Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene.
Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene.
复制标题
DOI：
10.1016/j.omtm.2020.09.001
复制
发表时间：
2020-12-11
期刊：
影响因子：
--
通讯作者：
Rex TS
中科院分区：
文献类型：
--
作者：
Backstrom JR;Sheng J;Wang MC;Bernardo-Colón A;Rex TS
CRISPRi 在临床应用中降低靶向基因表达的能力因递送挑战而受到抑制。现有的构建体太大，无法满足腺相关病毒 (AAV) 约 4.7 kb 包装大小的限制，腺相关病毒 (AAV) 是 FDA 唯一批准用于临床的病毒载体。因此，我们优化了 CRISPRi 组件以生成包含所有功能元件并有效降低体内内源基因表达的单个 AAV 载体。首先，我们通过使用螺旋接头和 c-Myc 核定位信号，将金黄色葡萄球菌灭活 Cas9 (SadCas9) 的核靶向性提高了 4 倍。其次，我们确定氨基末端 Krüppel 相关盒 (KRAB) 构建体是在体外降低靶基因表达最有效的方法。第三，我们优化了指导RNA的启动子并评估了肝细胞中KRAB-SadCas9表达的微型启动子。我们的最终构建体在体外减少了蛋白质转化酶枯草杆菌蛋白酶/kexin 9 (Pcsk9) mRNA 并将蛋白质分泌量提高了 5 倍。相应的 AAV2/8 载体定位于肝细胞核，体内 Pcsk9 mRNA 和血清蛋白水平降低 30%。这种单一的 AAV 方法提供了一种潜在的临床可转化方法，用于减少体内 CRISPRi 的靶向基因转录。修饰基因转录的成分的正确细胞定位和最大功能是临床相关治疗干预的核心。作者使用失活的 Cas9 作为模型系统优化了核靶向和转录抑制。随后对单一腺相关病毒进行的体内研究表明，目标基因的基因组表达降低。
The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo. First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro. Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 (Pcsk9) mRNA and secreted protein 5-fold in vitro. The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo. This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo. Proper cellular localization and maximal function of components that modify gene transcription are central to clinically relevant therapeutic interventions. The authors optimized nuclear targeting and transcriptional repression using deactivated Cas9 as a model system. Subsequent in vivo studies with a single adeno-associated virus revealed decreased genomic expression of a target gene.
登录
查看更多内容
影响因子：
16.6
作者：
Thakore PI;Kwon JB;Nelson CE;Rouse DC;Gemberling MP;Oliver ML;Gersbach CA
通讯作者：
Gersbach CA
DOI：
10.1080/21541264.2017.1322170
发表时间：
2017
期刊：
Transcription
影响因子：
--
作者：
Gao Z;Harwig A;Berkhout B;Herrera-Carrillo E
通讯作者：
Herrera-Carrillo E
影响因子：
64.5
作者：
Nishimasu H;Cong L;Yan WX;Ran FA;Zetsche B;Li Y;Kurabayashi A;Ishitani R;Zhang F;Nureki O
通讯作者：
Nureki O
影响因子：
20.1
作者：
Spolitu, Stefano;Okamoto, Haruka;Ozcan, Lale
通讯作者：
Ozcan, Lale
DOI：
10.1073/pnas.1612075113
发表时间：
2016-09-20
影响因子：
11.1
作者：
Ye, Lin;Wang, Jiaming;Kan, Yuet Wai
通讯作者：
Kan, Yuet Wai