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Petrezselyova, S.* ; Kinsky, S.* ; Truban, D.* ; Sedlacek, R.* ; Burtscher, I. ; Lickert, H.

Homology arms of targeting vectors for gene insertions and Crispr/Cas9 technology: Size does not matter; quality control of targeted clones does.

Cell. Mol. Biol. Lett. 20, 773-787 (2015)

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Crispr/cas9 ; Genome Editing ; Reporter ; Targeting Vector ; Homology Arms ; Embryonic Stem Cell; Genome; Cells; Cas9; Specificity; Homology; Length

ISSN (print) / ISBN 1425-8153

e-ISSN 1689-1392

Journal Cellular & Molecular Biology Letters

Quellenangaben Volume: 20, Issue: 5, Pages: 773-787

Publisher Springer

Publishing Place Warsaw : Versita ; Berlin ; Heidelberg

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Diabetes and Regeneration Research (IDR)