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CRISPR/Cas9 短同源定向靶向整合产生的内源性斑马鱼神经前体细胞 Cre 驱动子。

Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration.

机构信息

Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.

Genetics and Genomics Interdepartmental Graduate Program, Iowa State University, Ames, IA, USA.

出版信息

Sci Rep. 2021 Jan 18;11(1):1732. doi: 10.1038/s41598-021-81239-y.

DOI:10.1038/s41598-021-81239-y
PMID:33462297
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7813866/
Abstract

We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre), Tg(olig2-2A-Cre), and Tg(neurod1-2A-Cre) expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.

摘要

我们之前曾报道过使用 CRISPR/Cas9 和短同源序列在斑马鱼和人类细胞中高效、精确地靶向整合报告基因。在这里，我们应用这一策略来分离斑马鱼 Cre 重组酶驱动子，其空间和时间限制的表达模拟内源性基因。带有 48bp 同源臂的 2A-Cre 重组酶转基因被靶向到前神经基因 ascl1b、olig2 和 neurod1 中。我们观察到从 10%到 100%（2/20 olig2；1/5 neurod1；3/3 ascl1b）的高生殖系传递率。Tg(ascl1b-2A-Cre)、Tg(olig2-2A-Cre)和 Tg(neurod1-2A-Cre)转基因系在预期的前神经细胞群体中表达功能性 Cre 重组酶。2A-CreERT2 的体细胞靶向到 neurod1 导致在神经系统中对他莫昔芬有反应的重组。结果表明 Cre 重组酶的表达受靶向基因的天然启动子和调节元件的驱动。这种方法提供了一种简单、高效和具有成本效益的方法来产生细胞类型特异性的斑马鱼 Cre 和 CreERT2 驱动子，克服了与启动子-BAC 和转座子介导的转基因相关的挑战。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/c9f02a34fa92/41598_2021_81239_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/43ea6739c53b/41598_2021_81239_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/809fefd8032e/41598_2021_81239_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/e493892de246/41598_2021_81239_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/b39efc38469a/41598_2021_81239_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/c9f02a34fa92/41598_2021_81239_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/43ea6739c53b/41598_2021_81239_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/809fefd8032e/41598_2021_81239_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/e493892de246/41598_2021_81239_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/b39efc38469a/41598_2021_81239_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1aa3/7813866/c9f02a34fa92/41598_2021_81239_Fig5_HTML.jpg

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