CRISPR-Cas13
Last update: Sep 2020
Introduction
Cas13 was first identified in 2015 when Shmakov et al. found what is now known as Cas13a. The previous name of this nuclease is C2c2, and until recently, this is what it was called in the literature. After the initial discovery of Cas13a, different members of the Cas13 family (Fig.1) were also discovered.
The key difference between Cas13 and Cas9 or Cas12a is that it targets RNA, not DNA. It can bind to RNA using gRNA, and cleave it using its two RNase domains called HEPN. If those domains are inactivated, "nuclease dead" dCas13 that can bind to, but cannot cleave RNA, is made.
Figure 1. Family of Cas13 proteins
Instead of PAM, Cas13 recognizes protospacer flanking sequences (PFS). For example, for LshCas13a, PFS consists of a single A, U, or C base pair located at the 3' end of the spacer sequence. Notably, in numerous Cas13 homologs, especially when employed in mammals, PFS is not needed, which further simplifies the experimental design.
Cas13 nucleases use around 64-nucleotides long guide RNAs. The target-specific part of the gRNA is 28-30 nt long, which is around 40%-50% higher than that of Cas9 and Cas12a. A notable and, in fact, a crucial feature of CRISPR-Cas13 is that it does not only cleave its target RNA sequence, thus protecting the bacteria from viral RNA, but it also cuts other RNA molecules. Once the target RNA is cleaved, Cas13 adopts an enzymatically "active" state (instead of becoming inactive like Cas9 and Cas12a) and can thus still cleave RNA. In fact, it binds to and cuts all RNA molecules around, regardless of whether they contain the target sequence or PFS (you can imagine this as a berserk state). The consequence of this is substantial collateral damage. The reason why this happens is not yet fully understood, but it is thought that this mechanism is part of the programmed cell death pathway in bacteria. It enables cells to commit suicide or become dormant until recovery. This feature is thought to limit the spread of infection to surrounding bacterial cells.
Table 1. Comparison between Cas9, Cas12a (Cpf1) and Cas13a
Interestingly, this collateral activity is negligible when Cas13 is used in mammalian cells and plants, which allows researchers to employ various RNA targeting methods using CRISPR-Cas13.
CRISPR-Cas13 has a plethora of current and potential applications, including RNA knockdown. One important feature of Cas13 that encourages its use in research is its short coding sequence, which facilitates in vivo delivery. Furthermore, Cas13 non-specific RNase activity can be employed to cleave fluorescent reporters upon cutting a specific (target) sequence, which shows great promise in diagnostics (SHERLOCK). The following sections discuss the applications of Cas13 in more detail.
RNA Knockdown
The most obvious application of CRISPR-Cas13 is RNA knockdown. The process is illustrated in Fig.2. Usually, mammalian codon-optimized Cas13 is used for this purpose. Dual HEPN RNase domains in Cas13 allow cleavage of target RNA sequence, but the efficiency of this process differs depending on the ortholog and subtype of Cas13 used.
Figure 2. RNA knockdown using Lwa-Cas13a
LshCas13a and Cas13b both exhibit sequence constraints in the form of the Protospacer Flanking Sequence (PFS) when used in bacteria. Conveniently, so far, no such constraints were identified for Cas13a, Cas13b, and Cas13d in mammalian cells.
It was observed that the knockdown efficiency decreases when secondary RNA structures are present at the target site both in the case of bacteria and mammalian cells. This is likely because Cas13 does not exhibit helicase activity required to open up a double-stranded RNA region, and thus can only bind to single-stranded RNA (ssRNA). Similarly, secondary RNA structures near the protospacer also affect the experimental results. To determine which sequence is in the low secondary structure region and thus is easier to target, tools such as RNAplfold algorithm, which predict the RNA folding, can be employed.
On top of the guidelines mentioned above, it is vital to choose the right Cas13 subtype for the model organism used. So far, LwaCas13a, PspCas13b, and RfxCas13d have all shown appropriate knockdown efficiencies for numerous genes in mammalian cells (Table 2). When researchers compared the activity of Cas13a, Cas13b, and Cas13d in HEK 293T cells, RfxCas13d displayed the most robust knockdown. In the case of plants, only LwaCas13a has been tested so far, demonstrating considerable knockdown in rice protoplasts.
