Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration.
Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration.
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DOI：
10.7554/elife.71478
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发表时间：
2022-06-17
期刊：
影响因子：
7.7
通讯作者：
McGrail, Maura
中科院分区：
文献类型：
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作者：
Liu, Fang;Kambakam, Sekhar;Almeida, Maira P.;Ming, Zhitao;Welker, Jordan M.;Wierson, Wesley A.;Schultz-Rogers, Laura E.;Ekker, Stephen C.;Clark, Karl J.;Essner, Jeffrey J.;McGrail, Maura
关键词：
空间和时间上调节基因活性的能力对于研究发育过程中以及胚胎后过程和疾病模型中的细胞类型特异性基因功能至关重要。 Cre/lox 系统已广泛用于对斑马鱼的基因功能进行细胞和组织特异性条件分析。然而，缺乏简单有效的方法来分离稳定的 Cre/lox 调节的斑马鱼等位基因。在这里，我们应用 GeneWeld CRISPR-Cas9 靶向整合策略来生成 floxed 等位基因，从而提供强大的条件失活和拯救。将通用靶向载体 UFlip 整合到 rbbp4 和 rb1 的内含子中，该载体在 floxed 2A-mRFP 基因陷阱两侧具有用于克隆短同源臂的位点。 rbbp4off 和 rb1off 整合等位基因导致强 mRFP 表达，内源基因表达减少 99% 以上，并重现已知的 indel 功能丧失表型。 Cre 的引入导致 floxed 盒的稳定倒置、mRFP 表达的丧失和表型拯救。 rbbp4on 和 rb1on 整合等位基因不会导致与功能丧失突变相结合的表型。添加 Cre 会通过盒的稳定倒置、基因捕获和 mRFP 表达以及预期的突变表型导致条件失活。神经祖细胞 Cre 驱动程序用于条件失活和表型拯救，以展示如何将这种方法用于特定细胞群。这些结果共同验证了一种在斑马鱼中有效分离 Cre/lox 响应条件等位基因的简化方法。我们的策略提供了一个用于生成遗传镶嵌的新工具包，代表了斑马鱼遗传学的重大进步。
The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.