Submitted by Luke Domanico
The Cas enzyme is a bacterial protein that works as an endonuclease to cleave DNA. Once the enzyme cleaves DNA, a sequence can be inserted in the “hole” the enzyme just made, or ligated, resulting in the deletion of a gene.
How does it work? The Cas enzyme is given written directions on where to go in the genome. This is capable via a provided double stranded sequence ligated in a plasmid transfected into the cell line. Once the plasmid has successfully made its way into the cell, the oligonucleotide that has been inserted in the plasmid works as a Guide RNA (gRNA) to instruct the Cas enzyme on where to go.
How it’s done:
Begin with the DNA coding sequence of the gene of interest. This can be obtained from NCBI.
Then, select 250bp from the sequence. This section will be used in a CRISPR design tool at CRISPR.MIT.edu.
The tool will spit out 20bp oligonucleotides that match the provided 250bp segment. These oligonucleotides are scored by the inverse of off-target binding probability (in gene and out).
Upon selection of the highest score oligonucleotide, this 20bp segment will now be placed in the IDT oligoanalyzer. This will provide a compliment to the 20bp oligonucleotide, as well as information about the sequence, such as melting temperature (Tm), GC content, etc.
The sequence and complement will be the dsDNA inserted in the Cas plasmid, which will act as the gRNA when transfected.