CRISPR-based RNA editing: diagnostic applications and therapeutic options

KEYWORDS:

• DNA/RNA editing
• diagnostic
• non-coding RNA
• programmable nucleases
• therapy

1. Current status

Gene therapy is currently in the front rows of biomedical research with an increased focus on direct nuclease-based editing. If until now the research was mainly concentrated on modifications at the DNA level, new adapted tolls are starting to gain momentum due to their ability to induce changes within the RNA sequence. The advantage consists in the half-life of the messenger RNA and implicit the non-permanent character of the editing strategy. Recently, the ability of nucleases like Cas12a and Cas13 to cleave DNA and RNA, respectively, was used for detection of low particle infectious agents with high sensitivity – e.g. DETECTOR and SHERLOCK Systems. The present article briefly presents the latest advancements from the gene editing therapeutic area, as well as the new diagnostic tools with great likelihoods within the clinical environment.

2. Latest advancements in editing

The last decade of biomedical research witnessed an unprecedented increase in the number of advances on non-coding RNAs [1,2] and non-coding DNAs [3]. Year 2017 has been considered the Year of Gene-Therapy Breakthroughs according to the researchers from MIT [4]. The leeway of specifically changing the DNA or RNA sequence in order to correct the causative-specific dysregulation converging to a pathological state has the potential to reshape the future medicine [4]. Programmable nucleases for genome editing like transcription activator-like effector nucleases (TALENs), meganucleases, zinc-finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas are now in the spotlight of experimental research (Table 1) [5–8]. Based on these achievements several gene-therapy based treatments get contour in the last couple of years. In March 2017, a young male patient entered into complete clinical remission for sickle cell disease after receiving ex vivo gene therapy by a lentiviral vector [9]; Kymriah (a chimeric antigen receptor CAR-T therapy for non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia) and Yescarta (a CD19-directed genetically modified autologous T cell immunotherapy), two types of autologous CAR-T cell therapies designed for cancer patients unresponsive to the standard of care received FDA approval in 2017 [10]; in November 2017 a 7-year-old child with junctional epidermolysis bullosa (JEB) had his skin layer replaced by cultured self-stem cells genetically engineered [11]; Luxturna (an adeno-associated virus vector-based gene therapy indicated for the treatment of patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy) received FDA approval in December 2017 for sight-restoring in patients with Leber’s congenital amaurosis related blindness [12,13].

Table 1. Clinical trials based on genomic editing (http://clinicalTrials.gov).

Tool	Pathology	Status
CRISPR/Cas	Solid and hematological malignancies, Infections (HIV and gastrointestinal), Sickle Cell Disease, Thalassemia	Out of 13 studies, 10 are currently recruiting patients, where 2 are not yet recruiting and one is withdrawn
TALENs	Human Papillomavirus-Related Malignant Neoplasm	Two studies, both not yet recruiting
ZFNs	Human Papillomavirus-Related Malignant Neoplasm, MPS I/II, HIV, Hemophilia B, Transfusion Dependent Beta-thalassemia, Sickle Cell Disease	Out of 13 studies, 5 are currently recruiting patients, 5 are not yet recruiting and 3 are completed

All these advancements are raising hopes regarding the potential eradication of previously untreatable diseases, but nevertheless, there are important concerns related to the ethical aspects. As per example, gene-edited crops could be subject to the same rigorous regulations as conventional genetically modified (GM) organisms, as Court of Justice of the European Union ruled on July 25^th, 2018. Also, the long-term effects caused by irreversible changes in the DNA structure are questionable when comes to patients’ involvement. In this sense, a new type of approach is currently emerging into preclinical testing, where instead of inducing permanent modifications within the DNA sequence, a more versatile type of nucleic acid is modified – the RNA. The advantage arises from the half-life of the messenger RNA (mRNA), where the modifications are still translated functionally into modified proteins, but the effect is time and also disease stage manageable.

