• 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 and the time is pressing.
|Service contents||Provided by customers||Delivery time||Comments|
|• Construction of Common Cas9 vectors or lentiviral transfer vectors
The gRNAs slao can be constructed into the customer's vector (or that customer specify).
• Preparition of lentiviruses for gene knockout
• Design and preparion of donor vectors for HDR
|Gene names of a species or gene IDs||~2 weeks
(If gRNAs need be validated by cell transfection, it will take a total time of 6-8 weeks depending on the difficulty levels of the transfection.)
|Please inquire support staff if gRNAs need be validated by cell transfection.|
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KO vectors with gRNAs were transfected into cells. After 3 days single colnes with fluorescence were obtained by limited dilution. The genomes of each single clone were extracted to perform PCR and sequencing analysis.
Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacteriophage virus or plasmid.
The CRISPR/Cas system is an immune system that confers resistance to foreign genetic elements such as viruses and plasmids, and provides a form of acquired immunity in many bacteria and archaea species. The system recognizes and cuts these exogenous genetic elements to prevent repeated attacks in a manner analogous to RNAi in eukaryotic organisms. Bacteria employing CRISPR/Cas systems collect bits of genetic material from viruses or other invaders, and save those sequences as "spacer DNA" in specific regions of their genomes. Those foreign DNA-containing regions are called "clustered regularly interspaced short palindromic repeats(CRISPR)", When the cell undergoes a later attack, the spacer sequences stored among the CRISPR elements are expressed as CRISPR-RNAs (crRNAs). crRNAs binding with trans-activating crRNAs (tracrRNAs) are processed into complexes with CRISPR-associated proteins (Cas proteins). These complexes have the specificity of each spacer sequence. Target double-strand break(DSB) by these complexes requires base pairing between the crRNA and the target DNA. Target recognition is facilitated by the presence of a short sequence called a protospacer adjacent motif (PAM) that conforms to the sequence NGG (spCas9 recongnizes).
CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea.
crRNA contains the RNA used by Cas9 to guide it to the correct section of host DNA with tracrRNA forming an active complex with Cas9.
tracrRNA binds to crRNA and forms an active complex with Cas9.
gRNAs are a combined RNA consisting of a tracrRNA and a crRNA.