دورية أكاديمية
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice.
| العنوان: | Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice. |
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| المؤلفون: | Lee AY; Mouse Biology Program, University of California, Davis, 2795 2nd Street, Suite 400, Davis, CA 95618, USA., Lloyd KC; Mouse Biology Program, University of California, Davis, 2795 2nd Street, Suite 400, Davis, CA 95618, USA. |
| المصدر: | FEBS open bio [FEBS Open Bio] 2014 Jul 01; Vol. 4, pp. 637-42. Date of Electronic Publication: 2014 Jul 01 (Print Publication: 2014). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: John Wiley & Sons Ltd Country of Publication: England NLM ID: 101580716 Publication Model: eCollection Cited Medium: Print ISSN: 2211-5463 (Print) Linking ISSN: 22115463 NLM ISO Abbreviation: FEBS Open Bio Subsets: PubMed not MEDLINE |
| أسماء مطبوعة: | Publication: Jan. 2016- : West Sussex : John Wiley & Sons Ltd. Original Publication: Amsterdam : Elsevier, 2011- |
| مستخلص: | 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. |
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| معلومات مُعتمدة: | P30 CA093373 United States CA NCI NIH HHS; UM1 OD023221 United States OD NIH HHS; U54 HG006364 United States HG NHGRI NIH HHS; U42 OD012210 United States OD NIH HHS; U42 OD011175 United States OD NIH HHS |
| فهرسة مساهمة: | Keywords: CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat; Cas, CRISPR-associated protein; Cas9 nickase; Cas9n, Cas9 nickase; Conditional allele; DSBs, double stand breaks; ES, embryonic stem cells; Gene targeting; HDR, homology directed repair; Ispd; Mouse; TALENs, transcription activator-like effector nucleases; ZFNs, zinc finger nucleases; indel, insertion and deletion mutations; lspd, isoprenoid synthase containing domain; sgRNA, single guide RNA |
| تواريخ الأحداث: | Date Created: 20140828 Date Completed: 20140827 Latest Revision: 20211021 |
| رمز التحديث: | 20221213 |
| مُعرف محوري في PubMed: | PMC4141200 |
| DOI: | 10.1016/j.fob.2014.06.007 |
| PMID: | 25161872 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 2211-5463 |
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| DOI: | 10.1016/j.fob.2014.06.007 |