Abstract
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.
Highlights
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively
Compared with protein-based genome editing tools with customizable DNA binding specificities, such as Zinc Finger Nucleases (ZFNs) and TALENS, the newer clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] platform is based on simple base-pairing between an engineered RNA and the targeted genomic site, which enables rapid design, ease of use, and low costs[3]
The Cas[9] endonuclease is ushered to the specific site of interest by a single guide RNA, an engineered fusion molecule of the targeting CRISPR RNA and the trans-activating crRNA, to generate double-stranded DNA breaks (DSDBs) at the target site[4]
Summary
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively . Transgenic animals with reporter gene expression in specific tissues or cell types are valuable experimental tools Programmable endonucleases, such as Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and especially the CRISPR, the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system, were shown to be a kind of revolutionary technologies for targeted genome editing[1]. The sheep Rosa[26] (sRosa26) locus was characterized and its locus tagged with a knock-in reporter gene using a CRISPR/Cas9-mediated method Using this approach, transgenic sheep stably over-expressing a gene of interest were created through RMCE12. Fluorescent proteins such as GFP have been used for the characterisation and quantification of homologous recombination events as well as for endogenous gene-tagging and determination of subcellular localisation of expressed proteins[16]
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