Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

• Published in: Nature communications Vol. 5; no. 1; p. 5560
• Main Authors: Nakade, Shota, Tsubota, Takuya, Sakane, Yuto, Kume, Satoshi, Sakamoto, Naoaki, Obara, Masanobu, Daimon, Takaaki, Sezutsu, Hideki, Yamamoto, Takashi, Sakuma, Tetsushi, Suzuki, Ken-ichi T.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 20-11-2014; Nature Publishing Group; Nature Pub. Group
• Subjects: 42/41; 631/1647/1511; 631/1647/1513/1967; 631/337/1427/2190; Animals; Base Sequence; Bombyx; CRISPR-Cas Systems; Deoxyribonucleases; DNA - metabolism; Gene Knock-In Techniques - methods; Genetic Engineering; Genetic Vectors; Homologous Recombination; Humanities and Social Sciences; Humans; Molecular Sequence Data; multidisciplinary; Plasmids; Saccharomyces cerevisiae; Science; Science (multidisciplinary); Xenopus
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms6560

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.
One challenge facing the use of programmable nucleases in genome engineering is the requirement for homologous recombination. Here, Nakade
et al.
harness microhomology-mediated end-joining as a means of inserting exogenous coding sequences into the genome using both TALEN and CRISPR/Cas9 technologies.