In solving the mystery of gene function, there is no more important clue than the phenotype of inactivating the gene of interest. With a plethora of methods available, researchers must first determine what approach is best for their specific scientific questions and experimental systems.
For over a decade, RNA-interference-based methods of gene knockdown (i.e. RNAi & shRNA) have provided a wealth of insight into gene function, but in recent years the advent of CRISPR- and TALEN-based methods now allow genome editing to be used to quickly and efficiently test the effect of gene knockouts. Here, we review the advantages and disadvantages of these approaches, and describe some experimental situations in which one approach is better than another, focusing primarily on CRISPR/Cas9 and shRNA. For a discussion of the relative advantages of CRISPR/Cas9 versus TALEN, see our prior Newsletter on this topic (http://www.cyagen.com/us/en/community/newsletters/issue-1.html).
shRNA Knockdown:In this method, hairpin-forming RNAs are expressed from vectors introduced into cells. These RNAs are cleaved into short (20-25nt) double-stranded RNA molecules, which are then processed and incorporated into the RNA-induced silencing complex (RISC) which then complexes with target mRNAs to mediate mRNA degradation or translation inhibition. The end result is down-regulation of gene expression, without any change to the gene itself. Invariably, some fraction of functional mRNA remains in the cell, so gene function is not completely eliminated.
- High transduction efficiency vector systems such as lentiviral shRNA vectors can be used to treat populations of cells, and for many studies, data can be acquired directly, without the need for cloning.
- Due to efficient transduction and high shRNA expression from many shRNA vector systems, experiments with shRNA vectors are generally scalable and highly consistent when repeated.
- Some vector systems, such as regular plasmid shRNA vectors used in transient transfections or piggyBac-based shRNA vectors, can be removed from cells, making the knockdown reversible.
- Incomplete loss of gene function due to remaining functional mRNA.
- Some vector systems, such as regular plasmid transfection, have transient effects, rather than mediating permanent knockdown.
CRISPR Knockout:In this method, a guide RNA (gRNA) homologous to an 18-22nt target sequences in the genome is used to direct the coexpressed Cas9 nuclease to the target site. Hybridization of the gRNA localizes Cas9, which then cuts the target site in the genome, generating double-strand breaks (DSBs). Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) generates stochastic errors at the cut site. This usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout.
- Changes to the genome introduced by CRISPR/Cas9 are permanent and stable.
- Some clones should have a complete loss of gene function due to frameshifts or premature stop codons in the open reading frame of all copies of the gene.
- Different gRNAs can target Cas9 to different sites on the gene, potentially generating a variety of informative mutations, such as truncations.
- The mutations introduced are stochastic in the cell population, so individual clones need to be isolated and evaluated (e.g. by PCR or sequencing) to determine the nature of mutations in each clone.
- In many cases, cells will have no loss-of-function mutations or heterozygous mutations. In some cases this could be an advantage, providing useful data for interpreting the knockout phenotype.
Cyagen Biosciences recently introduced VectorBuilder, an award-winning online platform for custom design and cloning of DNA vectors for all of your experimental needs, including a full line of shRNA and CRISPR vectors, plus many other vector systems such as lentivirus, adenovirus, AAV, piggyBac, regular plasmid and more! Cyagen Biosciences also provides custom virus packaging and mouse/rat model generation, including transgenics, knockouts and knockins.