ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes
説明
<jats:title>Abstract</jats:title><jats:p>The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat <jats:italic>Thy1</jats:italic> locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the <jats:italic>Rosa26</jats:italic> locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human <jats:italic>SIRPA</jats:italic> locus, concomitantly knocking out the rat <jats:italic>Sirpa</jats:italic> gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat <jats:italic>Cyp2d</jats:italic> cluster with a 6.2-kb human <jats:italic>CYP2D6</jats:italic> gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.</jats:p>
収録刊行物
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- Nature Communications
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Nature Communications 7 (1), 10431-, 2016-01-20
Springer Science and Business Media LLC
