CRISPRi Frequently Asked Questions

How does CRISPRi differ from RNAi? 

The primary difference is that RNAi silences gene function at the mRNA level by preventing translation of mRNAs into proteins, while CRISPRi silences gene function at the level of the genome by recruiting DNA methylation and repressive histone markers effectively blocking transcription.

Do I need to use lentivirus, or can I use plasmid DNA or RNA?

Because knockdown detection requires waiting for natural decay of mRNAs and proteins after inhibition of transcription, it important to have stable expression of the CRISPRi components for at least 6 days. For this reason, lentivirus is the preferred choice.

How many genes can I study at one time?

From individual or combinations of single guides, to off-the-shelf and custom pools, up to whole genome resolution with the Sigma-Aldrich® CRISPRi pooled library.

How many sequencing reads do I need?

We recommend maintaining 300-500x coverage per guide throughout your screen and during NGS.

How many gRNAs per gene do I need?

At least two guides per gene, and up to 10 guides for increased sensitivity. The Sigma-Aldrich® CRISPRi libraries and pools provide 10 guides per gene.

What QC is done on the pools? 

Pooled QC is done by next generation sequencing for representation and distribution.

What MOI should I use?

We recommend using an MOI of <0.7 for making helper cell lines and an MOI of <0.2 for pooled guide libraries, to ensure that no cell receives more than one guide RNA.  This is very important for screens that will be analyzed by NGS.

How do you determine functional titer?   

As different cell lines may take up virus at different rates, functional titer must be determined in your cells. Detailed protocols for determining functional titer based on colony forming assays and FACS analysis can be found [HERE].

What about positive and negative controls? Do you have recommendations?

We have pre-designed negative and positive controls that will work in most cell lines; however, you may choose to design and use your own positive control guides that have been empirically validated.

How many gRNAs per cell?

A screen requiring NGS deconvolution should have no more than 1 guide per cell. Single-cell RNAseq with guide capture using 10x Genomics Feature Barcoding can detect multiple guides within each cell.

How do I follow up with my candidate genes?

You have several options to validate your hits, most commonly: Clones with alternate technologies: RNAi, ORF, CRISPRa. 10x Genomics scRNAseq, qRT-PCR, immunocytochemistry, and protein function assays.

For additional details and protocols please visit

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