Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)
Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)
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DOI：
10.3791/3304
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发表时间：
2012-06-01
影响因子：
1.2
通讯作者：
Briner, Dave
中科院分区：
文献类型：
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作者：
Hansen, Keith;Coussens, Matthew J.;Briner, Dave
基因组编辑是一种强大的技术，可用于阐明基因功能和疾病的遗传基础。传统的基因编辑方法，如基于化学的诱变或DNA序列的随机整合，以整体低效的方式赋予不加选择的遗传变化，并且需要掺入不需要的合成序列或使用异常的培养条件，这可能会混淆生物学研究。相比之下，细胞中的瞬时ZFN表达可以以高效的方式促进精确的、可遗传的基因编辑，而不需要施用化学品或整合合成的转基因。锌指核酸酶（ZFN）是结合并切割双链DNA（dsDNA）的不同序列的酶。功能性CompoZr ZFN单元由两个单独的单体蛋白组成，其结合约15 - 18个核苷酸的DNA "半位点"（参见图1）。当两个ZFN单体"归巢"到它们相邻的靶位点时，DNA切割结构域二聚化并在DNA中产生双链断裂（DSB）。(1)在基因组中引入ZFN介导的DSB为高效基因组编辑奠定了基础。通过非同源末端连接（NHEJ）DNA修复途径对细胞中DSB的不完美修复可导致小的插入和缺失（indel）。在细胞的基因编码序列内创建插入缺失可导致移码和随后以高效率对基因座进行功能性敲除。(2)虽然该方案描述了使用ZFN来创建基因敲除，但转基因的整合也可以通过ZFN切割位点处的同源定向修复来进行。CompoZr定制ZFN服务代表了使用ZFN技术为科学界进行靶向基因编辑的系统、全面和充分表征的方法。Sigma科学家与研究人员密切合作，1）根据项目目标进行尽职调查分析，包括分析相关基因结构，生物学和模型系统，2）应用这些知识制定合理的靶向策略，3）然后设计，构建和功能验证ZFN在相关细胞系中的活性。研究者接收阳性对照基因组DNA和引物，以及以质粒DNA和体外转录mRNA形式提供的即用型ZFN试剂。然后可以递送这些试剂用于在研究者的细胞系或选择的细胞类型中瞬时表达。然后通过标准分子生物学技术（包括PCR扩增、酶消化和电泳）测试样品在感兴趣的基因座处的基因编辑。在初始群体中检测到基因编辑的阳性信号后，对细胞进行单细胞克隆和基因分型以鉴定突变克隆/等位基因。
Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA. (1) Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency. (2) While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator's cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.