By Rebecca Delker, PhD
The bacterial defense system, CRISPR/Cas9, made huge waves in the biomedical community when the seemingly simple protein-RNA complex of Type II CRISPR systems was engineered to target DNA in vitro and in complex eukaryotic genomes. The introduction of double-strand breaks using CRISPR/Cas9 in a targeted fashion opened the portal to highly affordable and efficient site-specific genomic editing in cells derived from yeast to man.
To get a sense of the impact CRISPR technology has had on biological research, one simply needs to run a search of the number of publications containing CRISPR in the title or abstract over the past handful of years; the results practically scream in your face. From 2012, the year of the proof-of-principle experiment demonstrating the utility of engineered Cas9, to 2015, CRISPR publications rose steadily from a mere 138 (in 2012) to >1000 (at the time of this post). Publications more than doubled between the years of 2012 and 2013, as well as between 2013 and 2014. Prior to the use of CRISPR as a technology, when researchers studied the system for the (very cool) role it plays in bacterial defense, publications-per-year consistently fell below 100. In other words, it’s a big deal.
In fact, during my 10 years at the bench I have never witnessed a discovery as transformative as CRISPR/Cas9. Overnight, reverse genetics on organisms whose genomes were not amenable to classical editing techniques became possible. And with the increasing affordability of high-throughput sequencing, manipulation of the genomes of non-model organisms is now feasible. Of course there are imperfections with the technology that require greater understanding to circumvent (specificity, e.g.), but the development of CRISPR as a tool for genomic engineering jolted biological research, fostering advances more accurately measured in leaps rather than steps. These leaps – and those expected to occur in the future – landed the discoverers of CRISPR/Cas9 at the top of the list of predicted recipients of the Nobel Prize in Chemistry; though they didn’t win this year (the award went to researchers of the not-totally-unrelated field of DNA repair), I anticipate that a win lies ahead. The rapid success of CRISPR genome editing has also sparked patent battles and incited public debate over the ethics of applying the technology to human genomes. With all of the media attention, it’s hard not to know about CRISPR.
The transformative nature of CRISPR/Cas9 does not, however, end with genome editing; in fact, an even larger realm of innovation appears when you kill the enzymatic activity of Cas9. No longer able to cut DNA, dead Cas9 (dCas9) becomes an incredibly good DNA-binding protein guided to its target by a programmable RNA molecule (guide RNA, gRNA). If we think of active Cas9 as a way to better understand genes (through deletions and mutations), then dCas9 is the route to get to know the genome a bit better – a particularly enticing mission for those, including myself, invested in the field of Genomics. From high-throughput targeted gene activation and repression screens to epigenome editing, dCas9 is helping scientists probe the genome in ways that weren’t possible before. Here, I put forth some of the best (in my humble opinion) applications, actual and potential, of CRISPR technology that go beyond genome editing.
Cas9 and Functional (Epi)Genomics
For many years the genome was considered as the totality of all genes in a cell; the additional junk DNA found was merely filler between the necessary gene units, stitching together chromosomes. We’ve come a long way since this naiveté, especially in recent years. We understand that the so-called junk DNA contains necessary regulatory information to get the timing and position of gene expression correct; and now, more than ever, we have a greater appreciation for the genome as a complex macromolecule in its own right, participating in gene regulation rather than acting as a passive reservoir of genetic material. The genome, it has been shown, is much more than just its sequence.
The epigenome, consisting of a slew of modifications to the DNA and the histones around which the DNA is wrapped, as well as the 3D organization of the genome in the nucleus, collaborates with DNA binding proteins to accurately interpret sequence information to form a healthy, functional cell. While mutations and/or deletions can be made – more easily, now, with Cas9 – to genomic sequences to test functionality, it is much harder to conduct comparable experiments on the epigenome, especially in a targeted manner. Because of the inability to easily perturb features of the epigenome and observe the consequences, our understanding of it is limited to correlative associations. Distinct histone modifications are associated with active versus inactive genes, for example; but, how these modifications affect or are affected by gene expression changes remains unknown.
