CRISPR/Cas9 for Gene Knockout
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are DNA loci containing short repetitions of base sequences that are present within prokaryotes and function as a primitive immune system, cleaving foreign DNA (from invading viruses). The CRISPR system has been used as a gene editing tool in mammalian systems. When paired with the Cas9 nuclease, genomic DNA is cleaved in a site-specific manner and thus gene expression knocked out. Guide RNAs (gRNA or sgRNA) are designed to a specific genomic sequence, thus directing Cas9 to knock out the gene.
sgRNAs and Cas9 can be cloned into plasmids and then introduced into mammalian cells by transfection. For permanent expression (and hence permanent knockout), sgRNAs and Cas9 can be cloned into lentiviral vectors, packaged into viral particles, and transduced into target cells. Both the sgRNA and Cas9 are stably integrated into the host cell genome, and can be passed along to daughter cells when the cells divide. This provides for permanent expression of shRNA and Cas9, thus permanently knocking out target gene expression.
The rate and level of knockout is dependent upon the level of expression of Cas9. Cas9 levels can vary depending upon:
- Where Cas9 has integrated into the genome (if it has integrated into a transcriptionally inactive region, there will be less expression)
- The promoter used to express Cas9 (promoters are more or less effective depending on the cell type)
- The copy number of integrated Cas9
The CRISPR/Cas9 system can be used to knock out individual genes. In this case, Cellecta has a Single-Vector system expressing both sgRNA and Cas9, such that you only need to transduce your target cells one time. Cells can then be selected with puromycin to enrich for cells expressing the construct and having the desired gene knocked out.
The CRISPR/Cas9 system can be used for knocking out gene expression for loss-of-function screening experiments by using Cellecta’s 2-Vector CRISPR/Cas9 System. Cells are first transduced with a lentivirus expressing the Cas9 nuclease and selected to ensure a high level of expression of Cas9. Cells are then transduced with a lentivirus expressing a pooled sgRNA library, at an MOI such that each cell expresses no more than one sgRNA. This method helps to reduce noise for knockout screens.