Point mutations induce single-nucleotide substitutions that can further generate modified forms of proteins specifically associated with different diseases like cancer [14], cystic fibrosis [15], hemophilia A [16] and chronic granulomatous disease [17]. The correction of these point mutations can be made at either DNA or RNA levels with equivalent effects upon the translated protein [18]. RNA editing occurs at post-transcriptional level with a functional outcome mainly upon different forms of proteins [19]; the modifications in the RNA structure can occur through substitution, deletion or insertion [20,21]. In mammalian cell, two main families of enzymes are responsible for the RNA editing process, where adenosine is converted to inosine (guanosine-like complementarity) – adenosine deaminase acting on RNA (ADAR) family – and cytosine is converted to uracil – AID-apolipoprotein B mRNA editing enzyme(APOBEC) enzyme family [22–24]. These modifications can take place in different RNA types and regions; while AID-APOBEC activity has been detected mainly in the 3ʹ-UTRs [24], ADAR can undertake a wide range of RNAs, including primary microRNA transcripts with effects upon gene silencing [25,26]. Even though these correcting processes consist of a single-nucleotide change, the downstream effects can significantly alter the phenotype of a cell. Substitutions in target spots can influence processes related to signaling, catalysis and also post-transcriptional modifications, disrupt the open reading frames, microRNA seed sequences and also splicing signals [27]. Additionally, Shah et al. identified a distinct RNA editing mechanism, independent of APOBEC or ADAR, based on the allelic imbalances of guanosine and thymine alleles within the CCAT2 long non-coding RNA overlapping a functional cancer risk related SNP in myelodysplastic patients and a mouse model [28].

3. Is RNA-based genetic engineering the safer option?

RNA-based gene therapy could rapidly evolve into clinical practice if we take into consideration that for some pathologies, only partial restoration of protein homeostatic activity is sufficient for significant disease amelioration – e.g. Lesch-Nyhan syndrome can switch to a mild form of hyperuricemia after 8% improvement in enzyme activity [29]. Moreover, potential off-target effects in one RNA molecule will be much attenuated than in the case of DNA editing strategies due to the temporary nature of the RNA molecules and also the editing will take place only in the cells that actually express the targeted gene.

At the end of 2017, researchers revealed such a form of gene therapy through enrolment of a different Cas enzyme – Cas13 and CRISPR system entitled REPAIR – RNA Editing for Programmable A to I replacement (Table 2) [30]. Shortly, Cas9 within the classical CRISPR/Cas9 system was replaced with Cas13 that binds RNA instead of DNA; the latest was further fused with ADAR2, which catalyzes the deamination of adenine to inosine. The specificity of the cut is directed by a guide RNA (gRNA). As stated by the authors, the degradable character of the RNA transforms REPAIR into a tool with reversible consequences, being safer than those who alter the genome. Furthermore, a more targeted version – REPAIRv2 – developed by the same group, achieved up to 50% targeting efficiency without significant off-target events. The feasibility of the method was tested for Fanconi anemia and X-linked nephrogenic diabetes insipidus.

Table 2. Available nucleases for gene editing.

Tool	Recognition	Availability	Applicability	Applicability in library screening	Specificity	Improved versions	Costs	First time used in human cells	Multiplexing
MNs	Protein-DNA	Restricted	Gene editing	Low	High	MegaTEV, MegaTAL	+++	1994	Low
ZFNs	Protein-DNA	Available	Transcriptional adjustment, Gene knockout	Low	Low-moderate	AZP-SNase	++++	2003	Low
TALENs	Protein-DNA	Available	Transcriptional adjustment, Gene knockout	Medium	Moderate	Tev-mTALEN	++	2011	Moderate (toward high)
CRISPR/ Cas	RNA-DNA	Available	Transcriptional adjustment, Gene knockout, single base editing	High	Low-moderate (improved with eSpCas9 enzyme)	Cpf1, eSpCas9	+	2013	High
Targetrons	RNA-DNA	Restricted	Gene knockout	Unknown	Moderate	Thermotargetron	++	2015	Low
Others	DNA editing tools based on DNA-DNA recognition PNAs – peptide nucleic acids, TFOs – triplex-forming oligonucleotides, SGNs – structure-guided endonucleases
Others	RNA editing tools based on RNA-RNA recognition SNAP–ADARs (SNAP- adenosine deaminases acting on RNA) λN-ADARs (λN- adenosine deaminases acting on RNA) REPAIR (RNA Editing for Programmable A to I replacement)

*MNs – meganucleases, DSB – double-strand breaks.

