Starter kits

We recently 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 gRNAs to enable users to reproduce a subset of 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

Starter kits can be requested from the Center for Integrated Cellular Analysis website: https://www.multimodalintegration.org/techsharing/

The starter kit contains:

1. 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). Barcode sequences can be found here.

2. 8 x validated guide RNA DNA-oligo mixes (Store at -20°C/4°C; 100 µM each). Guide sequences can be found here.

   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

3. 2 x bcgRNA library generation oligos (Store at -20°C/4°C). Primer sequences can be found here.

   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)

Plasmids

Additional CaRPool-seq plasmids are available from AddGene.