CRISPR is now a common term, one that you see even in mainstream media. Yet it is incredible to think that its potential for editing genes was published just ten years ago. In a mere decade, the groundbreaking discovery has been successfully developed to the point that many clinical trials are underway.

Although the repeating sequences that make up CRISPR were first discovered in bacteria in 1987, it took more than two decades for researchers to discover its true usefulness. Then, 10 years ago, multiple papers were published proving that this intriguing system could be used to edit genes. Emmanuelle Charpentier and Jennifer Doudna went on to win a Nobel Prize in 2020 for their discovery (1), and similar results were published by the Broad team led by Feng Zhang (2) and Virginijus Šikšnys and George Church (3) and their teams.

Researchers quickly began investigating this incredible discovery to determine whether it could truly edit genes, cutting out the portions of DNA that result in disease-causing mutations. Scientists have been incredibly innovative and made astonishing progress in the field.

The swift advent of CRISPR technologies has brought urgency to a host of ethical considerations. For example, at what point, if ever, is it acceptable to edit a human embryo? If editing human embryos is ethical, then should it be confined to medically necessary edits? Or is it alright to make edits that improve quality of life? What about editing for traits that are deemed desirable? And the questions are much broader than the use of CRISPR in humans. Should CRISPR be used to drastically change a crop, bring back extinct species, or create hybrids of current and extinct animals?

Much change has occurred because of CRISPR in the past 10 years – in scientific and medical fields, as well as bringing up ethical and legal concerns. We have previously discussed how CRISPR works and the history of CRISPR, including the ongoing patent battle and potential effects on the industry and the controversy surrounding He Jiankui and babies whose genes had been edited with CRISPR while they were still embryos, in an attempt to make them resistant to HIV. Feel free to review those articles for a more in-depth look at any of those topics. Today we will focus on notable advances in the usefulness of CRISPR, and its future potential.

Turning Ideas into Reality

An astonishing amount of work has been done this past decade to build platforms for generating gene knockouts, rapidly creating knockout mice and other animal models, genetic screening, and multiplexed editing (4).

CRISPR is incredibly useful in the lab, as it is adaptable and precise. It is useful in drug discovery and diagnostics, allowing swift diagnosis of genetic diseases. A team in California even used it to create a diagnostic COVID-19 test and quickly detect new SARS-CoV-2 variants.

CRISPR is currently used to edit T cells (a type of immune cell) outside the body, so they can more effectively destroy the cancer cells. The edited cells are then injected back into the patient. Several Car T therapies have been approved to treat certain types of cancer.

It has drastically reduced the cost of genome sequencing, from $100,000 just over 20 years ago to less than $1,000. Genome sequencing can also be done with greater accuracy when using CRISPR and much more quickly – in 24 hours rather than the five years it originally took (4). This allows ideas such as sequencing the genomes of infants to determine whether they have disease-causing mutations to actually be feasible. With this capability, infants with rare diseases could receive treatment before they ever develop symptoms, perhaps allowing them to live a normal lifespan and enjoy a much improved quality of life than they can expect now.

While CRISPR has been developed at an astounding pace, there are still key challenges to overcome. Once developed and approved, it will be important to ensure both the affordability and accessibility of therapies, and it may be challenging to secure regulatory approval.

Two major challenges in the use of CRISPR lie in editing accuracy (binding to and editing only the target site) and precision (creating the exact desired edit). CRISPR Cas9 also creates a double-stranded break, which may have undesirable consequences. Another challenge is delivering the editors (4). Teams around the world are working to overcome the limitations of CRISPR.

For example, PASTE  (Programmable Addition via Site-specific Targeting Elements) is a new CRISPR tool created by researchers at MIT that can replace an entire defective gene with a functional version without inducing double-stranded DNA breaks. PASTE combines precise site-specific integrases with CRISPR-Cas9, avoiding the double-stranded break by adding first one DNA strand, then its complementary strand via a fused reverse transcriptase (5).