However, the concept of site-directed A-to-I RNA editing has been introduced before the identification of CRISPR/Cas based methods. The first engineered ribonucleoprotein for site-directed RNA modification has been implemented under the form of SNAP–ADARs (adenosine deaminases acting on RNA) [31] and is also the best-characterized method of its purpose at the current time. The strategy is based on the self-labeling protein SNAP-tag developed from human O6-alkylguanine-DNA alkyltransferase that binds in a covalent manner to a custom-made gRNA containing O6-benzylguanine (BG) tag. Competing with Cas13b-based methods, SNAP-ADAR was used for targeted adenosine to inosine editing within KRAS and STAT1, two crucial molecules within a large plethora of disease states, including cancer. The authors obtained up to 90% efficiency and also optimal precision and editing duration; specifically, off-target effects within the mRNA-gRNA duplex were diminished by genomic integration and showed superior specificity in comparison with the methods based on Cas13b where the off-target effects were amplified by the overexpressed editases [32]. Another approach, namely, λN-ADAR2, takes advantage of the non-covalent fusion of a gRNA-deaminase complex [33]. Potentiated efficiency was obtained within the same strategy by introducing a highly active mutant form of ADAR2, namely ADAR2 E/Q, further fused with up to four λN peptides (4λN-ADAR2 E/Q) [34]. The above-presented methods (CRISPR dependent and independent) were recently revised and compared [35]. In the constant attempt to reduce the off-target effects of RNA editing, several groups proposed the idea of taking advantage of the naturally occurring ADARs. Extending the idea of the early study of Woolf where the possibility of recruiting an endogenous ADAR in Xenopus was proved [36], recent studies developed principle-based similar approaches [37–39]. The first study of such manner in a human study models, managed to modify in Hela cells a mutation within PINK1 associated with loss of function effects and forced PINK1 connected mitophagy; this was done by designing a gRNA capable to capture human ADAR2 toward selected mRNA sites [37].

The ability of this RNA-based tool of introducing single-nucleotide changes can be adapted for numerous therapeutic purposes, including modification of primary microRNA sequences with the final purpose of impairing the ability of the ‘seed’ sequence to bind and inhibit complementary genes. MicroRNAs have evolved as important regulators of carcinogenesis [40–42], where numerous therapeutic attempts are made with exogenous mimic or inhibitor sequences for the attenuation of malignant hallmarks [43–46]. However, the delivery method and its specificity, sometimes the necessity of multiple administrations and potential immunogenicity and toxicity of the exogenous sequence are just several factors that are hampering the clinical implementation of microRNA therapeutics. Approaches such as RNA editing of primary microRNA sequence can limit the off-target effects and increase the specificity of the microRNA-based therapies.

4. Novel diagnostic value of genome editing tools

The use of CRISPR/Cas system for RNA-based gene therapy is currently raising numerous potential therapeutic applications; however, this new system can have also applications in the diagnostic sector. Several separate groups are repurposing the basics of CRISPR/Cas in novel diagnostic methods like viral infections difficult to detect by standard assays due to low numbers of circulating viral particles – eg. Zika virus.

