e2fac41b3b74c