1. What do you mean by “loss-of-function” and “loss-of-function screens”?
“Loss-of-function” refers to the loss of gene function—the disruption of a genetic element so that it no longer works. The phenotypic changes that occur when a gene is disrupted can suggest the function or role of that gene.
While there are several ways to inactivate a specific gene, only two current technologies—RNAi and CRISPR—provide convenient approaches to easily target and disrupt any gene with high efficiency. The two technologies do this differently. RNAi interferes with the gene’s transcript by inducing its rapid degradation, in which it “knocks down” the gene. CRISPR, on the other hand, induces a break in the DNA sequence for the gene which, when incorrectly repaired by the cells, “knocks out” the genomic template for the transcript. So, to disrupt a gene with either of these approaches, the only information required is the sequence of the target gene. Using this information, it is possible to design a short RNA sequence that will effectively interrupt expression of the gene. So, it is easy to design and make effectors to deactivate any gene just using information found in genetic databases. As a result, these technologies provide a practical way to target and deactivate thousands of genes to look for ones that are responsible for a certain phenotype. As such, they enable large-scale loss-of-function screening for genes involved in various biological responses.
- What is the major difference between shRNA screening and CRISPR screening? What is the advantage of CRISPR knockout constructs comparing to shRNA knockdown constructs?
As mentioned in the answer above, CRISPR and RNAi interrupt gene function using two different mechanisms. CRISPR technology allows gene knockout at the DNA level. To do this, though, it requires the introduction of an exogenous nuclease (Cas9) into the cells to cleave the targeted DNA. However, the result of the knockout is that the correct transcript for the gene is no longer expressed and a functional protein is not produced at all. Thus, CRISPR can induce a complete elimination of the protein for the target gene in the cell—a total inactivation of the gene. This process can take some time since it relies on the cell incorrectly repairing the target gene. To knock out a gene in a majority of cells across a population requires several days to a week. Also, once the gene is knocked out, the process is not reversible.
RNAi, however, induces degradation of the transcript for the gene using an endogenous pathway in the cells that modules mRNA expression. shRNA targets and reduces the level of the target gene’s transcript so that very little of the protein can be produced. The effect occurs relatively quickly, within a day or two. This knockdown of the transcript is reversible. If the shRNA is removed, then the transcript levels rise again and expression returns to normal.
Depending on the application, either approach may have advantages or disadvantages for individual targeted gene disruption. CRISPR is permanent and complete, so if you are interested in making a cell line where a gene is completely eliminated, CRISPR may be the best option. RNAi is reversible and still leaves some low level of expression of the gene. So, if you are interested in inducibly knocking down a gene’s expression, it may be more useful. Also, if the complete knockout of a target gene is highly toxic to cells, and shRNA knockdown may enable you to leave enough of the gene expressed so as to keep the cells viable but still see some effect on their proliferation.
For genome-wide scale screening with pooled effector libraries, both technologies are suitable. CRISPR, when introduced into cells with an optimized system, effectively knocks out targeted genes in the majority of the cells throughout a population in a week or two. The RNAi interference effect occurs more quickly, in just a couple of days. Thus, there are some differences in the screening approach using the two technologies.
RNAi-based screens have been used for a number of years with proven results. Conversely, the problems and pitfalls of RNAi have been reasonably well characterized. In particular, off target effects occur more often with shRNA mostly because some shRNA can mimic miRNA effectors.
CRISPR, as a newer approach, seems to also provide robust results and does not suffer from the same type of off targeting that shRNA does. However, it is clear off target effects can occur with CRISPR sgRNA and groups are looking into the nature of these. Also, since it is a relatively new approach, there may be other unknown issues that arise that can confound screening results.
From our work, we see that parallel screens with both technologies produce robust and largely overlapping results. Of course, there is not a complete match with regard to the hits generated from each screen on the same cell lines with libraries targeting the same set of genes. It may be that, as more data is generated, one or the other approach may be more suitable for uncovering certain types of genetic mechanisms or identifying particular classes of genes.