Taking advantage of the tight binding properties of dCas9, researchers have begun to use the CRISPR protein as a platform to recruit a variety of functionalities to a genomic region of interest. Thus far, this logic has most commonly been employed to activate and/or repress gene expression through recruitment of dCas9 fused to known transcriptional activator or repressor proteins. Using this technique, scientists have conducted high-throughput screens to study the role of individual – or groups of – genes in specific cellular phenotypes by manipulating the endogenous gene locus. And, through a clever extension of the gRNA to include a hairpin bound by known RNA-binding proteins, the targeted functionality has been successfully transferred from dCas9 to the gRNA, allowing for simultaneous activation and repression of independent genes in the same cell with a single dCas9 master regulator – the beginnings of a simple, yet powerful, synthetic gene circuit.
Though powerful in its ability to decipher gene networks, dCas9-based activation and repression screens are still gene-centric; can this recruitment technique help us better understand the epigenome? The first attempts at addressing this question used dCas9 to target histone acetyltransferase, p300, to catalyze the acetylation of lysine 27 on histone 3 (H3K27) at specific loci. The presence of H3K27 at gene regulatory regions has been known to be strongly associated with active gene expression at the corresponding gene(s), but the direction of the histone modification-gene expression relationship remained in question. Here, Hilton et al. demonstrate that acetylation of regulatory regions distal to gene promoters strongly activates gene expression, demonstrating causality of the modification.
More recently, recruitment of a dCas9-KRAB repressor fusion to known regulatory regions catalyzed trimethylation of lysine 9 on histone 3 (H3K9) at the enhancer and associated promoters, effectively silencing enhancer activity. Though there have only been a few examples published, it will likely not be long until researchers employ this technique for the targeted analysis of additional epigenome modifiers. Already, targeted methylation, demethylation and genomic looping have been accomplished using the DNA-binders, Zinc Finger Nucleases and TALEs. With the increased simplicity in design of gRNAs, dCas9 is predicted to surpass these other proteins in its utility to link epigenome modifications with gene expression data.
Visualization of Genomic Loci
When you treat dCas9 as a bridge between DNA and an accessory protein, just as in the recruitment of activators, repressors and epigenome modifiers, there are few limits to what can be targeted to the genome. Drawing inspiration from the art of observation that serves as the foundation of scientific pursuit, researchers have begun to test whether dCas9 can be used to visualize genomic loci and observe their position, movements, and interactions simply by recruiting a fluorescent molecule to the locus of interest.
This idea, of course, is not entirely new. In situ hybridization techniques (ISH, and its fluorescent counterpart, FISH) have been successfully used to label locus position in fixed cells but cannot offer any information about the movement of chromosomes in living cells. Initial studies to conquer this much harder feat made use of long tracts of repetitive DNA sequence bound by its protein binding partner fused to fluorescing GFP; though surely an advance, this technique is limited because of the requirement to engineer the repetitive DNA motifs prior to imaging.
To circumvent this need, researchers have recently made use of TALEs and dCas9 (and here) carrying fluorescent tags to image unperturbed genomic loci in a variety of live cell cultures. The catch is that both TALEs and dCas9 perform much better when targeting repetitive regions, such that multiple copies of the fluorescent molecule are recruited, enhancing the intensity of the signal. Tiling of fluorescent dCas9 across a non-repetitive region using 30-70 neighboring gRNAs (a task made much more feasible with CRISPR versus TALEs) can similarly pinpoint targeted loci, albeit with much higher background. As is, the technique lacks the resolution desired for live imaging, but current advances in super-resolution microscopy and single-molecule tracking, as well as improvements in the brightness of fluorescent molecules available, will likely spur improvements in dCas9 imaging in the coming years.
Finally, dCas9 is not only useful in live cells. CASFISH, an updated Cas9-mediated FISH protocol, has been successfully used to label genomic loci in fixed cells and tissue. This updated version holds many benefits over traditional FISH including a streamlined protocol; but, most notably, CASFISH does not require the denaturation of genomic DNA, a necessary step for the hybridization of FISH probes, eliminating positional artifacts due to harsh treatment of the cells. Unfortunately, as of now, CASFISH also suffers from a need for repetitive sequences or tiling of gRNAs to increase signal intensity at the locus of interest.
