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关注球盟会生物基因，可及时获取更多优惠内容！
CRISPR那些事儿
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FAQ
如何提高Cas酶的检测灵敏度？
1）设计高效的crRNA序列。顺利获得合理的设计以及在线网站结构预测，选择合适的crRNA,以取得良好的Cas酶反式切割活性。
2）选择合适的信号报告底物。有关研究指出，使用15 nt的单链DNA（ssDNA）作为报告底物时，Cas12a的切割反应速率达到最大值，相较于常用的5-nt ssDNA，反应速率显著提升。
3）优化反应条件和缓冲液。顺利获得优化CRISPR反应的条件如Cas酶与crRNA比例、Cas酶使用浓度、反应温度等可以在一定程度上提高检测效果。
2）选择合适的信号报告底物。有关研究指出，使用15 nt的单链DNA（ssDNA）作为报告底物时，Cas12a的切割反应速率达到最大值，相较于常用的5-nt ssDNA，反应速率显著提升。
3）优化反应条件和缓冲液。顺利获得优化CRISPR反应的条件如Cas酶与crRNA比例、Cas酶使用浓度、反应温度等可以在一定程度上提高检测效果。
如何设计crRNA？
可根据以下步骤进行设计
1）明确目的基因序列。
2）明确使用的Cas蛋白。不同的Cas蛋白，需要识别相应的PAM（Protospacer Adjacent Motif）序列，例如Cas12a需要依靠“TTTV”PAM序列进行靶标识别。
3）选择crRNA靶向区域。在目标基因上选择一个20nt核苷酸序列，该序列紧邻PAM位点，且与crRNA的互补链配对。
4）选定的20nt靶向区域作为 target sequence（可变部分）加上scaffold sequence （固定部分）以设计crRNA序列。
5）使用在线工具如CRISPR design tool（例如CRISPOR、 Benchling等）来帮助设计crRNA。这些工具可以预测sgRNA的效率和特异性，避免可能的脱靶效应。
6）设计完成后，可以顺利获得合成生物学公司订购合成的crRNA序列。
1）明确目的基因序列。
2）明确使用的Cas蛋白。不同的Cas蛋白，需要识别相应的PAM（Protospacer Adjacent Motif）序列，例如Cas12a需要依靠“TTTV”PAM序列进行靶标识别。
3）选择crRNA靶向区域。在目标基因上选择一个20nt核苷酸序列，该序列紧邻PAM位点，且与crRNA的互补链配对。
4）选定的20nt靶向区域作为 target sequence（可变部分）加上scaffold sequence （固定部分）以设计crRNA序列。
5）使用在线工具如CRISPR design tool（例如CRISPOR、 Benchling等）来帮助设计crRNA。这些工具可以预测sgRNA的效率和特异性，避免可能的脱靶效应。
6）设计完成后，可以顺利获得合成生物学公司订购合成的crRNA序列。
什么是基因过表达？
基因过表达指的是顺利获得各种技术手段使某个特定基因在细胞或生物体中表达水平显著高于正常水平。这通常是顺利获得引入额外的基因拷贝或使用强启动子驱动基因表达来实现的。
如何选择合适的细胞进行文库筛选？
细胞选择可以遵循以下原则：
1）与研究目的相符
2）sgRNA文库靶向的基因要与细胞的属源相符
3）细胞可以稳定传代
4）转染效率高
尽量不选用原代细胞。由于原代细胞无法稳定传代，可能在文库筛选实验的过程中，原代细胞已经出现大量死亡，无法完成实验。如果选用原代细胞进行文库筛选，为分析决上述的风险，可以顺利获得降低细胞覆盖度和选择gRNA数较小的文库进行筛选，尽可能降低文库细胞池的数量和缩短实验周期。
1）与研究目的相符
2）sgRNA文库靶向的基因要与细胞的属源相符
3）细胞可以稳定传代
4）转染效率高
尽量不选用原代细胞。由于原代细胞无法稳定传代，可能在文库筛选实验的过程中，原代细胞已经出现大量死亡，无法完成实验。如果选用原代细胞进行文库筛选，为分析决上述的风险，可以顺利获得降低细胞覆盖度和选择gRNA数较小的文库进行筛选，尽可能降低文库细胞池的数量和缩短实验周期。
球盟会生物基因载体构建的宿主菌是什么？顾客可以使用什么类型菌株来扩增质粒载体？
载体在进行扩增时，通常会使用大肠杆菌（E. coli）菌株来进行。在实验操作中，常用的大肠杆菌菌株是DH5α。DH5α适用于大多数非重组型载体。对于重组型载体，如慢病毒载体和转座子载体等，可以使用Stbl3菌株进行扩增。Stbl3是一种特殊的大肠杆菌（E. coli）菌株，来源于，HB101 E. coli strain，其基因组含有重组酶recA13突变，这可以有效地抑制长片段末端重复区的重组，从而降低错误重组的概率。
What are the potential applications of iPSCs in clinical practice?