Multiple teams are working on smaller Cas systems. Notably, a miniature Cas system (CasMINI) that consists of only 529 amino acids has been engineered by researchers at Stanford University. CasMINI can delete, activate, and edit genetic code, allows for base editing and robust gene editing, and is highly specific (6).

These and many other advances have been made to overcome various challenges, allowing for some CRISPR therapeutics to be in clinical trials, but much more can be done in these arenas that will help realize the full potential of CRISPR.

In Development – Building on the Foundation

Although an incredible amount of progress has been made in the past decade, the next decade is likely to bring the fulfillment of many ideas, as teams across the globe learn from and build upon the work that has already been done. Here are some notable developments in the field.

Intellia Therapeutics (which was co-founded by Dr. Doudna) and Regeneron Pharmaceuticals conducted the first-ever human study using a systemically administered CRISPR drug (NTLA-2001) in an attempt to treat ATTR amyloidosis. The study showed that NTLA-2001 was safe and provided deep reductions in TTR serum levels.

Intellia was just cleared by the US FDA to begin enrolling patients from the United States in their global ongoing Phase 1/2 clinical trial of NTLA-2002. An in vivo genome editing candidate, NTLA-2002 targets and inactivates the kallikrein B1 (KLKB1) gene, allowing it to treat hereditary angioedema (HAE), permanently reducing plasma kallikrein protein activity after a single dose, preventing HAE attacks.

PACT Pharma and the University of California, Los Angeles (UCLA) used CRISPR to alter the genes of immune cells,  adding certain cancer-specific immune receptors. In a small clinical trial, the team combined CRISPR gene editing with engineering T cells (a type of immune cell) to create personalized treatments for the 16 people who participated in the trial. The study “demonstrated early proof-of-concept that a patient’s immune system can be reprogrammed to recognize their own cancer” (7).

Caribou Biosciences, which was co-founded by Dr. Jennifer Doudna, is working on an off-the-shelf version of Car T therapy that will cut weeks of preparation time. They have developed CB-010, an allogeneic anti-CD19 CAR-T cell therapy designed for patients with relapsed or refractory B cell non-Hodgkin lymphoma. CB-010 was engineered using Cas9 CRISPR hybrid RNA-DNA (chRDNA) technology to insert a CD19-specific CAR into the TRAC gene and knock out PD-1 to boost the persistence of antitumor activity.

It is currently in Phase 1 clinical trial. In December 2022, Caribou shared 12-month clinical data from cohort 1, displaying promising initial safety and efficacy data. After a single dose, all six patients had complete response (no detectable signs of cancer), three of the six maintained a durable complete response (CR) at 6 months, and two maintained a long-term CR at the 12-month scan. Based on the promising initial data, the FDA granted Regenerative Medicine Advanced Therapy (RMAT) and Fast Track designations to CB-010.

CRISPR Therapeutics, co-founded by Dr. Emmanuel Charpentier, now has multiple therapies in clinical trials, treating beta-thalassemia, sickle cell disease, cancer, and diabetes. CRISPR Therapeutics and Vertex are conducting clinical trials of Exa-cel (CTX001), an investigational, autologous, ex vivo CRISPR/Cas9 gene-edited therapy that is being evaluated for patients with beta-thalassemia (TDT) or sickle cell disease (SCD), in which a patient’s own hematopoietic stem cells are edited to produce high levels of fetal hemoglobin (HbF) in red blood cells. Exa-cel elevates HbF, potentially alleviating transfusion requirements for patients with TDT and reducing painful and debilitating sickle crises for patients with SCD. They announced that Exa-cel would be submitted to the U.S. FDA for rolling review beginning in November, with completion of the U.S. submission package in Q1 2023, and that EMA and MHRA submissions were on track.

KSQ Therapeutics uses CRISPR to identify and validate the most selective and potent targets for new cancer therapies and identify and validate in vivo the best tumor-killing targets in T cells for new cancer monotherapies. KSQ utilized its proprietary CRISPRomics® platform to identify the deubiquitinating enzyme USP1 as an attractive cancer target, as is essential for the growth of certain tumors. KSQ-4279 is a first-in-class small molecule inhibitor of USP1 currently in a Phase 1 clinical trial in patients with advanced solid tumors.