Table 2. Genome editing efficiency of three Cas13 orthologs
CARVER
There are several ways in which we can use CRISPR-Cas13-mediated RNA knockdown, and the most obvious one is to employ it in the same way bacteria do- to protect ourselves from viral infections. Considering that around two-thirds of viruses that can infect humans, including Ebola, Zika, and influenza, have an ssRNA genome, Cas13 could be employed to destroy many invaders. Researchers at the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard have recently designed a method called CARVER, which stands for Cas13-assisted restriction of viral expression and readout. CARVER can be used to detect and destroy RNA-based viruses in human cells.
While technically Cas13 could be programmed to attack any part of a virus, a high rate of mutation of viruses means that the target section has to be chosen carefully in order to affect the possibly largest number of virus variants. Scientists computationally identified thousands of possible target sequences for Cas13. They then investigated the activity of Cas13 in human cells infected with lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), and vesicular stomatitis virus (VSV).
First, Cas13 and its gRNAs were introduced into human cells. 24 hours later, the cells were infected with the respective viruses. According to experimental results, viral RNA levels in cell cultures were reduced as much as 40 times, thanks to Cas13. Further investigations revealed that 8 hours after viral exposure, the infectivity of the influenza virus was reduced 300 times.
SHERLOCK
Cas13 can be used not only to destroy viruses but also to non-invasively detect their presence, which is of paramount significance in diagnostics. Apart from CARVER, scientists at MIT and Harvard have also designed a system called SHERLOCK, which stands for high-sensitivity enzymatic reporter unlocking. SHERLOCK exploits the ability of Cas13 to cleave all RNAs, regardless of complementarity, once it cleaves its target sequence. It is used to detect minute quantities of DNA and RNA in a sample, corresponding to femtomolar (10-15 M) concentrations.
First, the DNA (or RNA) that we suspect contains the sequence of interest (e.g., a viral sequence) is amplified using Recombinase Polymerase Amplification (RPA), which can proceed even at room temperatures. In the case of RNA, Reverse-transcription RPA (RT-RPA) is used. Next, amplified DNA is transcribed to RNA (which Cas13 can target) by T7 RNA polymerase. The RNA sequence is combined with Cas13 loaded with gRNA, and with an inactive fluorescent RNA reporter. If the target sequence, for example, the viral sequence, is present in the amplified RNA transcripts, the Cas13a enzyme is activated. It cuts the target RNA, and other RNAs surrounding it. This means that it also cleaves the RNA reporter present in the mixture. Upon cleavage, the fluorophore in the reporter is activated, and a detectable signal is produced. This way, we know whether the sequence of interest is present in the sample. It is a straightforward, inexpensive, and undemanding way to diagnose multiple viral infections. SHERLOCK system has so far been used to differentiate strains of Zika, detect dengue virus, genotype human DNA, and identify tumor mutations in vitro.
Figure 3. Detecting a target sequence with SHERLOCK
SHERLOCK can be even used to detect COVID-19. In February 2020, Broad Institute has released a simple research protocol that can be used to detect COVID-19 in the purified RNA sample. This is not a widely used diagnostic test just yet, so more research is certainly needed before it can be employed in mainstream diagnostics.
For a visualization of CRISPR-Cas13 activity, watch this Youtube video released by McGovern Institute in 2018.
Sources
Content
[1] Joel McDade. 2017. CRISPR 101: Targeting RNA with Cas13a (C2c2). https://blog.addgene.org/crispr-101-targeting-rna-with-cas13a-c2c2. [Accessed 23 July 2020].
[2] Omar Abudayyeh and Jonathan Gootenberg. 2020. Tips and Tricks for Cas13. https://zlab.bio/cas13. [Accessed 23 July 2020].
[3] Genetic Engineering & Biotechnology News. 2019. CRISPR-Cas13 Developed as Combination Antiviral and Diagnostic System. https://www.genengnews.com/news/crispr-cas13-developed-as-combination-antiviral-and-diagnostic-system/. [Accessed 23 July 2020].
Graphics
Figure 1-3. Omar Abudayyeh and Jonathan Gootenberg. 2020. Tips and Tricks for Cas13. https://zlab.bio/cas13. [Accessed 23 July 2020].
Table 7. Joel McDade. 2017. CRISPR 101: Targeting RNA with Cas13a (C2c2). https://blog.addgene.org/crispr-101-targeting-rna-with-cas13a-c2c2. [Accessed 23 July 2020].
Table 8. Omar Abudayyeh and Jonathan Gootenberg. 2020. Tips and Tricks for Cas13. https://zlab.bio/cas13. [Accessed 23 July 2020].