Question: How do I design custom probes for CRISPR Guide Capture for use with the Single Cell Gene Expression Flex Assay?
Answer: To design custom probes for CRISPR guide capture for use with the Single Cell Gene Expression Flex assay, the guide scaffold and sgRNA sequences must be known in advance. Custom probe design for CRISPR guide capture should follow the probe design principles provided in Custom Probe Technical Note (CG000621) with the following modifications:
- LHS probe has a TruSeq R2, a Probe Barcode and a 25-bp sequence that is the reverse-complement of the guide scaffold.
- TruSeq handle allows for separate indexing of CRISPR library.
- CRISPR LHS Probe Barcode sequences are different from those used in the WTA probes. Please see Table 2, below for specific probe barcode sequences.
- RHS probe contains a 5’ phosphate (/5Phos/) followed by TAAAC, a 20-bp sequence that is the reverse-complement of the protospacer being targeted, and partial capture sequence 1 (pCS1).
- Note: Guide scaffold sequences vary between vectors. The LHS and RHS probe sequence should be changed to match the guide scaffold used.
Table 1: CRISPR Guide Capture Custom Probe design for Single Cell Gene Expression Flex. The LHS probe has a TruSeq R2, a Probe Barcode, and a 25-bp sequence that is the reverse-complement of the guide scaffold. RHS probe contains a 5’ phosphate (/5Phos/) followed by TAAAC, a 20-bp sequence that is the reverse-complement of the protospacer being targeted, and pCS1.
| CRISPR LHS Probe Barcodes | |
| Barcode | Sequence |
| CR001 | ACCGTCCA |
| CR002 | CAACCTGT |
| CR003 | GTTAGATG |
| CR004 | TGGTAGAC |
| CR005 | ACTGTGCG |
| CR006 | CGACAAGC |
| CR007 | TTGTCCAT |
| CR008 | AATCAGGC |
| CR009 | CGGAGTCT |
| CR010 | AGCTGAAG |
| CR011 | CAGCTCCA |
| CR012 | TTAGAGGC |
| CR013 | CCATAACT |
| CR014 | TTTCGCGG |
| CR015 | AGACCGCT |
| CR016 | GCTTATTC |
Table 2: Unique Probe Barcode sequences for integrating in the LHS probe for CRISPR multiplexing experiments using the Single Cell Gene Expression Flex assay.
The first four Probe Barcodes (CR001-CR004) are base-balanced and are recommended when performing a 4-plex experiment. Note that for Singleplex experiments, the Probe Barcode may be omitted.
Additional Flex + CRISPR Resources:
- For details on workflow modifications required when performing CRISPR screening with the Flex assay please see: What workflow modifications are required when performing CRISPR Screening with the Single Cell Gene Expression Flex assay?
- For details on how to pool custom probe pools for a Flex + CRISPR experiment please see: How do I pool custom probes for a Single Cell Gene Expression Flex + CRISPR experiment?
- For details on how to analyze CRISPR screening and Flex data please see: How do I analyze CRISPR screening + Single Cell Gene Expression Flex data in Cell Ranger?
While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. The additional guidance and resources that have been provided in this article are intended to help enable these experiments and improve customer success.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex