As news regarding CRISPR/Cas technology and its potential in medical applications continues to garner significant attention and pervade even mainstream outlets, clinicians and laboratorians are anticipating the impending challenges the laboratory will face in incorporating what may quickly become an invaluable technology.
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology and its CRISPR Associated proteins (Cas9 or Cas13a) essentially offer a programmable, sequence-specific means to target the DNA or RNA sequence of your choosing. This is accomplished through standard base-pairing of the CRISPR guide RNA bound by the Cas protein to the targeted DNA or RNA, which then can be predictably cleaved or bound in the case of catalytically inactive Cas exonuclease (see FIGURE 1). This technology is the gateway to the implementation and vast utilization of genetic editing in a clinical setting. With this in mind, it is worth investigating the pending therapeutic and diagnostic applications of a CRISPR/Cas system in the clinical lab.
Current Uses for CRISPR
The ease with which CRISPR/Cas can be programmed produces several design advantages over other similar systems, such as zinc finger nucleases (ZFNs). In contrast to CRISPR/Cas, ZFNs must be engineered specifically for each target, whereas with CRISPR/Cas, only the guide RNA requires target-specific design. The ease-of-use and specificity of this methodology has positioned CRISPR/Cas for some remarkable clinical prospects including genome editing and other molecular diagnostic applications that were formerly intractable. Given that, many clinicians may be surprised to hear that CRISPR/Cas genome editing applications are closer to real impact in the laboratory than previously thought.
Currently, clinical trials are in place involving CRISPR modifications to patient T-cells for immuno-oncology by removing certain genes targeted by checkpoint inhibitors. Similarly, patient harvested T-cells have been modified by CRISPR/Cas for HIV treatments by targeting either the latent genomic virus or by removing genes encoding viral cell-surface receptors to prevent additional T-cell infections. Regardless, such modifications of patient derived T-cells do not come cheap. As one example, engineered Chimeric Antigen Receptor (CAR) T-cell based therapy is forecast to cost approximately $475,000.1
Proof of Concept
While extracted cells could be modified and infused in more proximal time frames, there are other proposals for genome editing of chronic infections or inherited disorders. For example, the mini-chromosomes (covalently closed circular DNA, or cccDNA) responsible for chronic hepatitis B infections have been targeted by CRISPR/Cas for intrahepatic HBV clearance. Other proof of concept experiments have edited disease causing genes for conditions such as Fanconi anemia or cystic fibrosis in cell lines or human organoids raising the prospect for future CRISPR/Cas-based treatment options. There is even the prospect of zygotic CRISPR/Cas editing to remove treatment concerns or carrier status by modifying the germline of patients and their children.
Medical Ethics and Technical Issues
Further clinical incorporation of these types of applications will require significant thought and debate as to the ethical issues involved in addition to the clinical and technical concerns. There are myriad concerns and informed consent issues that must be addressed before any potential therapeutics advance into the clinic.
From a technical implementation standpoint, there are three major technical CRISPR/Cas issues:
- Incidence of off-target effects lead to potential second-site cleavage events and potential undesirable alterations. There are a few strategies vying to suppress any detrimental off-target CRISPR/Cas cleavage events that are showing promise.
- Concerns exist over repair of any CRISPR/Cas cleavage; CRISPR is great at removing targets, but repairing the excised region is less efficient. There are two competing mechanisms that can either lead to the desired excision and repair for a successfully edited genome, or undesired fusion events resulting from non-homologous end joining, that in turn lead to off-target gene fusion events with potential deleterious effects.
- There is concern over the delivery of the CRISPR/Cas system. While the potential modification of harvested marrow, cells, or zygotes tends to inspire less concern, delivering any ribonucleic acid protein complex in vivo has definite and wide-ranging technical, oversight, consent, and management challenges.
These issues notwithstanding, many clinical labs are likely to see CRISPR/Cas incorporated into their molecular diagnostic workflows much sooner than originally expected. These diagnostic systems rely on the same programmable specificity to essentially either detect a nucleotide target through cleavage or capture a sequence-specific nucleotide target or targets using the catalytically dead version of Cas.
With the first option, nucleotide target detection via cleavage, there are currently two predominant CRISPR/Cas based diagnostic (CRISPR-Dx) approaches that use an active Cas exonuclease.
- In the first approach, CRISPR/Cas is used to specifically target the reference sequence (or wild-type sequence) of KRAS while leaving the versions commonly mutated in somatic cancers intact. This system, termed DASH (Depleted of Abundant Sequences by Hybridization), depletes unwanted sequences prior to polymer chain reaction (PCR) amplification. This prevents amplification of the targeted sequences and thereby enhances the amplification of rare somatic variants that may be driving a cancer. The prospects for DASH in enhancing next-generation sequencing for detection of rare DNA variants in applications such as liquid biopsies that hunt for rare and dilute circulating free DNA (cfDNA) certainly has the potential to reach clinical use given the current proof of concept publications.
- The second CRISPR/Cas exonuclease system termed SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing) uses a Cas13a coupled with isothermal amplification to provide attomolar detection of DNA (less than 1 part per quadrillion). In proof of concept studies, the investigators were able to detect viral and bacterial DNA, in addition to genotyping human cfDNA. They furthered their work by lyophilizing reagents to make them cold-chain independent and proposed costs of as little as $0.61/test.2
With the second CRISPR-Dx technique, termed CATCH (Cas9 Assisted Target of CHromosomal segments), catalytically dead Cas9 (dCas9) is used to enrich targeted chromosomal segments for sequencing. The potential of these systems for further target enrichment, especially for direct molecule sequencing for so-called 3rd generation sequencers, is intriguing for direct measure of DNA methylation or long-range sequencing needs.
The introduction of diagnostic CRISPR/Cas applications in the clinical lab is likely to happen in the short-term and carries amazing prospects for clinical genome editing in due course after more rigorous examination of potential off-target effects. That said, existing immuno-oncology applications and other such immune-modifications might make a more expeditious debut in the clinical lab.
There are currently 14 CRISPR/Cas-based clinical trials with most involving T-cell modifications. In the end, CRISPR/Cas is an invaluable clinical enzyme that has potential to revolutionize clinical labs in terms of laboratory developed tests, in addition to therapeutics involving genome editing.
- Hagen T. Novartis Sets a Price of $475,000 for CAR T-Cell Therapy. OncLive. 8/30/2017. www.onclive.com/web-exclusives/novartis-sets-a-price-of-475000-for-car-tcell-therapy. Accessed February 15, 2018.
- Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442.
Aaron E. Atkinson, PhD, is an instructor of pathology and laboratory medicine at the Laboratory for Clinical Genomics and Advanced Technology (CGAT) in the department of pathology and laboratory medicine, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire.
Claire E. Nerenz, JD, is senior manager of research compliance in the department of compliance and audit services, Dartmouth Hitchcock Medical Center.
Yvette R. Seger, PhD, is director of science policy at the Federation of American Societies for Experimental Biology in Bethesda, Maryland.
Gregory J. Tsongalis, PhD, is director of the Laboratory for Clinical Genomics and Advanced Technology (CGAT) in the department of pathology and laboratory medicine, Dartmouth Hitchcock Medical Center. He also is a professor in pathology and laboratory medicine at Geisel School of Medicine at Dartmouth in Hanover.
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