Question: Is 5’ CRISPR compatible with my guide of interest?
Answer: The CRISPR RT primer was designed in a highly conserved region of the guide backbone for high compatibility across Cas9 systems. This sequence (5’-CAAGTTGATAACGGACTAGCC-3’) found in the 5' CRISPR Screening User Guides is complementary to the guide backbone. As a best practice, it is recommended to BLAST the reverse complement of the non-poly(dT) CRISPR primer sequence found in the appendix of the 5’ CRISPR Screening User Guides against the sgRNA guide library of interest to ensure compatibility.
The reverse complement of the primer sequence is: 5'-GGCTAGTCCGTTATCAACTTG-3'.
If the backbone does not have the sequence listed above, it may be possible to design custom enrichment primers to amplify outlier guides. We have provided some general guidance on designing a custom primer (see ‘My guide is not compatible with the 5' CRISPR, how do I design a custom primer?’). Custom primer design is not officially supported, and we cannot guarantee assay performance.
Product: Single Cell Immune Profiling