|CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression or activation of gene expression in prokaryotic and eukaryotic cells.
Based on the bacterial genetic immune system - CRISPR (clustered regularly interspaced short palindromic repeats) pathway, the technique provides a complementary approach to RNA interference. The difference between CRISPRi and RNAi, though, is that CRISPRi regulates gene expression primarily on the transcriptional level, while RNAi controls genes on the mRNA level.
1. CRISPRi can silence a target gene of interest up to 99.9% repression.
2. Since CRISPRi is based on Watson-Crick base-pairing of sgRNA-DNA and an NGG PAM motif, selection of targetable sites within the genome is straightforward and flexible. Carefully defined protocols have been developed.
3. Multiple sgRNAs can not only be used to control multiple different genes simultaneously (multiplexing gene targeting), but also to enhance the efficiency of regulating the same gene target.
4. While the two systems can be complementary, CRISPRi provides advantages over RNAi. As an exogenous system, CRISPRi does not compete with endogenous machinery such as microRNA expression or function. Furthermore, because CRISPRi acts at the DNA level, one can target transcripts such as noncoding RNAs, microRNAs, antisense transcripts, nuclear-localized RNAs, and polymerase III transcripts. Finally, CRISPRi possesses a much larger targetable sequence space; promoters and, in theory, introns can also be targeted.
One-stop technical services are provided from gene information, cells, sequencing, analysis to construction of cell lines by our company. Our CRISPR genome editing technology applies to targets of any genes and mammalian cells. Professional researchers have plentiful CRISPR genome editing experience, including gRNA design, cell transfection, monoclonal cell culture and passages, solving difficulty in transfection and constructing tumor cell lines. After services are finishes, we will provide gene-edited monoclonal cells and detailed project reports.
1. Choose transfection methods of the specific cell: Try all kinds of methods, including liposome-mediated transfection, electroporation, nucleofection, lentivirus-mediated transduction, et al.
2. Determine antibiotic concentration: Decrease difficulties in selecting positive cells and checking them.
3. Culture monoclonal cells: Determine if monoclonal cells are cultured.
Three gRNAs are designed in common exons of different transcriptional products, generally. Target positions are as far as possible in the first third of the gene, and targets can destroy important domains of the protein and all the alternatively spliced transcripts. Six gRNAs are designed if the effects are not good or the time is pressing.
Construct vectors or pack lentiviruses
Common Cas9 vectors or lentiviral vectors are choosed according to the results of pre-experiments, then vector plasmids and lentiviruses are prepared.
Transfect the cells with vector plasmids or transduct the cells with lentivirus.
Analysize cell pool
Genomic PCR amplicons are sequenced to confirm if genome editing is produced.
Select single clones and bank positive cells
Single positive clones are selected according to the sequence results and these cells are banked.
Present final data
Provide clients with single cell clones and detailed reports.
Generally, cell lines are provided by customers. An additional charge is collected if our company provide them. In addition, 200 tumor cell lines are offered except general cell lines.
Additional single clone: The other single clone are selected from the same cell bank and presented to clients with analysis of target sequences included in final report. Functional identification of single clones: qPCR and western blot of the target protein.
1. Provide real one-stop service from gene synthesis, design of gRNAs and donors, cell transfection, preparing of single clones to sequencing analysis of single clone, only needing customers to present target genes
2. Do not use antibiotic to select cells and fuse tags in target genes(only in gene knock-out project)
3. Possess rich cell culture experiences
4. Do not add antibiotic in cell culture. Ensure aseptic operation and sterile environment strictly.
1. Target gRNA sequences are designed(free of charge), synthesized and cloned into any vectors according to target sequences provided by customers.
2. Provide construction service of stable overexpression cell lines and help you to develop stable cell lines expressing foreign genes.
In order to provide quotation efficiently and accurately, please download and complete the inquiry sheet, then fill out the whole inquiry sheet and email it to us, we will reply to you in time.
The product can only be used for scientific research purposes, NOT be used in any human or clinical experiments, NOT be used to modify any human reproductive system, including the human embryo and reproductive cells.