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.
Highlights
Genetic changes are introduced via a process called homologous recombination that occurs between an engineered exogenous DNA template and the genome of the mouse embryonic stem cells
By Southern blot analysis we demonstrated that Cas9 nickases in combination with targeting vectors with short homology arms led to insertion of foreign vector DNA by non-homologous integrations, resulting in additional genomic alternations
Tcf7L2 mini-targeting cassette was released by digestion with NotI, KpnI and 3256 bp fragment was introduced into the pL254-Tcf7L2 via bacterial homologous recombination in EL350 bacteria containing retrieval vector, resulting in the final targeting construct, which was confirmed by sequencing
Summary
Prize in Medicine in 2007 for their work in the development of a technique to target and introduce specific gene modifications in mice by the use of embryonic stem cells. We asked whether mESCs will repair a CRISPR/Cas9-induced DSB by homologous integration of a small or large DNA fragment with similar efficiencies For this purpose, we prepared two types of targeting vectors that served as templates for homologous recombination: i) classical targeting vectors with homology arms of 2 to 5 kb in length and ii) mini-targeting vectors carrying homology arms long only ~ 0.3 kb. By Southern blot analysis we demonstrated that Cas nickases in combination with targeting vectors with short homology arms led to insertion of foreign vector DNA by non-homologous integrations, resulting in additional genomic alternations These phenomena were not observed at similar extents when using targeting vectors with long homology arms
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