Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

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 kn...

Full description

Saved in:
Bibliographic Details
Published in:BMC genomics 2016-11, Vol.17 (1), p.979-979, Article 979
Main Authors: Aida, Tomomi, Nakade, Shota, Sakuma, Tetsushi, Izu, Yayoi, Oishi, Ayu, Mochida, Keiji, Ishikubo, Harumi, Usami, Takako, Aizawa, Hidenori, Yamamoto, Takashi, Tanaka, Kohichi
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Cell Line
Chromosomes
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Exodeoxyribonucleases - metabolism
Gene expression
Gene Knock-In Techniques
Gene Targeting
Genetic aspects
Genetic Loci
Genomics
Homologous Recombination
Humans
Methodology
Mice
Physiological aspects
Transcription Activator-Like Effector Nucleases
Zygote
Zygotes
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. 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. Our results provide a technical platform for high-throughput knock-in.
ISSN:1471-2164
1471-2164
DOI:10.1186/s12864-016-3331-9