Question: My guide is not compatible with the 5' CRISPR RT Primer Mix. How do I design a custom primer?
Answer: We recommend using a sgRNA that is complementary to the primer in our 5' CRISPR RT primer mix with 5' CRISPR Screening. This said, it might be possible to perform custom sgRNA capture.
Please note that custom sgRNA capture approaches have not been tested or validated by 10x Genomics and are not supported. We cannot provide specific experimental guidelines and can only provide the general recommendations included here.
First, check that the protospacer sequence is located at the 5’ end of the sgRNA. This ensures that it is feasible to design a custom primer downstream of the protospacer.
We also recommend consulting the 'Introduction' and 'Appendix: Oligonucleotides' sections of the Chromium Single Cell V(D)J Reagent Kits User Guides, which detail the 5' CRISPR screening assay scheme, and primer sequences, for guidance:
Custom sgRNA design considerations
- There are a few considerations for custom primer design, which will be used as a spike-in primer to the master mix used for chip loading and reverse transcription. The custom primer will have two parts: a non-polydT PCR handle followed by a sequence complementary to the guide of interest:
5- [non-poly(dT) sequence]-[reverse complement guide sequence]-3’
The non-poly(dT) sequence is:
A sample primer would be:
- The _* is a phosphorothioated DNA base that renders the oligo resistant to nuclease degradation and should be included in the primer design (as shown above).
- The custom primer would then be spiked into normal poly-dT RT primer (PN-200007) in the GEM Generation and Barcoding master mix.
- For primer concentration and volume, the methods section of the Weissman lab CRISPR publication can be used as a reference.
CRISPR Screening Library Construction
For custom Guide RNA cDNA amplification, it is possible that a custom Feature SI Primer will need to be used in place of Feature SI Primers 4. This custom primer would theoretically contain a mix of forward and reverse primers.
The forward primer would be:
The reverse primer would be in the following format:
5- [Partial Read 2 Sequence]-[Feature Primer]-3’
The feature primer sequence is complementary to the guide sequence and contains a few additional bases in comparison to the RT primer to increase specificity. Please see the 5' CRISPR User Guides for more details on primers and assay scheme.
A sample primer would be:
- Once a custom primer is designed, the following QCs should then be performed:
- The Tm of the primer should be in the same range as the primer provided. This can be determined by using an Oligo Tm calculator. Higher annealing temperatures are expected to be incompatible.
- Search the primer in your plasmid and genome of interest. We recommend selecting a primer with minimal interaction with other genome regions.
As custom primers have not been tested in-house, we cannot guarantee assay performance. In addition, modifying GEM reverse transcription conditions is not supported.
Product: Single Cell Immune Profiling