How Close Is CRISPR to Eradicating Disease and Correcting Genetic Defects?

Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing is among the most promising and exciting advancements in modern medicine in the past decade. In popular usage, CRISPR is shorthand for CRISPR-Cas9. Cas9 is an enzyme that acts like a pair of molecular scissors,¹ capable of cutting strands of DNA at defined sites, allowing existing genes to be removed and/or new ones added in living cells.

CRISPR-Cas9 editing is simple yet powerful, allowing for precise genetic modification of living organisms, with practical applications ranging from correcting genetic defects to treating and preventing the spread of diseases, and even to improving crops. The ability to target specific stretches of genetic code and edit DNA at precise locations, permanently modifying gene function in living cells and organisms, has researchers enthusiastic about the notion of curing over 6,000 known genetic diseases, which directly and/or indirectly affect just about every human on earth.

CRISPR Origins

CRISPR technology was derived from the 2012 discovery of natural defense mechanisms of bacteria and archaea that use CRISPR-derived RNA and various Cas proteins, including Cas9, to thwart attacks by viruses and other pathogens, lysing and destroying the DNA of the foreign invader.¹ This groundbreaking discovery led to the practical application of rewriting human genes, with a host of medical applications and scientific optimism. While CRISPR-Cas9 is perhaps the most discussed of the CRISPR family of technologies, many variants have been generated with the aim of increasing efficiency and precision while limiting errors and off-target mutations.

CRISPR-Cas9 in Clinical Trials

In 2016, Chinese researchers announced they had treated the first person with a CRISPR-Cas9 therapy to fight cancer, disabling the gene that encodes PD-1, which can shield cancer from the immune system.² As of January 2020, ClinicalTrials.gov listed over a dozen active CRISPR-Cas9 studies,³ with new research proposals being drafted around the globe.

In a 2017 Chinese study, gene-edited blood cells were transplanted into a patient to treat HIV, but the recipient showed no signs of clinical improvement.Although the transplanted cells thrived in the bone marrow of the recipient without any major adverse effects, the treatments failed to produce a clear medical benefit. Researchers reported that only 5% of the transplanted cells were edited, which they concluded was not enough to cure the disease, but they are continuing to explore ways to increase the efficacy.⁴

Clinical trials utilizing CRISPR technology in the United States began in 2018, led by researchers at the University of Pennsylvania, who showed that a CRISPR treatment designed to boost the cancer-fighting power of T cells was safe. The results are from three participants — two with multiple myeloma and one with sarcoma, whose T cells were removed and edited in the lab by disabling three genes, with the goal of directing these T cells to tumor cells via a specific cell-surface antigen.⁵

The findings were presented at the 2019 American Society of Hematology meeting, showing that no participant had side effects associated with the treatment, unlike past T cell treatments that have caused high fevers, low blood pressure, seizures, and other side effects. The study also showed that the CRISPR-edited T cells reproduced in the patients. While the safety of the procedure and the successful replication of the T cells in the patients represent positive clinical momentum, the experimental treatment did not slow the growth of the participants’ cancers.⁵

CRISPR Therapeutics and Vertex Pharmaceuticals have treated two individuals with sickle-cell anemia and beta-thalassemia, genetic disorders that deplete oxygen-carrying hemoglobin molecules in the blood. The treatment uses CRISPR to reactivate production of fetal hemoglobin, which is usually only produced shortly after birth. The initial results suggest that the edited cells transplanted into a woman with sickle-cell anemia and another woman with beta-thalassemia are safe, and the treatments seem to have relieved symptoms of the disorders. However, the participants will require follow-up observation over a longer period of time to determine the ultimate outcome. Researchers are optimistic that the treatment could prove to be a one-time curative therapy for patients with these blood disorders.⁵

In another clinical trial being conducted by Editas Medicine in partnership with Allergan, researchers are using CRISPR-Cas9 to delete a gene from cells in the eyes of participants who suffer from Leber congenital amaurosis 10, a form of inherited blindness. This pioneering study is the first aiming to edit cells in vivo, a practice that researchers have previously avoided owing to concerns about off-target effects. As a result, early participants will receive low populations of CRISPR-edited cells under the retina to test for safety, and, if proven safe, later participants will get higher doses.⁶

While no definitive cures have been identified for any of the diseases being studied in CRISPR clinical trials, there is no shortage of researchers and companies who believe in its future outlook, and clinical trials and patent applications should continue to increase annually.

