FAQ

How do I clone guide RNA oligos into the provided, barcoded and pre-digested plasmids?

A detailed protocol alongside a full list of reagents needed for arrayed guide RNA cloning is provided in the cloning protocol.

How do I design guide RNAs?

We have previously developed software for RfxCas13d gRNA design described here: (Wessels, Méndez-Mancilla et al. 2020). While our (like any other) prediction software, generally helps to find high-efficacy gRNAs, there are no garanties. Therefore, it may be useful to evaluate gRNA activity before running a CaRPool-seq experiment. In our manuscript, we ran a pooled CRISPR screen with single gRNAs to select active gRNAs.

Do you provide a template for Cas13 guide RNA ordering?

Yes, our template sheet can be downloaded here. Note: Please, first install a required Microsoft Excel plugin for reverse complementing of sequences.

Where can i find the plasmid map for pLentiRNAGuide_003 provided with the kit?

You find the plasmid map on Addgene.

Which plasmids are not included in the starter kit?

You will need to purchase plasmids for virus production, and for the generation of a stable RfxCas13d expressing cell line from addgene (pMD2.G, psPAX2, pLentiRNACRISPR_007).

How to generate a stable RfxCas13d expressing cell line?

A protocol for stable cell line generation is provided in the protocols section. Note, generating and verifying monoclonal Cas13d cell lines may take 4-6 weeks. Helpful tips for ensuring monoclonality are included in the protocol. Importantly, uneven Cas13 expression may lead to differences in gRNA efficacy, non-specific off-target activity and bcgRNA detection. Cells not expressing Cas13 will fail to process CRISPR arrays and stabilize bcgRNAs, leading to profilling of cells without perturbation and bcgRNA assignments. These cells resemble unperturbed cells and usually cluster with non-targeting control cells in CaRPool-seq experiments.

Which positive control experiments can I perform to ensure my system is working?

coming soon

How do I analyze the CaRPool-seq data?

coming soon

Can I use other cell lines than the suggested DOX-inducible monoclonal RfxCas13d expressing cell line?

Yes, other Cas13d systems can be used. (Examples include: Wei et al. using piggybac. Noviello et al. using degron systems. Legnini et al. using optogenetics.). Importantly, the provided guide RNA plasmids match RfxCas13d protein requirements and may not function for other Cas13 effectors.

Where can I find additional information?

See our main CaRPool-seq manuscript for more information.