The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a prokaryotic immune response that bacteria use to fight off invading viruses, has been adapted over the last few years for genetic engineering. The basic system consists of a DNA nuclease and RNA molecules that target the nuclease specifically to the foreign DNA from the virus.
A few years ago, the CRISPR system from S. pyogenes was modified so that a single RNA molecule could be used to both target a DNA sequence and recruit the S. pyogenes Cas9 (spCas9) nuclease to cut the DNA at the target site. This single-guide RNA (sgRNA) molecule combined with the spCas9 protein rapidly became the system of choice for gene knockout. However, the utility of the CRISPR system extends well beyond targeted gene knockout.
In an effort to take advantage of the highly-specific genomic DNA targeting ability of the CRISPR system for other applications, groups have engineered the Cas9 protein to alter its activity. The first examples of this came from the Weissman lab as UCSF, where they modified the Cas9 protein to eliminate the nuclease activity and produce a deactivated Cas9 (dCas9). However, the dCas9 protein still bound the sgRNA (Chen, et al., Cell 2013) and the complex could be targeted to essentially any location in the genome.
To this dCas9 protein, researchers have fused a gene repressor protein (i.e., the KRAB protein) to make a complex that could inhibit gene expression (CRISPRi), and also a gene transcription activator (i.e., VP16) to make a second complex that would induce gene expression (CRISPRa) (Gilbert et al., 2013) . By using these engineered dCas9 variants in conjunction with sgRNA targeting the promoter region of a gene, transcription of that target gene can be shut down or up-regulated. Also, since the effect does not alter the underlying DNA structure, like a CRISPR knockout does, the induction or repression is reversible. Using these systems, broad based screens that assess the effects of activation or repression of thousands of different genes on cell growth or other screenable phenotypes can be done using pools of sgRNA constructs that target the promoter or transcript start regions of genes. In particular, the CRISPRa system, enables gain-of-function screens to identify genes that are activated to induce a particular phenotype.
Other labs, including our group at Cellecta, have made versions of CRISPRa by fusing different transcription regulators to the dCas9 protein. Since CRISPRi works by blocking the transcription complex to shut down gene transcription, it is more robust than CRISPRa which requires the recruitment of factors or chromatin changes to increase gene transcription levels. Depending on the basal level of expression and regulatory elements controlling transcription of the target gene, activation levels for different genes will vary and/or require different combinations of activators.
Different variations of the CRISPRa dCas9 activation complex.
(A) The original dCas9-VP16 (Gilbert et al., 2013)
(B) dCas9 fused to the E1A-associated protein p300 histone acetyltransferase (HAT) (Klann, et al., Nat Biotechnol. 2017)
(C) The elements of the dCas9-VPR systems used by Chavez, et al., Nature Methods 2015 and redesigned by Cellecta so the whole complex is expressed from a single lentiviral construct.
(D) The Cas9 activation complex developed by Konermann, et al., 2015. This variant requires a modification of the sgRNA structure to incorporate an RNA aptamer stem-look to bind the MS2 element.
(E) Cellecta’s Cas9-activator which incorporates the transcription activation elements from the SAM complex, without the MS2, and is all expressed from a single lentiviral construct to enable it to be more easily used for genetic CRISPRa screens.
Since our group is primarily interested in genetic screening with pooled lentiviral libraries, we focused on dCas9 fusions that function with standard sgRNA designs. As a result, we decided to avoid the SAM activator which requires a different sgRNA structure than the standard (see figure). Instead, we created a dCas9 fusion that included the main activation elements from the SAM construct but organized in such a way that they could be expressed as a single protein. We dubbed this the dCas9-VPH since it is similar to dCas9-VPR (which we also modified so that it would express from a single lentiviral construct). Finally, we tested activation of several endogenous genes using it with sgRNA directed to their promoter regions.
While the data showed clearly that dCas9-VPH significantly activated expression for a number of endogenous genes, the real question was how well would it work with pooled sgRNAs in a genetic screen. For this analysis, we made a few variations of sgRNA designs and ran these in screens with both the dCas9-KRAB inhibitor and dCas9-VPH activator.
For these CRISPRi and CRISPRa test screens, we used the sgRNA designs from the publication from Horlbeck et al., (eLife, 2016). This publication indicated that it is best to target sgRNA for CRISPRi and CRISPRa to slightly different locations in the 5’ region of a gene for optimal repression or activation. We were also interested to test this finding since there would be a benefit to being able to use the same sgRNA library for both CRISPRa and CRISPRi screens. To test this possibility, we designed a third set of sgRNAs that we thought might be suitable for use with both CRISPRi and CRISPRa systems.