Prime Editing

In November 2019, Dr. David Liu of the Broad Institute of MIT and Harvard introduced an upgrade to CRISPR-Cas9 that could, in theory, correct almost 90% of all disease-causing genetic variations. His research resulted in prime editing, which allows for the deletion and/or the swapping of individual nitrogenous bases, as well as the insertion of new bases into the genome, with minimal DNA damage. In traditional base-editing, the Cas9 protein “scissors” don’t actually cut out a gene — rather, editing occurs when a cell detecting damage to both sides of the snipped double helix initiates DNA repair. However, the process is error prone, leading to unwanted, unexpected, and potentially dangerous mutations. It also makes it difficult to make more subtle modifications to DNA, such as replacing a single thymine base with an adenine.⁷

Prime editing improves this process by increasing the precision and specificity by which a gene is edited, while reducing potential errors in DNA repair. The prime editing system can precisely and efficiently swap any single base for any other, as well as delete and insert nucleotides or larger regions of DNA. The process involves a more efficient use of RNA and reverse transcriptase to direct highly targeted changes. The system comprises a modified version of Cas9 fused with reverse transcriptase and a specially engineered guide RNA, called pegRNA, which replaces the shorter single-guide RNA (sgRNA) used in traditional CRISPR-Cas9. PegRNA contains the required gene edit and steers the editing machinery to the correct genomic site.⁸ Once at the site, Cas9 nicks one strand of the double helix — as opposed to traditional CRISPR-Cas9 tools, which snip both sides. Then, reverse transcriptase uses one DNA strand to prime replication using the pegRNA sequence into the nicked spot, much like the search-and-replace function of word processing software. The prime editing system then makes a second snip of the opposing, non-edited strand, prompting the cell to remake the other strand to match the new genetic information using the new gene as a template.⁷

Ethical Concerns in Science and Society

The scientific world was sent into an uproar when Chinese researcher He Jiankui claimed that he had edited the genes of two human embryos at the Second International Summit on Human Genome Editing in Hong Kong in November 2018, without the consent of ethics and oversight bodies. While there seems to be general enthusiasm among researchers concerning somatic gene editing, germline gene editing contains considerable ethical nuance as safety concerns abound, given the possible ramifications to an infinite number of generations to come. Although he claimed that his work was intended to prevent the transmission of HIV from parents to newborns, the creation of the first genetically edited babies⁹ highlighted the slippery slope of ethical considerations for germline gene editing. CRISPR pioneer Feng Zhang of the Broad Institute responded immediately to He’s announcement by calling for a moratorium on implanting edited embryos in humans, stating, “Society needs to figure out if we all want to do this, if this is good for society, and that takes time. If we do, we need to have guidelines first so that the people who do this work can proceed in a responsible way, with the right oversight and quality controls.”¹⁰

Safety risks, legality, environmental issues, oversight and regulation, moral dilemmas (such as accessibility among various socioeconomic classes), and unintended consequences — both in the present and future — are among the concerns that question the world’s readiness to explore germline gene editing. With continued innovations to create safe, precise, predictable, and inexpensive tools, CRISPR could quite possibly be the future of medicine.

Cas9 Alternatives and Variants Provide Insight into the Competitive Future of CRISPR

While CRISPR-Cas9 is the most popular and widely used CRISPR variant, it is not without limitations and challenges.¹¹ CRISPR research remains in its infancy, and, while researchers are making strides, we are far from a perfectly safe and reliable gene-editing, disease-curing solution. As CRISPR-Cas9 relies on both the Cas9 nuclease and an engineered sgRNA for target location, delivery to the nucleus of the targeted cell can be difficult, causing unwanted effects.¹² As a result, there is no shortage of variants and alternatives that seek to accomplish more precise targeting with reduced potential for unwanted or unexpected results.

