Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems

Abstract

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

“…In mammalian cells, however, MMEJ was observed in the presence of normal NHEJ function when using truncation mutants of the V(D)J recombination proteins Rag1 and Rag2 during recombination [54,55]. Several recent studies suggested that MMEJ may be utilized to repair TALENor CRISPR/Cas9-induced DSBs in different animal models [56,57]. While this manuscript was under review, it was reported that precise in-frame knock-in could be achieved in zebrafish by using short homologous sequences (10-40 bp) [58], although it is unknown whether MMEJ, SSA or both were involved in that process.…”

A CRISPR view of development

“…Since each cell has a choice of whether to repair a break through NHEJ or HDR, a variety of different repair events will be present in the injected organism (and in individual cells). The frequency at which both alleles of a gene are affected has been reported to be high enough to visualize null phenotypes in developing mice and zebrafish (Jao et al 2013;Wang et al 2013a;Yasue et al 2014;Yen et al 2014). For example, zebrafish and mice injected with a gRNA targeting the tyrosinase gene resulted in embryos displaying mosaic pigmentation (Jao et al 2013;Yen et al 2014).…”

Magic wands of CRISPR—lots of choices for gene knock-in

“…As described earlier in this review, MMEJ has been utilized for the efficient gene knockout. The availability of MMEJ in genome editing applications has also been proven by various studies reporting the frequent observation of the traces of MMEJ repair on the edited alleles (Yasue et al 2014;Li et al 2015). The first example of MMEJ-dependent integration of donor plasmid was reported in 2014, where microhomologous ends between the genomic target site and exogenous DNA fragment were generated by simultaneous cleavage of genomic DNA and the donor vector (Nakade et al 2014).…”