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. 2016 Apr 11;6:24360.
doi: 10.1038/srep24360.

Rosa26-targeted Sheep Gene Knock-In via CRISPR-Cas9 System

Free PMC article

Rosa26-targeted Sheep Gene Knock-In via CRISPR-Cas9 System

Mingming Wu et al. Sci Rep. .
Free PMC article


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.


Figure 1
Figure 1. Characterization of sRosa26 and highly efficient gene knock-in and replacement at the sRosa26 locus.
(a,b) sRosa26 was expressed in a variety of organ tissues as determined by (a) RT-PCR and (b) quantitative RT-PCR. For RT-PCR, the designed primers annealed in sRosa26 sequence and amplified a correctly spliced product of 485 bp. Ovis GAPDH served as a control (234 bp). For qPCR, primers were specific to the sRosa26 sequence. The PCR product of the Ovis ACTB gene served as the reference control. Data are presented as the average expression levels of three individual RT/qPCR reactions.
Figure 2
Figure 2
(a) Analysis of sgRNA activity. 5 guide RNAs (sgRNAs) were designed to target the sequence of the sRosa26 locus. All 5 sgRNAs efficiently guided Cas9 for genome editing.
Figure 3
Figure 3
(a) Diagram of Cas9-mediated knock-in of LA-CAG-tGFP-polyA-RA into the sRosa26 locus. LA, left arm (1.0 kb); RA, right arm (1.0 kb). (b) CAG promoter-driven tGFP expression in sheep fibroblasts (CAG promoter region is shown in supplementary information).
Figure 4
Figure 4
(a) PCR analysis of tGFP in tissues knock-in lambs. M = marker, N = negative control, P = postive control The size of target band is about 650 bp. (b) Southern blot analysis of tGFP–KI mutant sheep, Both the negative control and tGFP-KI sheep can be detected the target sequence. (c) RT- PCR analysis of tGFP expression in tissues knock-in lambs. All the lambs were 1 month old. tGFP gene expression quantity is 4.5 times that of the negative control. (d) Western blot analysis of tGFP–KI mutant sheep. Total protein from tGFP-KI sheep was subjected to SDS-PAGE on a 12% acrylamide gel. GADPH was used as a loading control. The tGFP gene expression cann’t be detected in controls.

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