التفاصيل البيبلوغرافية
| العنوان: |
An Optimized CRISPR/Cas9 Adenovirus Vector (AdZ-CRISPR) for High-Throughput Cloning of sgRNA, Using Enhanced sgRNA and Cas9 Variants. |
| المؤلفون: |
Statkute E; Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom., Wang ECY; Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom., Stanton RJ; Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom. |
| المصدر: |
Human gene therapy [Hum Gene Ther] 2022 Sep; Vol. 33 (17-18), pp. 990-1001. Date of Electronic Publication: 2022 Apr 18. |
| نوع المنشور: |
Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: |
English |
| بيانات الدورية: |
Publisher: M.A. Liebert Country of Publication: United States NLM ID: 9008950 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1557-7422 (Electronic) Linking ISSN: 10430342 NLM ISO Abbreviation: Hum Gene Ther Subsets: MEDLINE |
| أسماء مطبوعة: |
Original Publication: New York : M.A. Liebert, c1990- |
| مواضيع طبية MeSH: |
CRISPR-Cas Systems* , RNA, Guide, CRISPR-Cas Systems*/genetics, Adenoviridae/genetics ; Cloning, Molecular ; Gene Editing ; Genetic Vectors/genetics |
| مستخلص: |
Recombinant adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anticancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high-throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. In this study, we adapt this vector system to enable high-throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimized to ensure efficient cloning, and high editing efficiency using spCas9 and single guide RNA (sgRNA) sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity, but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large-scale CRISPR/Cas9 projects to be undertaken easily and quickly. |
| معلومات مُعتمدة: |
HCRW_HS-20-30 United Kingdom HCRW HCRW_; MR/P001602/1 United Kingdom MRC_ Medical Research Council; MR/S00971X/1 United Kingdom MRC_ Medical Research Council; MR/V000489/1 United Kingdom MRC_ Medical Research Council |
| فهرسة مساهمة: |
Keywords: CRISPR; Cas9; SniperCas9; adenovirus vector; recombineering; sgRNA |
| المشرفين على المادة: |
0 (RNA, Guide, CRISPR-Cas Systems) |
| تواريخ الأحداث: |
Date Created: 20220224 Date Completed: 20220923 Latest Revision: 20240104 |
| رمز التحديث: |
20240104 |
| DOI: |
10.1089/hum.2021.120 |
| PMID: |
35196879 |
| قاعدة البيانات: |
MEDLINE |
