The Evolution of CRISPR-Cas9 Systems

By Crystal C. Lipsey, Ph.D.

It is well known that scientists revel in using their skills to resolve problems and move technology forward. A stellar example of this concept is the evolution of CRISPR technology throughout its discovery and application. Clustered regularly interspaced short palindromic repeats (CRISPR), which were initially named short regularly spaced repeats, were first characterized by Francisco Mojica, Ph.D. in 1993 (1). Mojica’s continued studies in microbes posited that CRISPR functions as an adaptive immune response in 2005 (2). In the almost 20 years since Mojica and his colleagues hypothesized the CRISPR immune mechanism, many scientists from several countries have published their findings and harnessed their collaborative efforts to shift the focus of CRISPR research from discovery to mechanisms of action. Throughout the 2000s, scientists from around the world have added to the growing body of knowledge that has led to the continuous improvement of CRISPR as a genome editing technology. Luciano Marraffini and Erik Sontheimer demonstrated that CRISPR targets DNA in 2008 (3). A second piece of the puzzle elucidating CRISPR and its association with DNA was published by Sylvain Moineau in 2010. Moineau proved that Cas9 is the protein in the CRISPR system that is responsible for cleaving DNA (4).  

Arguably, the most recognizable names associated with CRISPR are Emmanuelle Charpentier and Jennifer A. Doudna, Ph.D. Charpentier and Doudna were awarded the 2020 Nobel Prize in Chemistry for their contributions that led to “the development of a method for genome editing” (5). Charpentier’s research shows that an RNA duplex composed of tracrRNA and crRNA is necessary to guide Cas9 to its target. Doudna and Charpentier conducted collaborative research demonstrating a simplification of the CRISPR system by creating a single guide by fusing a crRNA and the tracrRNA (6).  The collective efforts of each of the researchers who have studied components of the CRISPR system culminated in what could be called the modern applications of CRISPR systems in academia, industry, and medicine.

CRISPR developments have moved beyond the bench and are now discussed in broader society. One major example is using CRISPR in commercial food and agricultural industries. Genetic modifications to food have long been a topic of discussion in the United States and several crops including apples, corn, potato, soybean, and wheat (7). The Food and Drug Administration (FDA) reports that most of the corn grown in the U.S. is GMO (genetically modified organism). In the 1990s, genetic traits were identified that made plants more resistant to pests leading to the development of a variety of commercially available GMO produce (7). However, until the most recent developments in CRISPR technology, selecting and modifying desired traits in these crops was a years-long process. Currently, commercial agriculture and consumer foods companies in numerous countries hold patents for CRISPR-Cas-modified plants (8; Figure 1). 

As commercial applications for CRISPR rise in industry, the prominence of CRISPR research continues to grow in academia and medicine. In 2013, Feng Zhang, Ph.D. was the first to successfully use CRISPR-Cas9 to edit eukaryotic cells by targeting and cleaving specific genes in human and mouse cells. Scientists have conducted CRISPR-Cas9 research using information from Zhang’s group at the Broad Institute to study different diseases, including albinism, cleft lip, diabetes, HIV, and muscular dystrophy (9; 10). CRISPR-Cas9 technologies are prevalent in cancer research, with many studies focusing on combating chemotherapeutic drug resistance (11). Since 2016, multiple studies have successfully applied CRISPR-Cas technology to advance beyond bench research to clinical trials (10), and the current trajectory of CRISPR-Cas9 clinical trials does not appear to be slowing down. Thirteen active, non-recruiting, interventional studies were returned in a “CRISPR/Cas 9” specific search using the online clinical research database ClinicalTrials.gov in 2024 (12). The trials span a range of different disease states and include hereditary and non-hereditary illnesses (Table 1). Of the 13 trials, four focused on CRISPR-Cas9 cancer interventions for multiple myeloma, renal cell carcinoma, T-cell lymphoma, and B-cell lymphoma. Most of the trials evaluated in Table 1 are Phase 1 clinical trials being conducted at locations in the United States with the remaining trials taking place in Australia, China, France, Italy, New Zealand, and the United Kingdom. While it remains to be seen if these trials will result in approved therapeutic approaches to combat diseases, Casgevy® made history as the world’s first CRISPR-Cas9 therapeutic in November 2023 (13). 

Casgevy® use was approved in the U.S. the following month and is the first FDA-approved CRISPR-Cas9 genome editing therapeutic (14). It is used to treat sickle cell disease in patients affected by vaso-occlusive sickle cell crisis. The CRISPR-Cas9 directed therapy targets and edits the DNA of blood stem cells (15). After editing, hematopoietic stem cells are transplanted back into the patient to increase the production of fetal hemoglobin, prevent red blood cell sickling, and stop vaso-occlusive crisis (14,15). Casgevy®’s landmark approval may spur an increase in clinical trials focused on developing the next generation of CRISPR-Cas9 therapeutics for a myriad of diseases. Commercial use of this drug may also further spark debates on the ethical use of CRISPR gene editing in medicine as technology advances rapidly. It is impossible to be sure where CRISPR will go next, but it is safe to posit that CRISPR’s future will be as storied as its past.

Figure 1. Global perspective of CRISPR-Cas editing agricultural and food industries

Academic institutions and commercial food producers have used gene editing via CRISPR-Cas9 to modify crops. Companies and/or academic institutions hold CRISPR agricultural patents for the following foods and are depicted on the map (map is not to scale): Wheat  (Australia); Cotton (Bulgaria); Brassica Plant/Cabbage family of plant (Canada); Plants (China); Potato (France); Plants (Germany); Cucumber, Carrot, Watermelon (Netherlands); Tobacco (Saudi Arabia); Banana (UK); Cannabis, Consumer Crops, Corn, Plants, Poblano Pepper, Seeds, Soybean, Tomato, Wheat (USA). Source:  Figure 1 was created using the results from a LexisNexis Cipher search query that was published online in Ref. 8 of this article.

Table 1. Interventional CRISPR/Cas9 Clinical Trials – (table source, clinicaltrials.gov; search terms listed reference 12)

References