What a guide needs
For the common SpCas9, the guide is 20 bases that match the target DNA, and the target must be followed directly by an NGG PAM on the same strand. Cas9 cuts 3 bases upstream of the PAM. The PAM itself is not part of the guide.
Sites can be on either strand: an NGG on the bottom strand shows up as CCN on the top.
Choosing among sites
- Position. To knock out a gene, target an early exon shared by all transcripts, so frameshifts disrupt the protein. For a precise edit, the cut should be within about 10 bases of the change.
- GC content of 40 to 80%. Very low GC binds weakly; very high GC can bind too easily elsewhere.
- No TTTT. Four Ts in a row end transcription from the U6 promoter that most guide plasmids use.
- A G at the start helps U6 transcription; if there is none, many protocols add one.
Off-targets
A guide can also cut sites that differ by a few bases, especially when the mismatches are far from the PAM. Check your shortlisted guides against the whole genome with a genome-wide search tool before ordering them, and pick those with no close matches elsewhere.
Find candidate sites
Paste your target sequence into the CRISPR Guide Finder. It lists every site on both strands with its position, strand and GC content, for NGG and other PAMs. Use the Restriction Map to plan a screening digest around the cut.