title: Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does.
abstract: 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.
authors.forename: Silvia
authors.initials: S
authors.lastname: Petrezselyova
authors.forename: Slavomir
authors.initials: S
authors.lastname: Kinsky
authors.forename: Dominika
authors.initials: D
authors.lastname: Truban
authors.forename: Radislav
authors.initials: R
authors.lastname: Sedlacek
authors.forename: Ingo
authors.initials: I
authors.lastname: Burtscher
authors.forename: Heiko
authors.initials: H
authors.lastname: Lickert
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D055879:Gene Knock-In Techniques / Q000592:standards; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D000066450:Mouse Embryonic Stem Cells; D016254:Mutagenesis, Insertional; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011786:Quality Control; D058118:Transcription Factor 7-Like 2 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051557:Hepatocyte Nuclear Factor 4; D009419:Nerve Tissue Proteins; D058118:Transcription Factor 7-Like 2 Protein; C496593:Hnf4a protein, mouse; C407737:Neurog3 protein, mouse; C544895:Tcf7l2 protein, mouse