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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
- Source :
- BMC Genomics
- Publication Year :
- 2016
- Publisher :
- BioMed Central, 2016.
-
Abstract
- Background Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Results Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions Our results provide a technical platform for high-throughput knock-in. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3331-9) contains supplementary material, which is available to authorized users.
- Subjects :
- 0301 basic medicine
Knock-in
Mouse
Zygote
Gene cassette
Biology
Exo1
Chromosomes
Cell Line
03 medical and health sciences
Exonuclease 1
Mice
CRISPR/Cas
Gene knockin
Transcription Activator-Like Effector Nucleases
Genetics
High throughput
CRISPR
Animals
Humans
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knock-In Techniques
Enhancer
Homologous Recombination
Reporter
Transcription activator-like effector nuclease
Base Sequence
Methodology Article
Gene targeting
Cloning-free
Flox
Cell biology
030104 developmental biology
Exodeoxyribonucleases
Genetic Loci
Gene Targeting
MMEJ
Expression cassette
CRISPR-Cas Systems
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14712164
- Volume :
- 17
- Database :
- OpenAIRE
- Journal :
- BMC Genomics
- Accession number :
- edsair.doi.dedup.....5208bc21c57057b5b924b1cdb9fdd5b8