Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Division of Cardiology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
Dev Cell. 2018 Jul 2;46(1):112-125.e4. doi: 10.1016/j.devcel.2018.06.003.
Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.
斑马鱼是正向遗传学的强大模型。反向遗传学方法受到产生稳定突变系所需时间的限制。我们描述了一种用于基因敲除的系统，该系统可在 G0 斑马鱼中持续产生缺失表型。针对单个基因的四组 CRISPR/Cas9 核糖核蛋白复合物的卵黄注射重复地重现了 8 个测试基因中每个基因的种系传递的敲除表型，在 >90%的 G0 胚胎中。产生了早期胚胎（6 hpf）和稳定的成年表型。同时进行多基因敲除是可行的，但在某些情况下会出现毒性。为了便于使用，我们生成了一个包含 21,386 个斑马鱼基因的四向导组的查询表，并验证了其中的几个。使用此资源，我们针对 50 个心肌细胞转录调节剂进行了靶向处理，发现了 zbtb16a 在心脏发育中的作用。该系统为在斑马鱼发育、生理学和疾病模型中快速筛选感兴趣的基因提供了一个平台。
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