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The previous blog post ” Two-Vector CRISPR System Is Better Approach for Knockout Screens” discussed the advantages of expressing the Cas9 nuclease on a vector separate from the one used for the guide RNAs (sgRNA)—a two-vector CRISPR system—when using the system for complex pooled loss-of-function screens.  In addition to being a more economical approach, since constructs with the Cas9 lentivirus vector package poorly, the article also noted that starting a screen with a population of cells already selected and adapted to expressing relatively consistent levels of Cas9 will produce more robust screens.

The reason the two-vector approach gives better screening results is straightforward. The Cas9 construct can be introduced into a small population of cells, then these can be selected strongly to express high levels of the nuclease so that, when the sgRNA are introduced, gene knockout occurs more quickly.

Obviously, the fact that cells pretransduced with a lentiviral Cas9 construct have time to initiate expression of the Cas9 nuclease before the sgRNAs are introduced enables the knockout reaction to ramp up sooner when the library is introduced. Also, with the two-vector system, the selection for the Cas9 relies on a different antibiotic (e.g., hygromycin) than the sgRNA library (e.g., puromycin), so cells can be stringently selected for the presences of the construct which will generally result in higher levels of the Cas9 protein. However, while both of the pretransduction and stringent selection help produce faster knockout across more cells, the biggest impact on knockout rates with the two-vector approach may be easily overlooked.

The real advantage of the two-vector system for CRISPR knockout screens stems from the characteristic that cells can take up and integrate more than one copy of a lentivirus. As a result, a high multiplicity of infection (MOI), for example 2-4 fold more virus than cells, can be used to produce a population where most of the cells have multiple copies of the Cas9. This approach is not an option when using a system where the Cas9 gene is expressed on the same construct as the sgRNA.

The data below demonstrate the significant enhancement in CRISPR knockout in cells expressing multiple Cas9 genes.  You can see the cells expressing 3 copies on average of Cas9 show a 5-fold improvement in knockout of the target GFP gene after 9 days, as compared with cells containing, on average, just one copy of Cas9. An antibody assay also showed that cells with the higher copy number of Cas9 were, in fact, expressing more Cas9 protein, as expected.

In a screen with a single Cas9-sgRNA vector, most cells will only pick up one Cas9 gene. The Cas9 on the same lentiviral construct as the sgRNA sequence enforces a one-to-one relationship where each cells only gets one Cas9 for each sgRNA. Since, when screening complex heterogeneous pooled libraries, it’s necessary to use a low MOI with approximately twice the number of cells as compared to viral particles, most cells will only receive one copy of each. The only cells that will get more than on Cas9 gene are ones that also pick up more than one sgRNA—an event to be avoided. As a result, the proportion of Cas9 to sgRNA in cells in a one-vector screen is not optimal. Putting Cas9 on a separate construct, though, avoids the problem and enables a researcher to modulate the amount of Cas9 to sgRNA.

Cas9 copy number influences CRISPR knockout percentage

Figure legend: Cells expressing GFP were transduced with Cas9 at high MOI producing a population with approximately 3 Cas9 per cell on average, or a low MOI generating a population of approximately 1 cell on average. After selecting the Cas9 transductants with hygromycin, each population of cells were then transduced with the same sgRNA to GFP and grown for 9 more days in media containing puromycin. Cells with a higher number of integrated copies of Cas9 have 5-fold fewer GFP-positive cells.