CRISPR: As Powerful a Tool in Finding Disease As It Is in Treating It

CRISPR: As Powerful a Tool in Finding Disease As It Is in Treating It

In the world of genetic research, the major breakthrough of the decade is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), a gene editing methodology with vast potential in treating devastating disease. CRISPR has been demonstrated successfully, showing that genes that transcribe for potentially lethal conditions can be edited out; when the technique is performed in the germline, the edits are hereditary, which would ostensibly eliminate the risk in future generations, perhaps potentially eradicating hereditary diseases. Kalorama has produced a white paper focusing on gene editing and a full report on gene editing markets.

 The term CRISPR itself refers to specific repetitions found in prokaryotic DNA – found in 40% of all sequenced bacterial genomes and 90% of sequenced archæa. Each repetition is followed by short segments of “spacer” DNA from previous exposures to bacterial plasmids or viruses. The methodology called CRISPR/Cas refers to a system of two components. A guide RNA (gRNA) is inserted into a cell, where it travels along the genome, searching for a specific nucleotide sequence. Once the sequence is found, the accompanying Cas9 enzyme cleaves the DNA molecule; here, the gene could be shut down and removed, or a new sequence can be inserted.

 Some potential problems exist, from unknown deleterious effects of editing the genome, problems that could arise if the target sequence is somehow missed and a mosaic is created, and of course, the inevitable ethical questions that come with altering the genetic foundation. There have also been questions as to CRISPR’s scalability, but the process is being made more systematic even at this relatively nascent point in its existence, with the widening production of tools and reagents that resemble those of other genetic methodologies, from oligo libraries to assay kits – the entire human genome is even available as a synthetic CRISPR RNA in a 96- or 384-well plate format for use in arrayed screens.

 The most obvious applications for CRISPR lie in gene therapy, while consideration is being made as well into other avenues of development, such as pharmaceuticals and agriculture, and there is incredible potential in etiological research by way of easily creating new genetic animal models. But a variant of the technique, called SHERLOCK, could become a vital and versatile tool in diagnostics and outbreak management. SHERLOCK, or Specific High-sensitivity Enzymatic Reporter unLOCKing, is a CRISPR-based system in that it uses a gRNA and an enzyme to find and cut DNA, respectively. A crew led by bioengineer James Collins and CRISPR pioneer Feng Zhang developed the technique at the Broad Institute at MIT and Harvard, based on work by structural biologist Jennifer Doudna of UC Berkeley, who was among the first to see the diagnostic potential of CRISPR.

 The method is a bit different from the CRISPR/Cas we all know, as it uses a different enzyme, Cas13a (or C2c2, using Doudna’s nomenclature), and instead of cutting at the specific sequence, multiple cuts are made in the surrounding area, an effect referred to as “collateral cleaving” in the original article describing the technique. It was concluded in Doudna’s findings that, for the technique to be effective, a much greater degree of sensitivity would be necessary; to achieve such sensitivity, Zhang’s team first used an isothermal amplification method to boost the resulting DNA amplicon, and the sample was expanded further by splitting the DNA into RNA. Finally, included in the process is an RNA probe that fluoresces when cleaved, which provides a sort of a signal boost, indicating that the sequence was indeed detected. The result: a diagnostic assay that can detect its target at the attomolar level; that is, a test 1000 times more sensitive than a previous CRISPR-based Zika diagnostic Collins and team designed in 2016, and a million times more sensitive than the commonly used ELISA.

 SHERLOCK has vast potential in molecular point-of-care diagnostics. It can detect Zika in blood within hours; better yet, it could do so with urine samples, which is safer and less invasive. Users can distinguish between different bacterial and viral species, such as E. coli from E. ærogenes or Dengue from Zika from Chikungunya, and between variations of pathological agents, such as African and American strains of Zika or DNA from tumors released into the bloodstream – which could differ by just a single nucleotide. SHERLOCK can also detect genes that confer antibiotic resistance, identify cancerous mutations in simulated cell-free DNA fragments, and rapidly read genetic information from saliva samples to detect things like heart disease risk factors.

 Of course, there are plenty of powerful solutions capable of producing relatively rapid and accurate results. However, the reagents used in SHERLOCK can be freeze-dried and carried out on glass fiber paper, meaning that SHERLOCK can be highly portable, and very inexpensive – a paper test implementing the technology could be designed and used for well under a dollar per test. Between the low cost and the ability to immediately process raw samples to facilitate a rapid response, SHERLOCK is potentially the ideal solution for disease outbreaks in resource-poor regions.

 Collins says that he and his team at the Broad Institute is planning a startup company once investment funding has been confirmed, though there are regulatory hurdles to pass from the United States Food and Drug Administration as well.