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Further experimental planning
The table below highlights the key features and requirements of Cas9 targeted sequencing using S. pyogenes Cas9. Although other CRISPR/Cas enzymes, such as Cas12a, are available, we recommend that new users begin developing their own targeted sequencing assay using S. pyogenes Cas9 for the following reasons:
- Cas9 enzymes, including a wide variety of high-fidelity mutants, are more widely available from third parties;
- A far wider library of verified probe designs is already available for Cas9 via the scientific literature;
- The rules for predicting the efficiencies (i.e. the proportion of target predicted to be cut) and off-target effects of Cas9 crRNAs are better understood than for other enzyme crRNAs, i.e., the design criteria are better-defined;
- The Cas9 cleavage reaction yields a blunt-ended double-stranded break, so the biochemistry required to generate an end to which an adapter can be ligated is simpler.
Parameter S. pyogenes Cas9 (1.) Protospacer length 20mer RNAs required crRNA and tracrRNA (or sgRNA) Cost per crRNA at 2 nmol scale (2.) $95 (US) £48 (UK) Cost of tracrRNA at 5 nmol scale (3.) $95 (US) £48 (UK) PAM NGG, 3' Structure of double-strand break blunt-ended End-preparation method dA-tailing Table footnote:
1. Streptococcus pyogenes species
2. Based on pricing of Alt-R™ synthetic crRNA at the lowest synthesis scale in vials, provided by Integrated DNA Technologies (IDT). 2 nmol is sufficient for ~200 reactions. Workflows involving multiple crRNAs require preparing an equimolar mix of crRNAs. crRNAs may be ordered in bulk in multi-well format at lower cost. Pricing correct as of September 2018.
3. Based on pricing of Alt-R™ synthetic tracrRNA from IDT. One-time purchase: the same tracrRNA is used for all Cas9 cleavage reactions. 5 nmol is sufficient for ~500 reactions. Pricing correct as of September 2018.