Microhomology-mediated End-joining-dependent Integration of Donor DNA in Cells and Animals Using TALENs and CRISPR/Cas9

Overview

Journal Nat Commun

Specialty Biology

Date 2014 Nov 21

PMID 25410609

Citations 240

Authors

Shota Nakade

Takuya Tsubota

Yuto Sakane

Satoshi Kume

Naoaki Sakamoto

Masanobu Obara

Takaaki Daimon

Hideki Sezutsu

Takashi Yamamoto

Tetsushi Sakuma

Ken-Ichi T Suzuki

Affiliations

Soon will be listed here.

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.

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