Question: Can you generate CRISPR Screening libraries with the GEM-X Universal 3’ Gene Expression v4 assay?
Answer: It may be possible to generate CRISPR Screening libraries with the GEM-X Universal 3' Gene Expression v4 assay. However, this is not officially supported as it has not been validated by 10x Genomics. Protocol modifications would be required, as outlined below. Assay performance cannot be guaranteed. For GEM-X CRISPR screening applications, we highly recommend utilizing the fully validated and supported 5’ CRISPR workflow: Universal GEM-X Single Cell 5' v3 Gene Expression with Feature Barcoding technology for CRISPR Screening.
sgRNA design
To enable capture of single-guide RNAs (sgRNAs) in the GEM-X Universal 3' Gene Expression v4 assay, the sgRNAs should be designed to contain Capture Sequence 1.
Note that this differs from the previous Next GEM 3’ v3.1 assay version, which enables capture of sgRNAs that contain either Capture Sequence 1 or Capture Sequence 2. The gel beads in the GEM-X Universal 3' Gene Expression v4 assay differ from the gel beads in the Next GEM 3’ v3.1 assay and do not contain Capture Sequence 2.
For further details regarding gel bead oligos, refer to the Appendix of the following User Guides:
- Next GEM 3’ v3.1: Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for CRISPR Screening
- GEM-X 3’ v4: Chromium GEM-X Single Cell 3' v4 Gene Expression with Feature Barcoding technology for Cell Surface Protein User Guide.
cDNA amplification
To enable amplification of sgRNA-derived fragments in the GEM-X Universal 3' Gene Expression v4 assay, a Read 1N forward primer and TSO reverse primer would be required during the cDNA Amplification step. Untested and unsupported workarounds would be to purchase custom primers from a third party vendor, or to use Feature cDNA Primers 1 (PN-2000096) from the Next GEM 3’ Feature Barcode Kit (PN-1000262).
The oligonucleotide sequences of Feature cDNA Primers 1 (PN-2000096) can be found in the Appendix of the following Next GEM User Guide:
Please note that the primer concentrations in 10x Genomics assays are proprietary.
CRISPR library generation
To enable CRISPR library generation in the GEM-X Universal 3' Gene Expression v4 assay, primers would be required for the Feature PCR step prior to the Sample Index PCR. The Chromium GEM-X Single Cell 3’ Feature Barcode Kit v4 (PN-1000702) does NOT contain the appropriate primers. Untested and unsupported workarounds would be to purchase custom primers from a third party vendor, or use Feature SI Primers 3 (PN-2000263) from the Next GEM 3’ Feature Barcode Kit (PN-1000262). After the Feature PCR step, the Dual Index Kit NT (PN-1000242/PN-3000483) would be required for the Sample Index PCR step.
The oligonucleotide sequences of Next GEM Feature SI Primers 3 (PN-2000263) can be found in the Appendix of the following User Guide:
Please note that the primer concentrations in 10x Genomics assays are proprietary.
Data analysis
Cell Ranger v9.0.1 and above can be used to analyze GEM-X 3’ v4 CRISPR Guide datasets. However, please note that this is not officially supported analysis and performance is not guaranteed. Additionally, a Gene Expression library is required for CRISPR Guide Capture analysis.
For details on supported library combinations, refer to
Libraries Supported by Cell Ranger - Official 10x Genomics Support
Related Article: Troubleshooting CRISPR issues on low fraction of guide reads and high fraction of unrecognized protospacers – 10X Genomics
Products: Universal 3' Gene Expression