The Future of CRISPR: Three Areas The Nobel Prize Winning Tech Will Be Most Impactful
By Ruchi Jhonsa, Ph.D.
“CRISPR is a wonderful tool that allows one to ask and answer questions that were impossible to address previously and will help solve problems for humanity” –Dr. Jennifer Doudna, Nobel Laureate in Chemistry, 2020.
Declared as one of the most important discoveries of the 21st century, CRISPR technology has revolutionized the field of gene editing. The technology derived from bacterial defense mechanisms is now used for treating serious genetic diseases, restore eyesight in people with inherited blindness, engineer crops that are disease resistant and tolerant towards environmental stress, and eliminate disease-causing organisms.
Moreover, the technology is rapidly evolving and has found a place in many other applications besides gene editing. The impact of CRISPR in the biological world is so immense that the pioneers of the technology were recognized and granted the prestigious Nobel Prize in Chemistry for the year 2020.
While the technology has proven itself in the lab, many caveats need to be filled before reaching its true potential in the future. One of them is the delivery of CRISPR machinery to its destined location. According to Dr. Jennifer Doudna, this is one “place where the most innovation is needed.” Currently, CRISPR modifications are done in the cells extracted from the body. However, Dr. Doudna envisions a day when this can be done inside the body. “That would be absolutely transformative,” she opined.
Besides its application in medicine, CRISPR can have a global impact, especially in mitigating climate change. In recent years, its impact has become evident, particularly in agriculture. Several researchers are trying to solve this problem by making plants resilient to climate change. But the other way to look at it is through the lens of microbiology. Making genomic changes to soil microbiome to fix carbon could help lessen the impact of climate change.
Creating Resilient Plants and Animals
“(The) Power of CRISPR in agriculture is enormous. In the biomedical field, CRISPR will help hundreds of thousands of people, in agriculture CRISPR will help billions of people,” said Brian Staskawicz from UC Berkeley at the World CRISPR day virtual conference.
Supporting billions of mouths with conventional plant or animal breeding is practically impossible as challenges like plant or animal diseases keep emerging. The Food and agriculture organization estimates that up to 40% of food crops are lost due to plant pests and diseases annually. However, by combining conventional breeding with genome editing technology such as CRISPR, it is possible to achieve goals that may not have been possible in the past.
To construct durable resistance for plant diseases, scientists are using multiple approaches. One of them involves combining multiple layers of plant immunity via generating mutations in alleles that confer bacterial susceptibility and expressing pattern recognition receptors that confer natural immunity to plants.
Similarly, in animal farming, CRISPR, combined with traditional breeding, is improving animal health and, in return, animal productivity. It does so by knocking down the genes that make animals susceptible to respiratory viruses and tuberculosis, increasing male progeny by introducing segments of Y chromosomes on autosomal chromosomes, and increasing muscle yield by knocking down the myostatin gene.
CRISPR Based Functional Screens to Enable Drug Discovery
“Human genetics can be an important guide for drug discovery and drug targets,” said Dr. Martin Kampmann, UCSF, which can be studied using vast genomics, proteomics, and transcriptomics data sets. However, a different approach to studying the human genome is by perturbing the function of a large number of genes and observing the phenotype.
One way to do that is by inactivating or activating the genes by CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) methods. This method utilizes the power of guide RNA to locate the desired gene. However, instead of active Cas9 that nicks the target site, this method utilizes inactive Cas9 fused with a gene’s repressor or activator. The perturbation of many genes in this manner, along with selection pressure, facilitates the identification of genes relevant for specific cellular functions and can help uncover important drug targets.
They are particularly powerful in combination with induced pluripotent stem cell technology, which enables differentiated cell types, such as neurons and glia, from cells obtained from patients. This helps develop models of neurological diseases and facilitates the identification of potential disease mechanisms and relevant therapeutic targets.
