CICA Tech Sharing: CaRPool-seq

On the CaRPool-seq website you’ll find additional information on protocols, reagents, sequencing, computational tools, and FAQ.

Each CaRPool-seq Starter Kit contains the following reagents:

• 96 pre-barcoded and ready-to-ligate (BsmBI/Esp3I-digested) guide RNA vectors (Addgene #192505; Per well: 10 µl of 20ng/µl; store at -20°C/4°C).

• 8 x validated guide RNA DNA-oligo mixes (Store at -20°C/4°C; 100 µM each).

  1 x 10 µl non-targeting control oligos

  3x 10 µl single target oligos

  3 x 10 µl dual target oligos

  1 x 10 µl triple target oligos

• 2 x bcgRNA library generation oligos (Store at -20°C/4°C).

  20 µl of bcgRNA cDNA amplification additive primer (0.4µM)

  20 µl of Feature SI Primers 2 (10µM)

  20 µl of RPIx bcgRNA library indexing primer (10µM)

  20 µl of P5 primer (10µM)

  20 µl of P7 primer (10µM)

• 96 pre-barcoded and ready-to-ligate (Esp3I-digested) guide RNA vectors (Addgene #192505) - Store at -20°C/4°C

  • Per well: 10 µl of 20ng/µl (1 µl/ rxn)

For lentivirus generation of guideRNAs plasmids and CRISPR effector plasmids, the user may want to order the following plasmids from Addgene:

• Dox-inducible RfxCas13d expression plasmid (Addgene #138149)

• psPAX2 packaging plasmid (Addgene #12260)

• pMD2.G packaging plasmid (Addgene #12259)

Protocol

The full protocol is detailed in the CaRPool-seq manuscript with supplementary information located on CaRPool-seq.com.

The starter kit includes materials to generate a CaRPool-seq plasmid library using arrayed ligation cloning for up to 96 constructs, plasmids to generate a Cas13d expressing cell line, and validated guide RNAs to test the Cas13d cell line before performing CaRPool-seq. 

Please note, a successful CaRPool-seq experiment depends on the activity of the guide RNAs. It is not uncommon to see variability between gRNAs for the same target, and it can be useful to first test individual gRNAs for activity before combining multiple gRNAs into single CRISPR array. A good starting point for the selection of guide RNAs can be to use this guide RNA design software.

In 2022, we introduced Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes.

The CaRPool-seq Starter Kit enables researchers to perform combinatorial gene perturbations screens with single-cell RNA-seq (or CITE-seq) readout. As part of the Starter Kit, we provide a set of cloned gRNA to enable users to reproduce the multi-modal CarPool-seq screen presented in Fig. 2 of the manuscript (which includes single, double, and triple perturbations). We also provide a pre-barcoded, and ready-to-ligate library of 96 plasmids for simplified arrayed ligation cloning. Using these reagents, users can easily setup CaRPool-seq screens for up to 96 separate combinatorial perturbations.