CRISPR-Cas9 Genome Editing in Drosophila.
CRISPR-Cas9 Genome Editing in Drosophila.
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DOI：
10.1002/0471142727.mb3102s111
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发表时间：
2015-07-01
影响因子：
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通讯作者：
O'Connor-Giles KM
中科院分区：
文献类型：
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作者：
Gratz SJ;Rubinstein CD;Harrison MM;Wildonger J;O'Connor-Giles KM
CRISPR-Cas9系统将模式生物的基因组工程从可能变为现实。CRISPR-Cas9可以很容易地编程生成序列特异性双链断裂，当通过易出错的非同源末端连接修复时破坏目标位点，或者通过同源定向修复（homology-directed repair， HDR）催化精确的基因组修饰。在这里，我们描述了一种通过crispr - cas9介导的HDR对果蝇基因组进行快速高效工程的简化方法。在这种方法中，向表达Cas9的转基因果蝇注射表达引导rna （gRNAs）和阳性标记的供体模板的质粒。我们详细介绍了目标选址；gRNA质粒生成；捐助模板设计与搭建；以及工程化品系的产生、鉴定和分子鉴定。我们还提出了替代方法，并强调了实验设计的关键考虑因素。本文概述的方法可用于快速可靠地生成各种工程修饰，包括基因组缺失和替换，精确的序列编辑和蛋白质标签的结合。
The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.