A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.
A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.
复制标题
DOI：
10.3390/ijms24098271
复制
发表时间：
2023-05-05
影响因子：
5.6
通讯作者：
Burwitz, Benjamin J.
中科院分区：
文献类型：
--
作者：
Wettengel, Jochen M.;Hansen-Palmus, Lea;Yusova, Sofiya;Rust, Lauren;Biswas, Sreya;Carson, Julien;Ryu, Junghyun;Bimber, Benjamin N.;Hennebold, Jon D.;Burwitz, Benjamin J.
CRISPR/Cas系统是治疗性基因组编辑最有前途的工具之一。这些系统的使用取决于导向器和同源定向修复（HDR）模板的最佳设计。虽然这种设计可以在计算机上实现，但验证和进一步优化通常是在报告系统的帮助下进行的。在这里，我们描述了一种新的报告系统，称为BETLE，它允许快速、敏感和细胞特异性检测基因组编辑和模板特异性HDR，方法是在不同的开放阅读框架中编码多个报告蛋白。框外非同源末端连接（NHEJ）导致可分泌的NanoLuc荧光素酶的表达，从而实现高灵敏度和低成本的编辑分析，或荧光mTagBFP2，允许基因组编辑细胞的枚举和组织特异性定位。BETLE包括一个验证CRISPR/Cas系统对感兴趣序列的站点，使其具有广泛的适应性。我们使用缺失39个碱基对的缺陷moxGFP来评估BETLE，并在具有单个和多个报告基因整合物的细胞系中显示了spCas9、saCas9和asCas12a的编辑以及序列特异性HDR和moxGFP的修复。综上所述，这些数据表明BETLE可以在体外快速检测和优化CRISPR/Cas基因组编辑和HDR，并代表了未来在体内应用的最先进工具。
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
登录
查看更多内容
DOI：
10.1126/science.abb1400
发表时间：
2020-07-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Pausch P;Al-Shayeb B;Bisom-Rapp E;Tsuchida CA;Li Z;Cress BF;Knott GJ;Jacobsen SE;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
16.6
作者：
Roidos, Paris;Sungalee, Stephanie;Mardin, Balca R.
通讯作者：
Mardin, Balca R.
影响因子：
16.6
作者：
Mishra AP;Hartford SA;Sahu S;Klarmann K;Chittela RK;Biswas K;Jeon AB;Martin BK;Burkett S;Southon E;Reid S;Albaugh ME;Karim B;Tessarollo L;Keller JR;Sharan SK
通讯作者：
Sharan SK
影响因子：
4.6
作者：
Liu WH;Völse K;Senft D;Jeremias I
通讯作者：
Jeremias I