کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1981726 1539420 2014 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice
چکیده انگلیسی


• We generated a floxed allele by using paired Cas9n (nickase), gRNAs and single DNA template in mouse.
• We confirmed that the floxed allele was germline transmitted and functional in F1 offspring.
• A floxed allele of the isoprenoid synthase containing domain (Ispd) gene in C57BL/6N background mice was created.
• This method can be used to generate knockout mice for genes that are potentially embryonic lethal.

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: FEBS Open Bio - Volume 4, 2014, Pages 637–642
نویسندگان
, ,