Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish.
Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish.
复制标题
DOI：
10.3389/fcell.2021.735598
复制
发表时间：
2021
影响因子：
5.5
通讯作者：
Matsuoka RL
中科院分区：
文献类型：
--
作者：
Quick RE;Buck LD;Parab S;Tolbert ZR;Matsuoka RL
斑马鱼是研究心血管形成和功能的一种有价值的脊椎动物模型，这是由于其外部生长的胚胎中的循环系统易于可视化和快速发育。尽管有明显的优势，但斑马鱼有许多重要基因的旁系同源物，使得反向遗传学方法效率低下，因为产生携带多个基因突变的动物需要大量的努力。在这里，我们提出了一种简单而稳健的基于CRISPR RNA/Cas9的合成诱变方法，用于产生双等位基因F0斑马鱼敲除。使用双指导合成CRISPR RNA/Cas9核糖核蛋白（dgRNP）系统，我们比较了在将每个基因注射一个、两个和三个dgRNP到细胞质或蛋黄中后双等位基因基因破坏的效率。我们发现，同时细胞质注射三个不同的dgRNP每个基因到一个细胞阶段的胚胎导致最有效和一致的双等位基因的基因中断。重要的是，这种三重dgRNP方法能够有效地灭活细胞自主和细胞非自主基因功能，这可能是由于双等位基因破坏的低嵌合性。为了支持这一发现，我们提供了证据表明，通过该方法产生的F0动物完全表型复制了在相应的稳定突变纯合子中观察到的内皮和血管周围缺陷。此外，这种方法忠实地概括了两个vegfr 2斑马鱼旁系同源物之间的遗传相互作用产生的干血管表型。从机制上讲，基因组编辑和mRNA衰变的研究表明，每个基因三个dgRNP的组合诱变作用导致移码突变的可能性增加，从而实现有效的双等位基因破坏。因此，我们的方法提供了一个高度稳健的遗传平台，以快速评估F0斑马鱼中的新基因和冗余基因功能。
The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.
登录
查看更多内容
影响因子：
4.5
作者：
Keatinge M;Tsarouchas TM;Munir T;Porter NJ;Larraz J;Gianni D;Tsai HH;Becker CG;Lyons DA;Becker T
通讯作者：
Becker T
影响因子：
14.9
作者：
Cade L;Reyon D;Hwang WY;Tsai SQ;Patel S;Khayter C;Joung JK;Sander JD;Peterson RT;Yeh JR
通讯作者：
Yeh JR
影响因子：
20.3
作者：
Bridge, Gemma;Monteiro, Rui;Boshoff, Chris
通讯作者：
Boshoff, Chris
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao
影响因子：
4
作者：
Bedell VM;Westcot SE;Ekker SC
通讯作者：
Ekker SC