Researchers at Duke University successfully used Class 1 CRISPR systems (Cas9 systems are categorized as Class 2) and a Cas3 protein to edit the epigenome of human cells. The Class 1 technique makes use of multiple proteins in a process called CRISPR-associated complex for antiviral defense (Cascade). This complex binds with high accuracy to the correct sites and uses a Cas3 protein to target and edit the DNA. The research team was also able to both activate and repress target gene expression. The team characterizes this system as an enhancement of CRISPR technologies, as it provides a potential alternative for CRISPR-Cas9 when there are complications such as immune responses to Cas proteins. It can also recruit various modifiers of gene regulation, including activators and repressors.¹²

In an effort to multiplex CRISPR systems to target numerous genes simultaneously, researchers at ETH Zurich in Switzerland swapped Cas9 for Cas12a, which allowed them to simultaneously edit genes at 25 target sites. Cas12a allows for shorter sgRNA address molecules compared with Cas9, permitting more to fit on a plasmid. While speed is one objective of the Cas12 system, it also allows for simultaneous upregulation of some genes and downregulation of others.¹²

Cas13a targets RNA as opposed to DNA. Its application can be seen in the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) tool, which is being used as a molecular detection platform to detect specific strains of Zika and Dengue virus. Its application further enables researchers to distinguish pathogenic bacteria and genotype human DNA and to identify mutations in cell-free tumor DNA.¹³ Cas14 is related to Cas9 but only one-third the size of the Cas9 protein. While Cas9 was isolated from bacteria, Cas14 was found in the genome of a group of archaea. Due to its size, it could be an alternative for editing genes in small cells or in some viruses. More practically, with its single-stranded DNA-snipping ability, it is likely to improve rapid CRISPR diagnostic systems¹⁴ and be utilized as a detection tool, working in tandem with Cas12 and Cas13 to enable detection of RNA, double-strand DNA, and single-strand DNA, with the potential to more quickly diagnose various diseases.¹⁵

Some researchers are also testing whether anti-CRISPR proteins (“kill switches”) have a place within the CRISPR family of tools. Given the unpredictable nature of the current systems, one can argue that the ability to turn off a CRISPR system may be an invaluable tool to prevent, slow, or stop unintended edits and/or unwanted outcomes and reduce off-target effects.¹⁶

There are a myriad of other CRISPR variants and alternatives being studied and tested in labs around the world. While current CRISPR tools are effective at cutting DNA, the possibility of random repair remains.¹² The future of genome editing will require new tools to enable more precise changes to the genome, while also eliminating random, unexpected, and/or unwanted outputs. Safety, precision, and predictability are the most important factors determining the future of a widespread application of CRISPR technologies in patients.

References

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3. ClinicalTrials.gov. U.S. National Library of Medicine. Jan2020. Web.

4. Ledford, Heidi. “Quest to use CRISPR Against Disease Gains Ground.” Springer Nature Limited. 6 Jan 2020. Web.

5. Saey, Tina Hesman. “The First U.S. Trials in People put CRISPR to the Test in 2019.” Society for Science & the Public. 16 Dec. 2019. Web.

6. Saey, Tina Hesman. “CRISPR Enters its Frist Human Clinical Trials.” Society for Science & the Public. 14 Aug. 2019. Web.

7. Fan, Shelly. “Everything You Need to Know About Superstar CRISPR Prime Editing” Singularity University. 5 Nov. 2019. Web.

8. Collins, Francis. “Gene-Editing Advance Puts More Gene- Based Cures Within Reach.” U.S. Department of Health and Human Services. National Institutes of Health. 5 Nov. 2019. Web.

9. Wee, Sui-Lee. “Chinese Scientist who Genetically Edited Babies Gets 3 Years in Prison.” The New York Times. 30 Dec.

10. Bergman, Mary Todd. “Perspectives on Gene Editing.” The Harvard Gazette. 9 Jan. 2019. Web.

11. “How to Choose the Right Cas9 Variant for Every CRISPR Experiment.” Synthego. Jan. 2020. Web.

12. Rees, Victoria. “How will CRISPR Evolve in the Future?” Drug Target Review. Russell Publishing Limited. 26 Nov. 2019. Web.

13. “Nucleic acid detection with CRISPR-Cas13a/C2c2.” National Center for Biotechnology Information. U.S. National Library of Medicine. 13 Apr. 2017. Web.

14. Sanders, Robert. “Smallest Life Forms have Smallest Working CRISPR System.” University of California, Berkley. 18 Oct. 2018. Web.

15. Giquel, Benoit. “CRISPR-Cas14: A Family of Small DNA- Targeting Enzymes Enabling High-Fidelity SNP Genotyping.” Addgene.org. 29 Nov. 2018. Web.

16. Dolgin, Elie. “Kill Switch for CRISPR Could Make Gene Editing Safer.” Scientific American. Springer Nature America, Inc. 17 Jan. 2020. Web.