CRISPR

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of bacteria. It contains a programmable protein which can cut, or edit, DNA and RNA. Scientists are using the technology as a robust gene-editing tool, editing and modifying genes in living cells and organisms.

CRISPR gene editing uses enzymes to cut, edit and replace DNA at specific places in the genome. It often is harnessed to alter a piece of DNA. CRISPR also can turn genes on or off without altering their sequence. But beyond the ability to treat disease, CRISPR also is a platform for diagnostics. CRISPR proteins can generate a signal that matches sequences of DNA or RNA. When they do this, CRISPR can detect the presence of specific nucleic acid sequences, becoming a novel diagnostic.

A CRISPR system is created by programming a Cas enzyme, which is involved in cellular apoptosis and proliferation, to bind to a certain DNA or RNA sequence. The programming involves designing a guide RNA complementary to the sequence the enzyme is to bind with. A guide RNA can be designed to target the Cas protein to a gene to be edited. When the protein finds this sequence, the built-in molecular scissors of the Cas protein cut the nucleic acid at that location.

The Gut

While CRISPR has been used as a research tool for the most part, the technology is moving toward commercial applications. Scientists at North Carolina State University have found that the CRISPR-Cas system can target and eliminate specific gut bacteria, such as Clostridioides difficile, the pathogen that causes colitis.

Microbiologists from two North Carolina State University teamed with NC State startup company Locus Biosciences to test the effectiveness of using a bacteriophage to carry a programmable CRISPR to specifically target and eliminate C. difficile. In 2019, Locus Biosciences inked a collaboration and license deal with Johnson & Johnson’s Janssen Pharmaceutical to develop precision antibacterial therapies based on CRISPR-Cas3-enhanced bacteriophages. Under the terms of the deal, Janssen is paying Locus $20 million up front. Locus will be eligible for up to a total of $798 million in development and commercial milestones, as well as royalties on any product sales. In July, Locus had raised approximately $12.5 million in debt from 31 investors, according to a recent securities filing.

Janssen has the license to develop, manufacture and commercialize CRISPR-Cas3-enhanced products which target bacterial pathogens for potentially treating respiratory and other organ infections. The removal of harmful bacteria while ensuring that the rest of a patient’s microbiome is intact is a significant step toward treating diseases that could be linked to bacterial health in the body.

Gene Edited Therapy

Meanwhile, in June, CRISPR Therapeutics, Zug, Switzerland, and Vertex Pharmaceuticals reported clinical proof-of-concept for CTX001 in patients with transfusion-dependent beta thalassemia (TDT), and progress in assessing the safety and efficacy of a single dose of CTX001 in patients aged 18 to 35 with severe sickle cell disease (SCD). The first patient with severe SCD who received a single infusion of CTX001 remains free of vaso-occlusive crises, a common painful complication, nine months after treatment, results of a Phase 1/2 clinical trial showed.

CTX001 is an investigational ex-vivo CRISPR gene-edited therapy being evaluated for patients suffering from TDT or severe SCD in which a patient’s hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells. HbF is a form of the oxygen-carrying hemoglobin that is naturally present at birth, which then switches to the adult form of hemoglobin. The elevation of HbF by CTX001 could alleviate transfusion requirements for TDT patients and reduce painful and debilitating sickle crises for SCD patients, the companies said in a statement.

Diagnostics Potential

On the diagnostics front, Mammoth Biosciences, based in South San Francisco, envisions CRISPR technology as a way to sense, or detect, biomarkers or disease using DNA or RNA. The company wants to develop easy and affordable POC tests. Mammoth Biosciences has received patents (10,253,365 and 10,337,051) which enable it to offer DNA and RNA detection as CRISPR diagnostics.

Employing Nucleases

Mammoth Biosciences says that its DETECTR testing system searches for the presence of specific nucleic acids in a sample that are indicative of disease. The system works by employing CRISPR nucleases that are programmed to find a defined gene sequence. When it locates the sequence of interest, the nuclease activates a cleavage capability, which generates a signal. The signal confirms the sequence has been found.

In early September, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a SARS-CoV-2 test developed by Mammoth Biosciences. The SARS-CoV-2 DETECTR Reagent Kit is a CRISPR-based reverse transcription and loop-mediated amplification (RT-LAMP) test for detecting the virus' N gene in upper respiratory specimens. Labs that are CLIA-certified to perform high-complexity tests can use the Mammoth Biosciences test.

Meanwhile, another startup also is applying CRISPR to diagnostics. Sherlock Biosciences, Cambridge, MA, is using CRISPR and engineered biological systems to develop new diagnostics. Sherlock Biosciences was named after one of its foundational platform technologies, Sherlock -- specific high-sensitivity enzymatic reporter unlocking -- which the company licensed exclusively from the Broad Institute, a biomedical and genomic research center that is partners with the Massachusetts Institute of Technology and Harvard University.

Sherlock technology was developed by scientists led by company co-founder and chair of Sherlock’s scientific advisory board, Dr. Feng Zhang, who collaborated with co-founder Dr. Jim Collins, to identify specific genetic targets using CRISPR. Sherlock detects the genetic fingerprints of virtually any DNA or RNA sequence in any organism or pathogen, according to the company.

Amplifying Genetic Sequences

Sherlock amplifies genetic sequences and programs a CRISPR molecule to detect the presence of a specific genetic signature in a sample. After locating the signature, the CRISPR enzyme activates signaling molecules for detection. The detection system might take the form of a paper strip test or a cell phone display.

In May, Sherlock Biosciences received an EUA from the FDA for its Sherlock CRISPR SARS-CoV-2 kit for the detection of the virus that causes COVID-19, providing results in approximately one hour. The test kit is designed for use in laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 to perform high complexity tests.

The kit works by programming a CRISPR molecule to detect the presence of the genetic signature for SARS-CoV-2 in a nasal swab, nasopharyngeal swab, oropharyngeal swab, or bronchoalveolar lavage specimen. When the signature is found, the CRISPR enzyme is activated and releases a detectable signal.

Detecting Opportunistic Viruses

And at the Max Delbrück Center for Molecular Medicine in Berlin, Germany, CRISPR is being harnessed with urine sampling to improve kidney transplant diagnostic testing. The diagnostic screens for two common opportunistic viruses that infect kidney transplant patients -- cytomegalovirus (CMV) and BK polyomavirus (BKV), and for CXCL9 mRNA, whose expression increases during acute cellular kidney transplant rejection.

Affordable urine-based diagnostic tests have not been widely adapted for nucleic acids, such as DNA or RNA. But CRISPR finds very small segments of a DNA or RNA sequence guided by a complimentary piece of RNA. Scientists used a specific Sherlock CRISPR-Cas13 protocol known to optimize the process for viral DNA. The test is similar to a home pregnancy test. After a paper strip is dipped in the prepared sample, if only one line appears on the strip, the result is negative. Two lines indicate a virus is present.

A patent application is pending. Scientists also are interested in larger clinical studies comparing the assay to conventional monitoring methods. They would also like to make the test more streamlined.

CRISPR technology, a lab development just a few years ago, is beginning to find commercial applications as the technology matures and makes significant inroads in the gene editing, therapeutic and diagnostic fronts.



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