iPSCs have broad clinical potential, including applications in cell therapy (e.g., for diabetes or heart disease treatment), tissue engineering (e.g., development of artificial skin or liver tissue), and personalized drug screening (e.g., selecting optimal treatments based on a patient’s specific cellular response). These applications may transform treatment methods, offering more effective and personalized medical services.
What are induced pluripotent stem cells (iPSCs)?
Induced pluripotent stem cells (iPSCs) are cells reprogrammed from adult cells to a pluripotent state. They exhibit similar characteristics to embryonic stem cells, capable of differentiating into nearly all cell types in the body. This technology allows scientists to generate various cell types in vitro for research and therapy without the need for embryonic stem cells.
What is the difference between iPSCs and embryonic stem cells (ESCs)?
Both iPSCs and embryonic stem cells (ESCs) are pluripotent, but iPSCs are derived from reprogrammed somatic cells, while ESCs originate from early embryos. iPSCs do not involve embryo use, making them a more ethically acceptable choice, and they also avoid immune rejection issues, as they can be generated based on a patient’s genetic background.
How do iPSCs help us understand complex diseases?
Cells from patients are isolated and reprogrammed into iPSCs, which are then induced to differentiate into specific cell types to create disease models. These models enable researchers to study disease mechanisms, uncover disease-related genes, and molecular pathways, thereby advancing the development of new therapies. By analyzing these cells, scientists can observe disease-related changes at the cellular level, providing new perspectives in disease research.
What role does gene knock-in play in drug development?
Gene knock-in plays a crucial role in drug development. It is used in target validation by introducing specific genes into cell lines or animal models to confirm drug target efficacy. It also aids in establishing disease models, testing drug efficacy and safety in these models, and supporting drug screening through high-throughput screening in knock-in cell lines to identify potential drug candidates. Additionally, gene knock-in helps uncover drug mechanisms, optimize drug structure, and improve dosing strategies, expediting drug development while enhancing efficacy and safety.
What is the core principle of gene knock-in technology?
Gene knock-in technology involves inserting an exogenous gene sequence into a specific within the genome for gene function studies or disease treatment. Edigene utilizes advanced gene editing tools, such as the CRISPR/Cas9 system, to guide nucleases to cut the target DNA, and employs homology-directed repair or non-homologous end joining to accurately insert the gene at the desired , achieving efficient and precise gene knock-in.
Why choose EDITGENE, and what are EDITGENE’s main advantages in gene knock-in technology?
EDITGENE’s advantages in gene knock-in technology include:
Guaranteed results: With 10 years of CRISPR gene editing experience and a team of PhDs from world-renowned institutions offering one-on-one support.
High precision: EDITGENE’s optimized tools reduce off-target effects, enhancing editing accuracy.
High efficiency: EDITGENE’s technology platform improves knock-in success rates, accelerating experimental progress.
Customized service: Tailored knock-in solutions to meet specific research or therapeutic goals.
Why choose EDITGENE to establish stable overexpression cell lines?
EDITGENE brings 10 years of CRISPR-based cell editing experience and offers one-on-one support from a team of PhDs from globally recognized institutions.
What is the difference between a stable cell line and a transient cell line?
The main difference lies in the duration and stability of gene expression:
Transient cell line – The target gene is expressed temporarily in cells, typically lasting hours to days, and is suitable for short-term experiments.
Stable cell line – The target gene is stably integrated into the cell genome, allowing long-term expression, suitable for extended research and production.
What unique advantages does EDITGENE offer for monoclonal screening services?
EDITGENE utilizes industry-leading 3D single-cell printing technology, which enables precise isolation and positioning of individual cells, significantly increasing the success rate and efficiency of monoclonal screening. This technology is widely applied in biomedicine research, antibody development, drug screening, and therapeutic selection, showcasing broad application prospects in cell research.
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