New CRISPR sensing systems that have a collateral activity – the cleavage of nearby sequences in an indiscriminately manner – were used as a signal amplifier for the identification of a specific molecule [47] (Figure 1). The first one, DETECTR (DNA endonuclease targeted CRISPR trans reporter) [48] uses type V CRISPR and Cas12a with collateral activity for the detection of viral DNA; Cas12a is specifically constructed to bind viral DNA where the collateral cleavage ability is disrupting a ssDNA ‘reporter’ marked with a fluorophore at one end and with a quencher molecule at the other designed to capture the fluorescence. Upon cleavage of the ssDNA, the emitted fluorescence (amplified through recombinase polymerase for viral DNA) can be detected for diagnostic of viral infection. This approach can detect minimal concentration of viral particles – one molecule per microliter of sample and also distinguish between different strains of HPV. Through a similar strategy SHERLOCK (specific high sensitivity enzymatic reporter unlocking) was implemented [49] by adapting the detecting system on the ability of Cas13 to cleave RNA sequences. Still, the second system has several advantages in terms of specificity since it uses simultaneously four Cas13 proteins, each with their reporter, that allow the detection of more than one virus at once (maximum four) and is also based on an improved recombinase polymerase amplification for more sensitive diagnosis. Finally, another group [50] based their method on the strategic parameters of SHERLOCK but designed the system to detect the viral infection in body fluids like urine and saliva upon enzyme inactivation. They named the system HUDSON (heating unextracted diagnostic samples to obliterate nucleases). Moreover, their method can distinguish between mutated strains at a sensitivity of a single nucleotide modification – detection of Zyka virus in different geographic areas and also with different mutational pattern.

Figure 1. Diagnostic systems based on the activity of specific Cas enzymes and their collateral activity. DETECTR system is composed of Cas12a enzyme, a specific gRNA for viral DNA recognition and an ssDNA construct loaded with a quencher and a fluorophore; in the OFF state of the system, the fluorescence is captured by the quencher (DETECTR system A). After target recognition, Cas12a cleaves the viral DNA, but also the ssDNA construct, liberating the fluorophore from the quencher (DETECTR system B). SHERLOCK system has similar principles, although the entire construct contains also an additional enzyme – Csm6 – that cleaves a secondary ssDNA construct (SHERLOCK system A). The cleavage products of Cas13 activate Csm6 that further disrupts the second ssDNA segment and amplifies the fluorescence signal (SHERLOCK system B).

These diagnostic tools are expending the possibilities around the CRISPR/Cas system, applications that are also susceptible to a more rapid implementation within the market due to the economical friendly status. Moreover, these instruments can be relatively easy adapted for the minimally invasive detection of other pathologies like cancer by sensing circulating tumor DNA in the body fluids of patients.

5. Conclusion

Until not long ago, targeted corrections within the DNA sequence using nucleases like Cas9 were considered the future of medicine and many studies were concentrated on minimizing the off-target effects of the genomic editing tools for possible implementation within the clinic. Even so, recent research has uncovered alternative possibilities of genetic manipulation by counteracting the RNA sequence with minimized long-term effects due to the degradable character of this type of nucleic acid. The debate between the genetic manipulation of DNA versus RNA is still on, where the future will reveal which strategy will finally achieve its place in the clinic. On the other side, similar strategies are used for other purposes: diagnostic of viral infections with at an unprecedented sensitivity. This type of research has minimal risks for the patient and can be also adapted for detection of other pathologies based on distinctive markers within liquid biopsies.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewers Disclosure

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Additional information

Funding

Dr. Calin is the Felix L. Haas Endowed Professor in Basic Science. Work in Dr. Calin’s laboratory is supported by National Institutes of Health (NIH/NCATS) grant UH3TR00943-01 through the NIH Common Fund, Office of Strategic Coordination (OSC), the NCI grants 1R01 CA182905-01 and 1R01 CA222007-01A1, an NIGMS 1R01 GM122775-0, a U54 grant #CA096297/CA096300 – UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, a Team DOD (CA160445P1) grant, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund (SINF) 2017 grant, and the Estate of C. G. Johnson, Jr. This work was also supported by Competitivity Operational Program, 2014-2020, entitled “Clinical and economical impact of personalized targeted anti-microRNA therapies in reconverting lung cancer chemoresistance” - CANTEMIR, no. 35/01.09.2016, MySMIS 103375.