Beam Therapeutics creates base-editing drugs. In contrast to CRISPR Cas9, which cuts both strands of DNA, base editing nicks one of the strands of DNA, allowing a single genetic letter of DNA to be precisely changed. Beam has both ex-vivo and in-vivo therapies in development. In November 2022, Beam enrolled the first patient in a Phase 1 clinical trial of BEAM-101, an ex-vivo base editing therapy candidate for the treatment of sickle cell disease.

World-Changing Applications Beyond Medicine

Although CRISPR is still very young, the CRISPR technology market is rapidly growing. This is due in part to its versatility. Not only do CRISPR based technologies have the potential to permanently treat disease, but they have the potential to profoundly impact crops, animals, our understanding of the past, and even humanity’s impact on the global climate and reducing carbon emissions.

This tool that is naturally found in bacteria is not only useful in repairing human DNA, but also in improving plants. It is possible to edit the genes of plants to boost yield, make them more nutritious and more resistant to disease or drought, grow faster, thrive in a different environment, or even taste better.

CRISPR is also being tested to determine if it can change the microbe populations in livestock, in an effort to reduce methane emissions, ultimately decreasing carbon emissions and improving the health of our planet.

Wonderous Future Potential

While not enough is known about CRISPR to edit embryos today with full confidence that it is safe and will not have lasting negative repercussions on the human germ line, it is possible that editing human embryos will provide safe, effective treatments for diseases in the future.

In fact, Dr. Tippi MacKenzie, a pediatric and fetal surgeon at the University of California San Francisco who also directs The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, is developing gene-editing approaches that can be used on a late-second or third trimester fetus. She hopes to address diseases that are easier to treat in utero that would harm the child as it develops further.

As a fetus is further along in development than an embryo, editing a fetus could correct a disease, without passing the edit to that individual’s future children, which was one of the major concerns that arose when He Jainkui announced that he had edited embryos.

Discovering What Cannot Be Imagined

As Joy Wang and Jennifer Doudna wrote, “CRISPR also serves as a notable example of the connection between curiosity-driven research, innovation, and technological breakthroughs. By continuing to explore the natural world, we will discover what cannot be imagined and put it to real-world use for the benefit of the planet” (4).

CRISPR is so versatile that it allows researchers to dream fantastic dreams – that could feasibly become reality in the future. Dr. Zhang of the Broad Institute of Harvard and MIT envisions using CRISPR to improve health by restoring cells to a more youthful, healthier state. This idea was pure science fiction just decades ago and, because of CRISPR, it now could prove to be a life-changing reality in our lifetime.

From reducing the effects of aging, to editing fetuses or embryos to heal disease before it can begin, to editing multiple genes at once to treat complex and common diseases, CRISPR could revolutionize the way humans combat disease, improve quality of life, and even change the health of planet earth.

References:

1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28. PMID: 22745249; PMCID: PMC6286148.
2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23. doi: 10.1126/science.1231143. Epub 2013 Jan 3. PMID: 23287718; PMCID: PMC3795411.
3. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033. Epub 2013 Jan 3. PMID: 23287722; PMCID: PMC3712628.
4. Wang JY, Doudna JA. CRISPR technology: A decade of genome editing is only the beginning. Science. 2023 Jan 20;379(6629):eadd8643. doi: 10.1126/science.add8643. Epub 2023 Jan 20. PMID: 36656942.
5. Yarnall, M.T.N., Ioannidi, E.I., Schmitt-Ulms, C. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01527-4
6. Xu X, Chemparathy A, Zeng L, Kempton HR, Shang S, Nakamura M, Qi LS. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell. 2021 Oct 21;81(20):4333-4345.e4. doi: 10.1016/j.molcel.2021.08.008. Epub 2021 Sep 3. PMID: 34480847.
7. Foy, S.P., Jacoby, K., Bota, D.A. et al.Non-viral precision T cell receptor replacement for personalized cell therapy. Nature (2022). https://doi.org/10.1038/s41586-022-05531-1

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