CRISPR-Cas9 Therapeutics: A Technology Overview

Author: Alfred Chin, Johns Hopkins University

In a controversial paper published in the journal Protein & Cell earlier this year, Chinese scientists at Sun Yat-sen University reported the world’s first genomic editing of human embryos (1). Using genomic editing technology known as CRISPR-Cas9, the researchers edited the β-globin gene in human ‘non-viable’ embryos to correct β-thalassemia, a genetic blood disorder. Far from being clinically applicable, the results of the study shed light on the myriad of complications the team encountered in harnessing CRISPR-Cas9 as a therapeutic agent. The study came at a time of intense debate among the scientific community over the ethics of genomic editing. Some leading biologists have called for a moratorium on human genome editing (2), while other researchers believe that withholding technology that can eliminate crippling genetic diseases such as cystic fibrosis is unethical (3). Because of the rapid advancement of CRISPR-Cas9 technology, discussion on the science, legality, and ethics of therapeutic genome editing has never been more pertinent.


 Origins and Biology

The CRISPR-Cas system was first described as a bacterial adaptive defense mechanism against foreign invaders such as plasmids or viruses. CRISPRs, ‘clustered regularly interspaced short palindromic repeats’, are short DNA repeats of viral origin found in the genomes of bacteria, and Cas (CRISPR-associated) refers to proteins with nuclease domains that can recognize and cut DNA (4). Upon viral infection, CRISPR loci are transcribed to form mature CRISPR RNAs. Complexed with Cas proteins, the mature CRISPR RNA recognizes the foreign nucleic acid and guides the Cas protein to cleave and disable the invading virus.

In a Science paper published in June 2012, American RNA biologist Jennifer Doudna and French microbiologist Emmanuelle Charpentier capitalized on the bacterial defense mechanism by engineering a novel, programmable CRISPR/Cas system that was site-specific (5). Doudna and Charpentier’s method was the first demonstration of site-specific DNA cleavage using the Cas9 protein in vitro. However, it was not until 2013 – when MIT bioengineer Feng Zhang engineered a novel version of CRISPR-Cas9 to edit genomes in human cells (6) – did genomic editing become a therapeutic possibility.


Therapeutic Applications

CRISPR-Cas9’s ability to correct genetic defects presents an effective solution for individuals afflicted with incurable genetic disorders. Researchers are racing to optimize CRISPR-Cas9 for specific genetic disorders as proof-of-concepts that may eventually lead to clinical applications, and landmark advancements in correcting hereditary diseases are made every year. In 2013, Breakthrough Prize in Life Sciences winner Hans Clevers and his colleagues corrected the CFTR (cystic fibrosis transmembrane conductor receptor) locus in intestinal stem cells of cystic fibrosis patients (7). In 2014, Daniel Anderson and his group at MIT successfully corrected the Fah mutation responsible for hereditary tyrosinemia type I – a fatal genetic disease characterized by an inability to metabolize amino acid tyrosine – in liver cells (8). In 2015, a team from Duke University led by Charles Gersbach restored proper dystrophin expression in human cells with Duchenne muscular dystrophy (9).

Realizing that CRISPR-Cas9 offers unparalleled genetic specificity, researchers are using the genomic editing technology to facilitate discoveries in cancer biology. Representative animal models are integral in medical research, and CRISPR-Cas9 has led to cancer models that better reflect the disease in humans. Last October, Feng Zhang and Nobel laureate Phillip Sharp successfully engineered a CRISPR-Cas9 system that is easily delivered into mice to model the deleterious effects of mutations in cancer, facilitating “rapid screening of causal genetic mutations in a variety of biological and disease applications” (10). This was possible due to the ability of their system to introduce loss-of-function mutations in tumor suppressor genes and gain-of-function mutations in proto-oncogenes. Earlier this year, Christopher Vakoc and his team at Cold Spring Harbor Laboratory extended CRISPR-Cas9 technology to comprehensively identify protein domains that sustain cancer cells (11). The broad screening capabilities of Vakoc’s approach may help identify novel cancer drug targets, which are of great interest to drug discovery efforts led by pharmaceutical companies.


Addressing Challenges and Limitations

Before CRISPR-Cas9 can modify the genome of patients, it needs to be efficiently delivered into the nucleus. Traditionally, the Cas9 protein used by researchers comes from Streptococcus pyogenes, but the protein’s large size limits its use in therapeutic applications. In a paper published in Nature earlier this year, Feng Zhang and Phillip Sharp discovered that Cas9 from Staphylococcus aureus – which is more than one kilobase shorter – can edit the genome with similar efficiency to the S. pyogenes Cas9 protein (12). Aside from employing a smaller Cas9 protein, CRISPR-Cas9 delivery can be enhanced further by attaching the system to molecules that facilitate uptake. David Liu and his group at Harvard University complexed CRISPR-Cas9 with cationic lipids, resulting in higher potency and specificity of genomic editing in vivo than conventional delivery methods (13).

Whether it is drug dosage or radiotherapy duration, effective therapeutic applications require precise control. Methods to temporally and spatially regulate CRISPR-Cas9 are an emerging focus of research in 2015, and they provide researchers with greater flexibility and options. Scott Lowe and colleagues at Memorial Sloan Kettering Cancer Center developed an inducible CRISPR (iCRISPR) system that can be activated by administering doxycycline in vivo, allowing researchers to activate CRISPR-Cas9 at will in mouse models (14). In a groundbreaking paper published in Nature Biotechnology earlier this month, Moritoshi Sato’s lab at The University of Tokyo combined CRISPR-Cas9 with another revolutionary genetic research method – optogenetics (15). Heralded as “Method of the Year” by Nature Methods in 2010, optogenetics refers to the ability to control cellular activity by light. Sato’s photoactivatable Cas9 protein comprises of fragments that activate and assemble when exposed to blue light. Not only is light non-invasive, but it also renders extremely high spatial, temporal, and reversible control.

While CRISPR-Cas9 is highly site-specific, off-target cleavages can occur. Understandably, genomic editing accuracy is a paramount concern because of the devastating effects of any unintended edits in humans, and methods to detect off-target cleavages are needed. J. Keith Joung’s lab at Massachusetts General Hospital developed a method called GUIDE-seq to identify errors in CRISPR-Cas9 edits across the entire human genome in living cells, and the method remains the most rigorous and sensitive in the field to date (16). In addition to allowing researchers to assess the accuracy of future CRISPR-Cas9 systems, error-checking tools such as GUIDE-seq may be used by clinicians in the future to verify successful therapeutic genome editing in patients.


Intellectual Property

Given that CRISPR-Cas9 has fundamentally changed the way biologists around the world approach genetics, it is no surprise that the patent office is flooded with CRISPR-Cas9 patents. Patent applications for CRISPR-Cas9 technologies in 2014 more than quadrupled those in 2013, and MIT, Broad Institute, and Feng Zhang collectively command a majority of filed CRISPR-Cas9 patents (17). Currently, Feng Zhang – a cofounder of CRISPR-Cas9 startup Editas Medicine – holds the first US patent for CRISPR-Cas9 editing of eukaryotic genomes, which has been licensed to several biotech and pharmaceutical companies (18). However, Jennifer Doudna and Emmanuelle Charpentier – cofounders of Caribou Biosciences and CRISPR Therapeutics respectively – believe that they should own the rights to CRISPR-Cas9 and have requested the US Patent & Trademark Office to reconsider the patents owned by Feng Zhang and Broad Institute.  With billions of dollars at stake, legal fights over CRISPR patents may prevent commercialization of CRISPR-Cas9 and reflect badly on the universities involved (19).


Ethical and Legal Concerns

The primary ethical concern for CRISPR-Cas9 therapeutics pertains to the prospect of human germline engineering. Last month, Nature Biotechnology published a feature article with inputs from researchers, ethicists, and business leaders around the world on the ethical issues raised by CRISPR germline engineering (20). The general consensus seems to be that germline engineering is inevitable because researchers will successfully troubleshoot any technical barriers sooner or later. However, one thing remains unanimously agreed – germline engineering must be carefully regulated. While the medical benefits of germline engineering are clear, many experts foresee societal risks. One obvious concern is that only rich families may be able to afford genomic editing, conferring an inherent advantage to babies born in wealthier countries. Another long-term concern is a genomic editing slippery slope that may lead to ‘designer babies.’  Where is the line drawn between therapeutics and enhancement?

In terms of the legality of germline engineering, most experts are in favor of a temporary moratorium to foster discussion since an international ban is unlikely to be entirely enforced. While researchers in China have already published studies on germline engineering, US National Institutes of Health director Francis Collins issued a statement earlier this year that banned NIH-funded research into genomic editing of human embryos (21). Due to the divergent medical research policies around the world, the issue of whether germline engineering should be regulated internationally or domestically has also been raised in the scientific community.

Perhaps the biggest biomedical discovery in decades, CRISPR-Cas9 holds enormous prospects for therapeutic intervention to cure devastating genetic diseases. It also allows humans to permanently change the course of evolution. The decisions made by governments, researchers, and biotech companies on the use of therapeutic gene editing in the next decade will undoubtedly change science and medicine forever.




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