Abstract
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.
Highlights
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] systems
We microinjected into rat zygotes these engineered protein nucleases along with a long donor DNA fragment encoding for GFP, previously used to obtain homology-directed recombination (HDR) using TALEN encoded by mRNA1
non-homologous end joining (NHEJ) analysis of mutated founders showed an increased rate of mosaicism using Cas[9] protein, since 42.1% of the fetuses were mosaics compared with 25% using Cas[9] mRNA. It tended to be higher using TALEN protein (33%) compared with TALEN mRNA (21.7%), as described in a previous work[1]. These results show that for the rat Rosa[26] locus Cas[9] protein but not TALEN proteins were more efficacious to obtaining HDR than the respective mRNA and this was associated to a higher rate of NHEJ
Summary
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas[9] systems. Our results indicate that the use of Cas[9] protein could represent a simple and practical methodological alternative to Cas[9] mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals Engineered nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) are key tools for the introduction of gene-inactivating mutations by non-homologous end joining (NHEJ) mechanisms, as well as precise modifications such as base mutation, exon exchange or insertion of expression cassettes by homology-directed recombination (HDR)[1,2]. Every other genetically engineered animal model, including rats and mice, using any type of nuclease, including meganucleases, ZFNs and TALENs, has been generated using DNA or mRNA2,3, TALEN proteins, in particular, have never been used in the generation of transgenic animals In this context, we recently described HDR in the rat Rosa[26] locus using TALEN mRNA and a large GFP expression cassette[1]. The use of Cas[9] protein may widen the application of genome-editing techniques in different species
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