CRISPR Knockout Rate Dependent on Cas9 Expression Levels

Cellecta recently developed a one-plasmid CRISPR lentiviral vector that expresses both the sgRNA and Cas9 protein. CRISPR enables convenient knockout of a target gene at the genomic level, as opposed to shRNA which knocks down the level of the RNA transcript for the target gene.

Due to the differences between the shRNA and CRISPR mechanisms, when a plasmid expressing shRNA is introduced into a population of cells, levels of the target gene transcript drop pretty quickly—within a couple days—across most of the cells. However, when cells pick up a CRISPR sgRNA-Cas9 construct, the target genes is disrupted in individual cells throughout the population over a period of days to weeks. In other words, the longer you wait, the more cells there will be with the gene knocked until, eventually, most all the cells with the CRISPR construct lose the functional gene. However, getting to a point where a majority of the cells have the target gene knocked out may take one to two weeks or longer.

A recent experiment we did indicates that the level of Cas9 expression affects the rate at which the target gene is knocked out in a population of cells. In one version of the Cellecta single plasmid CRISPR lentiviral construct, Cas9 expression is driven by the CMV promoter which produces high transcript levels in the HEK293 cells used for the experiment. However, another version of our CRISPR vector uses the UbiC promoter to drive Cas9 expression. Although both promoters are robust in HEK293 cells, expression levels from the UbiC promoter is not as high as those from CMV.

As can be readily seen from the data below, the stably integrated GFP target is lost more rapidly in the cells transduced with the stronger CMV version of the CRISPR construct. The results indicate that higher levels of Cas9 more rapidly knockout the target gene in more cells.

GFP Knockout Cells

The figure shows the percentage of GFP knock-out cells for 4 different GFP-targeting sgRNAs as measured by FACS when Cas9 is expressed from either the UbiC promoter or the CMV promoter. The cells used are a derivative of HEK293 cells containing a stably integrated and constitutively expressed GFP gene. After 9 days, 60%-70% of the cells transduced with constructs expressing Cas9 from the CMV promoter have GFP knocked out for all 4 sgRNAs. However, during the same period with the same sgRNAs, the GFP gene has been knocked out in only 35%-50% of the cells using the construct that expressed Cas9 from the UbiC promoter.

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