906
edits
Changes
updated with links to CRISPR design tool
==Designing the Targeting Strategy==
* Determine where you want to target the gene and copy that DNA sequence. It needs to be the genomic DNA from that particular species that is targeted, so if your region of interest spans several introns, you need to copy the genomic DNA not the mRNA sequence. In other words, if you can design your CRISPR pairs to be within one exon that is best. In that case submit the exon to the CRISPR tool. If your target site is close to an exon boundary then you need to submit the genomic sequence around that exon. You do not want your CRISPR pairs to span an intron.
* Paste this sequence into the CRISPR tool at http://crispr.mit.edu/ and select your target species. When complete select Double Nickase Design for Nickase or CRISPR Guides and Off-Targets for non-Nickase* Based on where you want to cut the DNA select the one or two guide DNA sequences. Name those sequences based on the nucleotide it cuts after (see cuts after position in the CRISPR tool output). Use the actual nucleotide from the reference mRNA sequence, not the nucleotide based on the arbitrary region that you pasted into the CRISPR tool.
* Print out, or sketch all of the targeting information into your notes.
