RRC ID 33118
Author Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT.
Title Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.
Journal Nat Commun
Abstract Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.
Volume 5
Pages 5560
Published 2014-11-20
DOI 10.1038/ncomms6560
PII ncomms6560
PMID 25410609
PMC PMC4263139
MeSH Animals Base Sequence Bombyx CRISPR-Cas Systems* DNA / metabolism* Deoxyribonucleases* Gene Knock-In Techniques / methods* Genetic Engineering Genetic Vectors Homologous Recombination Humans Molecular Sequence Data Plasmids Saccharomyces cerevisiae Xenopus
IF 12.121
Times Cited 214