- What technology has been shown to be more reliable?
With respect to genetic screens, at this point, I would have to say that there isn’t enough data to answer the question. “Reliable” is a broad term, so it may be that one approach is more reliable than the other for certain applications or types of screens. Given over ten years of screening studies by our group, I would say that RNAi has been more proven and is better understood. However, CRISPR seems to have some promising characteristics that may make it generally a better choice for these types of screens in the long run. It’s too early to say for sure though.
- Could you please comment on the off-target effects that are expected if using the CRISPR screening system? How do you design your sgRNA to mitigate the risk of off-target effects?
I commented a bit on the off target effects above. CRISPR off target effects are not yet really well understood. On the other hand, shRNA off target effects are well known and any screen needs to account for these.
With both approaches, however, Cellecta believes the best way to address off target effects in screens is with empirical results from the screen. This means that the libraries should have appropriate controls and, most importantly, include multiple shRNA or sgRNA to each gene target. Our standard genome-wide libraries have 8 shRNA/sgRNA to each gene, and our custom libraries can have as many as the customer wants—usually 10-15.
With multiple effectors to each gene, an investigator does not have rely on the results one or two shRNA or sgRNA to call hits. They can see the results across a range of effectors to the same target, use better statistics, and generate more reliable results.
- Is CRISPR efficiency dependent on the exon it targets? Can it target more than one exon in a given gene?
CRISPR sgRNA are designed to specifically target a 20 base sequence. It is unlikely this would target two regions in the same gene unless they both have the same sequence. It is possible to introduce two CRISPR sgRNA sequences into the same cell to target two regions of the same gene.
The 20-base target sequence for CRISPR can be located anywhere in the gene that is followed by an appropriate Protospacer Adjacent Motif (PAM)—a 3-base sequence motif directly following the guide target site. However, since knockout of the gene with CRISPR relies on mis-repair of the genomic DNA sequence so that it produces a faulty/nonsense transcript, the thinking is that targeting the earlier exons in the gene will generally be more effective. For any specific gene, this may depend on the structure. In designing libraries, however, we do focus on designing sgRNA to exons corresponding to regions in the first half of the transcript.
- If the MOI ratio is too high (CELL: LIBRARY COMPLEXITY), is it possible that one cell can be hit by multiple CRISPR or shRNA?
In setting up a screen, we normally recommend to infect 40-50% of the plated cells with the pooled library and to ensure that enough cells are infected so that, on average, each sgRNA or shRNA goes into 200 cells (infect at 200-fold the complexity of the library). With this many cells at this multiplicity of infection (MOI), some fraction of the cells certainly pick up more than one virus—and therefore express more than one effector.
With any screen, though, since the purpose of the screen is to find the few effectors that have a direct effect on the response, most of the effectors will not affect the phenotype. For example, with a dropout viability screen, most effectors will not be toxic. For a rescue screen where you are looking for effectors that confer resistance to added factor, most of the effectors will not save the cells.
Given this situation, then, one sgRNA or shRNA goes into 200 cells. Of these, maybe 40 cells also pick up a second sgRNA or shRNA, and possibly 5-10 with a third. However, the second (and third) sgRNAs or shRNAs in each of these 40 cells will all be different. Most of these secondary effectors will not have any effect. If one or two do have an effect, then it is only 1 or 2 out of 200—a couple percent at most. There is not enough overlap to significantly confound the results.
- Do you use a lentiviral-based system for CRISPR targeting the GFP? Or just plasmids?
We use a lentiviral-based system for almost all our work, including the GFP transductions.
- I am interested in learning more about the shRNA constructs
Our scientific group has been working with shRNA screens since 2003—4 years before our company was started in 2007. We have information on our website about both our shRNA and sgRNA libraries (both custom and premade genome-wide pooled), custom constructs, and cell lines. We’re always happy to answer any questions you might have about either CRISPR or RNAi screens, libraries, or related products and services.