Results from the genetic screens with three small sgRNA libraries, each targeting the same set of ca. 2000 genes with 3 sgRNA each. The set of sgRNAs for two of the libraries were designed using the published target sequences in Horlbeck et al., (eLife 2016). The sgRNAs in the third library were designed based on a novel strategy devised by Cellecta. Each pooled lentiviral sgRNA library was transduced into HEK293 cells expressing either dCas9-KRAB or dCas9-VPH. Cells were grown for a couple of weeks, then genomic DNA was isolated and the depletion or enrichment of specific sgRNAs was assessed.
Top Panel: The number of depleted sgRNA identified in each of the three library screens in cells expressing the dCas9-KRAB repressor. It is clear that only the sgRNA with designs based on the Horlbeck eLife publication worked in this CRISPRi screen.
Lower Panel: Screens with the three sgRNA libraries in cells with the dCas9-VPH activator. These screens were run with two concentrations of the MEK inhibitor U0126, and with no additional treatment. The chart shows the number of genes with sgRNAs either enriched or depleted under each of the screening conditions with each library.
As it turned out, our “dual function CRISPRi/CRISPRa” guide designs did not work well, especially for the CRISPRi screen with dCas9-KRAB. In fact, the sgRNA designs optimized for the dCas9-VPH activation did not work at all for the dCas9-KRAB inhibition screen either. Only the guides designed specifically for CRISPRi, based on the Horlbeck publication, were effective at inhibiting gene expression with the KRAB variant. In general, these guides target sites farther downstream of the promoter and into the first exon of the gene.
For CRISPRa, the story was more complicated. We screened each of the 3 libraries that had the 3 different versions of sgRNA in cells expressing the dCas9-VPH activator under 3 conditions:
Following these screens, we looked for guides to genes that were either significantly enriched or depleted. This would indicate that upregulation of their gene targets was either lethal to the cells or increased their viability. We found a handful of guides in each of the screens that were significantly enriched or depleted, but the results tended to be noisy across the screens.
Interestingly, without any treatment, more genes were identified with the library that had the Cellecta CRISPRi/CRISPRa guide designs. However, in response to treatment, more genes were identified with the libraries using the guide designs from the Horlbeck publication. Since this was a relatively small experiment and we did not do further follow up analysis, the mechanism by which the sgRNA altered the growth of the cells is unclear. Although the dCas9-VPH activator should up-regulate gene expression, it is possible that the complex may not work well in combination with the sgRNA for some genes. However, the complex may still bind to the target location and act to inhibit expression of a gene instead by simply blocking expression. Thus, while the screen provides an interesting list of candidates for analysis, CRISPRa screening is more complex than a typical gene disruption or loss-of-function screen using CRISPRi or CRISPR knockout. Post-screen characterization of individual constructs corresponding to the hits identified in a CRISPRa screen is required to understand how the target gene might be involved in the phenotype of interest.
What is clear from the CRISPRa screens with the different guide designs, however, is that the Horlbeck CRISPRa designs worked at least as well if not better than the others in all three screens. Given this, and the published evidence for these designs, we concluded the best approach would be to use the published CRISPRa designs for our libraries.
Of course, the utility of using a dCas9 variant in conjunction with sgRNA to target an activity to a specific site extends beyond gene transcription regulation. Other types of proteins or peptides with functional domains can be fused or otherwise bound to the dCas9 to produce other changes in the chromatin or DNA at the targeted locations.
For example, a few groups have developed dCas9 variants fused with base-modifying enzymes to make a complex to introduce single-base point mutations or broader substitution mutations around a target site. Other groups have used dCas9 with histone deacetylases and base methylases to alter the chromatin structure around the targeted gene.
There are many other examples in the literature of novel variations of the CRISPR system that expand its use. Since the initial use of CRISPR as a research tool only started in 2013, the technology is still very much a new trend. As it continues to develop, it is easy to see that this sort of fused-function CRISPR DNA targeting system provides a very flexible and potent platform. It seems clear that in the next several years we will see an explosion in the applications of CRISPR to address a diverse range of questions in biological research, as well as in diagnostic and therapeutic applications. Our goal is to try to keep up with this rapidly developing field and continue to optimize and make available to life science investigators useful developments that can help elucidate the genes regulating important biological responses.