Targeting RNA with Cas9
From cutting to tagging to modifying, it is clear that Cas9 has superstar potential when teamed up with double-stranded DNA (dsDNA); however, recent data suggests that this potential may not be limited to DNA. Mitchell O’Connell and colleagues at Berkeley found that Cas9 could bind and cleave single-stranded RNA (ssRNA) when annealed to a short DNA oligonucleotide containing the necessary NGG sequence. In addition, the authors made use of dCas9 and biotin-tagged gRNA to capture and immobilize targeted messenger RNA from cell extract. Though it remains to be shown, this proof-of-principle binding of dCas9 suggests that it is plausible to recruit a variety of functionalities to RNA as has been done for dsDNA. Recruitment of RNA processing factors through Cas9 could potentially enhance translation, generate known RNA editing events (deamination, e.g.), regulate alternative splicing events, or even allow visualization of RNA localization with conjugated fluorescent molecules. Again, each of these processes requires no modification to the RNA sequence or fixation, both of which can disrupt normal cell physiology.
Improving CRISPR Technology
The development of CRISPR technology, particularly the applications discussed here, is still in its infancy. It will likely take years of research for Cas9 and dCas9 to reach their full potential, but advances are underway. These developments pertain not only to the applications discussed here, but also genome engineering.
Specificity of Cas9
Cas9’s biggest flaw is its inability to stay focused. Off-target (OT) binding (and here) of Cas9 and DNA cutting have been reported and both present problems. With particular relevance to dCas9-based applications, promiscuous binding of Cas9 to regions of the genome that contain substantial mismatches to the gRNA sequence raises concerns of non-specific activity of the targeted functionality. Efforts to reduce OT binding are needed to alleviate these concerns, but progress has been made with the finding that truncated gRNA sequences are less tolerant of mismatches, reducing off-target Cas9 activity, if not also binding.
Temporal Precision of Cas9
One of the most exciting developments in dCas9 genome targeting is the potential to manipulate the genome and epigenome in select cell populations within a whole animal to gain spatial resolution in our understanding of genome regulation; however, as we have learned over the years, gene expression patterns don’t only change with space, but also time. A single cell, for example, will alter its transcriptome at different points during development or in response to external stimulus. The development of split versions of Cas9 (and dCas9), which require two-halves of the protein to be expressed simultaneously for function, will not only improve spatial specificity of Cas9 activity but holds the potential to restrict its activity temporally. Drug-inducible and photoactivatable (!) versions of split Cas9 restrict function to time windows of drug treatment or light activation, respectively. In addition, a ligand-sensitive intein has been shown to temporally control Cas9 activity by releasing functional Cas9 through protein splicing only in the presence of ligand.
Expanding the CRISPR Protein Repertoire
Finally, CRISPR technology will likely benefit from taking all of the weight off of the shoulders of Cas9. Progress toward designing Cas9 molecules with altered PAM specificity, as well as the isolation of Cas9 from different species of bacteria, has helped expand the collection of genomic sites that can be targeted. It has also enabled multiplexing of orthogonal CRISPR proteins in a single cell to effect multiple functions simultaneously. More recently, the Zhang lab isolated an alternative type II CRISPR protein, Cpf1, purified from Francisella novicida. Cas9’s new BFF is also able to cut genomic DNA (as shown in human cells), but in a slightly different fashion than Cas9, generating sticky overhangs rather than blunt ends. Cpf1 also naturally harbors an alternate PAM specificity; rather than targeting sequences upstream of NGG, it prefers T-rich signatures (TTN), further expanding the genomes and genomic sites that can be targeted.
CRISPR/Cas9 has already proven to be one of the most versatile tools in the biologist’s toolbox to manipulate the genomes of a variety of species, but its utility continues to grow beyond these applications. Targeting Cas9 to the mitochondria rather than the nucleus can specifically edit the mitochondrial genome, with implications for disease treatment. Cas9 has been used for in vitro cloning experiments when traditional restriction enzymes just won’t do. And, by directly borrowing the concept of Cas9 immunity from bacteria, researchers have enabled enhanced resistance to viruses in plants engineered with Cas9 and gRNAs. While we ponder what innovative technique will come next, it’s important to think about how this cutting-edge technology that promises to bolster both basic and clinical research came to be: this particular avenue of research was paved entirely by machinery provided by the not-so-lowly bacteria. That’s pretty amazing, if you ask me.