CRISPR/Cas is the abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats and the system evolved in prokaryotic adaptive immune response system as the acquired immunity. CRISPR/Cas9 is able to use non-coding RNAs to guide the nuclease Cas9 and cut the cells DNA at a desired location allowing genes to be removed or added the new one. The cell has two different ways how to fix the DNA damage created by Cas9 protein:
The sgRNA consists of crRNA what is specific to DNA target and tracrRNA that can interact with Cas9 protein. The Cas9 protein has the DNA endonuclease activity and if the sgRNA is binded to Cas9, together can cause the specific-site double-stranded DNA cleavage. The double-stranded damage is then repaired mostly by preferred NHEJ pathway.
The workflow includes following steps
1) Design sgRNAs: you need to identify your target sites and avoid the potential off-target effects and then synthetize the target sgRNA.
sgRNA In Vitro Transcription Kit
2) Transcribe and screen sgRNAs in vitro don´t waste your time by delivering the ineffective sgRNAs to your cells and test the individual sgRNAs in vitro.
Guide-it sgRNA In Vitro Transcription Kit
Guide-it sgRNA Screening Kit
3) Deliver sgRNA and Cas9 to cells by provided delivery systems as plasmid, viral systems, RNA, protein or gesicles.
4) Detect Cas9 and confirm gene editing. Takara Bio also offers monoclonal or polyclonal antibodies against recombinant Cas9 protein.
Guide-it Mutation Detection Kit
Guide-it Cas9 Antibodies
5) Genotype determination: You can identify the monoallelic and biallelic mutants after gene editing.
Guide-it Genotype Confirmation Kit
6) Identify indels: Determining the variety of insertions and deletions created using editing technologies including CRISPR/Cas9 and other assays for other methods.
Guide-it Indel Identification Kit
7) Detect any nucleotide substitution at any genomic locus. Use high-throughput SNP screening without the need for sequencing and rapid 4 hours workflow.
Guide-it SNP Screening Kit
Guide-it Knockin Screening Kit - detection of single-nucleotide substitutions to longer insertions
8) The knockin mutations are proved to be much harder to introduce then the knockout. For this application Takara provides special kit what is desighned to produce a long ssDNA oligo of up to 5 kb to be a repair template in knockin experiments.
Guide-it Long ssDNA Production System
Documents and resources
For more information visit producer's website.
The gesicles are the unique technology also used for transport Crispr/Cas9 complex into the cells
The Electroporation ready Cas9 system for gene editing mediated by CRISPR/Cas9
The unique and specific recombination system allows to edit genes in application as gene knockins or knockouts
The Cas9/sgRNA can be delivered into the target cells by AAV and lentiviral systems