The technology has helped many pharmaceutical companies to identify innumerous drug targets. Genentech, a member of Roche Group, uses such selection-based pooled CRISPR screens regularly to identify regulators of fundamental processes, including various forms of cell death.
Necroptosis is one of the cell death processes that work independently of caspase proteins. Using pooled screens, scientists could identify novel proteins that regulate the Necroptosis pathway. The company also routinely performs fitness-based pooled CRISPR screens for identifying modifiers of drugs or new synthetic lethality in cancer.
While these screens yield powerful synthetic lethal partners for the protein of interest, they hold true for only the model in which they are being tested. In such scenarios, Genome-wide CRISPR knockout screens across hundreds of cancer models are beneficial. With this, one can identify targets, which can work across a subset of models rather than just one.
Pooled screens are powerful yet are restricted to simple readouts, including cell proliferation and sortable marker proteins. But a step ahead and which, according to Dr. Martin, “everyone is leveraging” is a combination of pooled CRISPR screening with single-cell RNA sequencing that directly links guide RNA expression to transcriptome responses in thousands of individual cells.
CRISPR Technology in Disease Diagnosis
Although the CRISPR technology was developed to precisely edit the genome, scientists have developed new applications that could help with the quick diagnosis of disease pathogens or detect mutations in cells without the need for gene amplification.
Cardea Bio has developed a novel application of CRISPR, which is based on the ability of the guide RNA to detect pieces of the genome. The company has developed a CRISPR-chip that is an electric transistor fused with a CRISPR complex targeted against various genome mutations. When a sample containing the mutated DNA is passed through the transistor, it binds to the CRISPR guide RNA and generates an electrical signal. This application can detect deletions and SNPs in genomes and test the efficacy of various guide RNAs binding to target sequences and efficacy of various Cas versions in binding to target sequences.
In the real world, these electronic powered sensors fared in the detection of Duchenne Muscular Dystrophy mutation in DMD samples and sickle cell disease mutation in SCD samples-something that’s difficult to accomplish without gene amplification. These sensors are remarkable as they forego the need for DNA amplification-which takes hours-thereby, reducing the time of sample detection. This factor is especially important during a pandemic such as COVID-19 when fast and accurate detection is necessary to curb the virus.
CRISPR technology can also be used for sensitive and specific nucleic acid detection in clinical diagnostics and genotyping. In the last few years, scientists have utilized the Cas9 protein variant, Cas13a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the attomolar level.
Cas13 is an RNA-guided RNAse that can cleave ssRNA non-specifically in the samples derived from pathogens or viruses. Once Cas13 recognizes and binds to the programmed sequence, the non-specific RNAse activity of Cas13a becomes activated. This not only results in cleavage of pathogen ssRNA but also of fluorescently tagged reporter ssRNA added in the reaction.
Cleavage of the quenchable fluorescent RNA by the activated Cas13 produces a quantifiable signal that indicates the presence of target nucleic acid. This method is famously known as SHERLOCK, developed in the Zhang lab at the Broad Institute and licensed by Sherlock Biosciences. In the future, Sherlock can find use in the detection of viral DNA, antibiotic resistance genes, cancer detection, tracing traits in plants, and several other applications. This method was recently used in the development of an FDA approved CRISPR-based SARS-CoV-2 detection kit.
Another cool application derived from CRISPR technology is RNA editing, which involves making changes in the RNA to generate a transient phenotype. This technology finds potential application in processes that need only transient activation of a gene, such as in the case of damaged tissue repair, where transient activation of TGFbeta signaling by editing phosphorylation sites on beta-catenin allows for regeneration of the damaged tissue.
According to Dr. Jonathan Gootenberg from MIT, CRISPR-based RNA editing, which combines CRISPR tools with RNA modifying enzymes and can be used for multiple other applications, including modification of translation or localization of the protein and making epitrancriptomic modifications.
Editor: Rajaneesh K. Gopinath, Ph.D.
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