**Question**: What is MOI and how do I assess it?

**Answer**: Multiplicity of infection (MOI) refers to the number of viral particle(s) present relative to host cell(s). For example, if one million viral particles are added to one million cells, the MOI is 1.0. If 100,000 viral particles are added to one million host cells, the MOI is 0.1.

That being said, infecting at an MOI of 1.0 does **not** guarantee that all host cells will become infected/transduced.

To estimate the number of cells transduced, the Poisson distribution equation is needed:

*P(k) = e ^{-λ}λ^{k}/k!*

*is the MOI**k is the number of viral particles and takes values 0, 1, 2, etc.*

In this equation, *P(k)* is the fraction of cells infected by *k* viral particles. The equation can be simplified to calculate the fraction of uninfected cells (*k=0*), cells with a single infection (*k=1*), and cells with multiple infection (*k>1*):

*P(0) = e*^{-}^{λ}

*P(1) = **λ**e ^{-}*

^{λ}*P(>1) = 1-e ^{-}*

^{λ}*(*

*λ*

*+1)*

For example:

If we infect one million cells at a MOI of 1:

*P(0) = e*^{-}^{1}*= 0.37 = 37% of cells are not transduced*

*P(1) = 1**e ^{-}*

^{1}*= 0.37 = 37% of cells have 1 viral particle*

*P(>1) = 1-e ^{-}*

^{1}*(*

*1*

*+1)= 0.26 = 26% of cells have >1 viral particles*

An important assumption to consider here is that this equation relies on all cells in a culture being identical in their ability to be infected. This can become challenging if the cells are not stably expressing Cas9 or the cell population is highly heterogenous with differences in susceptibility to infection.

To calculate MOI, the viral titer of the cells needs to be determined.

As shown below, in this particular example the 'optimal MOI' resulted in the following:

- ~50% of the cells containing a single guide
- ~10% of the cells containing two guides
- ~40% of the cells lacking the presence of a guide

The cells containing two (or more guides) can be identified during downstream analysis and can be excluded, if desired, or can, depending on the application be analyzed to examine the impact of combinatorial perturbations on the transcriptome.

And finally, as not all cells will contain a one (or more) guides, we recommend enriching the transduced cell population to isolate only these cells (as only these cells provide useful information for the CRISPR Screening Assay). Please see the following article for tips: Do you recommend enriching for transduced cells?

Products: Single Cell gene Expression