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CRISPR那些事儿
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FAQ
How is Prime Editing 7 (PE7) different from traditional CRISPR/Cas9 technology?
Traditional CRISPR/Cas9 technology achieves gene editing by introducing double-strand breaks at the target DNA site and then using the cell’s homologous recombination repair mechanism. This approach carries multiple risks, such as lower editing efficiency, reduced homozygous mutation rates, and random insertions or deletions. Prime Editing, however, does not require double-strand breaks. With its Cas9n-RT editing enzyme system and pegRNA, Prime Editing achieves more accurate and safer gene editing with reduced off-target effects.
Are all types of genes suitable for KO cell lines?
Not all genes are suitable for knockout. Some gene knockouts may result in cell death or severe dysfunction, particularly for essential genes. In such cases, conditional knockouts or gene knockdowns (e.g., RNAi) may be used instead.
Why choose EDITGENE?
EDITGENE provides access to a comprehensive library of over 4,500 high-quality knockout (KO) cell lines, enabling researchers to save valuable time. Our custom gene knockout services are highly efficient, boasting a high positive rate while minimizing off-target effects. Clients also benefit from personalized, one-on-one support from a team of PhD experts from globally renowned institutions, ensuring top-tier service and results.
What is the difference between KO cell lines and gene knockout animal models?
KO cell lines are used for in vitro experiments, suitable for high-throughput screening and cellular studies, while gene knockout animal models are used for in vivo experiments to study gene functions within an entire organism and its interaction with the environment.
Why do researchers use KO cell lines?
Researchers use KO cell lines to investigate gene functions by observing the effects of gene deletion on cellular behavior. This helps in understanding the role of genes in various processes like cell growth, metabolism, and signal transduction. KO cell lines are vital for studying diseases like cancer, genetic disorders, and neurodegenerative diseases.
What is a KO cell line?
KO (Knockout) cell line is a cell line where a specific gene has been completely removed or rendered non-functional through gene editing technologies such as CRISPR-Cas9. These cell lines are critical for understanding gene functions and disease mechanisms.
Are KO cell lines applicable to all cell types?
KO cell lines can be applied to various cell types, including cancer cells, stem cells, and primary cells, but different cell types may have varying sensitivities to gene editing, and may vary among different cell types. In certain cell types, achieving gene knockout may require optimization of transfection conditions and selection of appropriate gene-editing tools.
What is gene overexpression?
Gene overexpression refers to using various techniques to significantly increase the expression level of a specific gene in cells or organisms. This is often achieved by introducing additional gene copies or using strong promoters to drive gene expression.
Why conduct gene overexpression?
Gene overexpression aids in studying the function of specific genes, revealing their role within the organism. It is also commonly used in drug screening, vaccine development, and protein production. For example, by overexpressing a therapeutic protein, researchers can evaluate its efficacy in disease models.
KO细胞系是否适合所有类型的基因？
虽然KO细胞系技术强大，但并非所有基因都适合敲除。某些基因的敲除可能导致细胞死亡或严重的生理功能障碍，特别是那些对细胞存活至关重要的基因。在这种情况下，可以选择条件性敲除或基因敲降（如RNAi）来研究这些基因的功能。
KO细胞系与基因敲除动物模型有什么区别？
KO细胞系和基因敲除动物模型都用于研究基因功能，但它们各自有不同的应用场景和优势。KO细胞系在体外实验中使用，适用于高通量筛选和细胞水平的基因功能研究。基因敲除动物模型则在体内实验中使用，适用于研究基因在整个生物体中的功能及其与其他基因和环境的相互作用。
一般CRISPR相关试剂以及Cas蛋白可以存放多久？
CRISPR检测相关试剂：
①RPA恒温扩增试剂盒于-20℃保存，可长期存放。
②靶标质粒可长期存放-20℃ ③ Cas蛋白反复冻融容易影响活性，建议分装多管，存放-80℃，根据实验需要取出使用。另外，短期内使用的，可放-20℃保存。
③crRNA容易降解，建议短时间内用不到的于-80℃保存。
④探针是DNA双链，相对来说没有那么容易降解，可放-20℃保存。
①RPA恒温扩增试剂盒于-20℃保存，可长期存放。
②靶标质粒可长期存放-20℃ ③ Cas蛋白反复冻融容易影响活性，建议分装多管，存放-80℃，根据实验需要取出使用。另外，短期内使用的，可放-20℃保存。
③crRNA容易降解，建议短时间内用不到的于-80℃保存。
④探针是DNA双链，相对来说没有那么容易降解，可放-20℃保存。
如何选择全基因组或亚基因组CRISPR文库？
CRISPR文库类型可分为全基因组文库和亚基因组文库。如果目标是进行全基因组范围内的筛选，那么全基因组文库是最佳选择。这类文库通常包含针对整个基因组的sgRNA。如果研究目标有偏向性，如只关注特定基因家族或特定信号通路，那么可以选择亚基因组文库，以减少不必要的筛选工作量和成本。
iPSC如何帮助我们理解复杂疾病？
从患者分离出体细胞并重编程为iPCS，然后诱导分化为特定的细胞类型，用于创建疾病模型。这些模型可以帮助研究人员研究复杂疾病的发病机制，揭示疾病相关的基因和分子通路，从而有助于新的治疗方法的开发。顺利获得分析这些细胞，科研家可以观察到疾病在细胞水平上的变化，提供了疾病研究的新视角。
什么是单克隆筛选，为什么它在基因编辑研究中如此重要？
单克隆筛选是从混合细胞池中分选出单个克隆，并由该克隆培养扩增成细胞系。单克隆筛选保证了使用的细胞系是来源于同一个细胞，并且确保了遗传背景的高度一致。在细胞被基因编辑或基因修饰后，初始细胞池的每个细胞的遗传背景的差异是巨大的，这样的细胞池用于后续的实验，实验结果无疑是不准确的。顺利获得单克隆筛选，可以取得遗传背景一致、具有稳定基因编辑的细胞群体，监测到的表型变化才是稳定准确的。
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