CRISPR—Deliverer or Disruptor?

Inborn errors of metabolism (IEM) cause hereditary multisystemic metabolic diseases (HMD), and classically they result from one or more dysfunctional enzymes or defects in the transportation of proteins. The dysfunctionality is caused by mutations in genes that code for the enzymes; enzyme deficiency or inactivity leads to accumulation of substrate precursors or metabolites, or to frank deficiencies of the enzyme’s products (Camp 2012) (Scriver, Sly, Childs, et al. 2000).

IEM are not rare diseases when we observe their cumulative incidence. Moreover, more than 300 diseases caused by IEM are known today, and this number is constantly growing as an artifact of novel and emerging molecular techniques for the various biochemical phenotypes. However, the clinical diagnosis of HMD incidence does not seem to be increasing in parallel, perhaps as a consequence of limited routine genetic workup (Weaver, Johnson, Singh, Wilcox, Lloyd-Puryear, and Watson 2010) (Camp 2012).

Apart from acute resuscitation and supportive care, present chronic management of IEM is largely limited to diets, medical foods, and dietary supplements formulated to exclude components that a patient is incapable of processing because of his condition. This strategy helps to minimize or eliminate the buildup of toxic metabolites that result from the block in metabolism while maintaining adequate nutrition for growth and development.

Some in the medical and plant biology fields consider that gene editing technologies will contribute considerably to improving the quality of life and assisting in providing safe and nutritious foods. One of those technologies is known as “clustered regularly interspaced short palindromic repeats” [CRISPR)/CRISPR-associated (Cas)], originally considered a bacterial and archaea adaptive defense system (Bhaya 2011) (Jinek, Chylinski, Fonfara, Hauer, Doudna, and Charpentier 2012). This technology represents a potential curative intervention for IEM.

The basic principles of CRISPR/Cas (such as -Cas9, -Cas12a) permit investigators to edit out diseases in humans, plants, and other organisms. Fundamentally, RNA templates transcribed from the CRISPR regions are closely associated with an enzyme system known as CRISPR-associated protein 9, or Cas9. If the RNA template finds a match in a viral invader’s DNA, the enzyme chops up the DNA to destroy it. In the same way, CRISPR/Cas9 and other related gene-editing endonucleases can be paired with locations in any genome for use as an editing tool.

CRISPR/Cas9 and CRISPR/Cpf1 technology has generated considerable interest in transforming plant cells (Yin, Gao, and Qio 2017). In recent research efforts, genes from the monocots corn, sorghum, sugarcane, and indica rice were successfully transformed, which suggests that these food crop transformations could contribute to advance plant growth and maturation (e.g., ripening), alter complex agronomic traits, increase nutritional value, expand plant varieties and crop yield, improve drought stress tolerance, and increase disease, pest, and herbicide resistance (Lowe, Wu, Wang, et al. 2016). Importantly, Cpf1, also known as Cas12a, is more precise in gene editing and yields more consistent DNA cleavage patterns, while generating larger indels (insertions/deletions) and minimizing off-target mutations (Strohkendl, Saifuddin, Rybarski, Finkelstein, and Russell 2018). Certainly, the natural cellular DNA repair process, like this gene-editing technology, can introduce indels, which in turn can disrupt the translation of a specific or targeted gene. Regardless of these encouraging observations where off-target effects appear to be very low, these effects remain challenging and call for attention in plant gene editing.

The relatively brief history of CRISPR/Cas9 is not without drama. A pair of papers published in 2018 in Nature Medicine showed that two kinds of cell lines are often resistant to CRISPR gene editing unless a protein called p53 is broken or missing (Haapaniemi, Botla, Persson, Schmierer, and Taipale 2018) (Ihry, Worringer, Salick, et al. 2018). P53 is recognized for its role in initiating DNA repair in broken genes, and since cells lacking functional p53 are prone to become cancerous, these manuscripts advised of the critical importance that CRISPR-edited cells should be screened for p53 before targeting them in human therapies.

Malignant transformation has always been a concern for any intervention that ruptures DNA. Cas9 snips across DNA’s double-stranded helix at a particular location specified by a guide RNA. Guide RNAs are designed to minimize off-target, unintended cutting, but there is a risk that unintended cutting may occur. One off-target cut does not necessarily equal malignancy, but as the probability of inadvertent snips increases, the odds of rupturing an important tumor suppressor gene, such as the gene for p53, increases.

Many clinical trials using CRISPR are underway in China, and the public’s fears of editing the germline seem inevitably to set the stage for strident and important controversy. For example, this technology was applied to mutant cells and 18 heterozygous embryos, thereby correcting 16 of the FBN1 mutations responsible for Marfan Syndrome (Zeng, Li, Li, et al. 2018). This was a follow-up germline editing study using CRISPR-Cas9 reported earlier where the MYBPC3 gene mutation responsible for hypertrophic cardiomyopathy was corrected in 42 out of 58 preimplantation embryos (Ma, Marti-Gutierrez, Park, et. al. 2017). FBN1 codes for fibrillin, a protein that is a critical component of connective tissue. Marfan Syndrome is an autosomal dominant disorder associated with abnormal aortic dilatation, which leads to subsequent aortic aneurysms and possible dissection. The incidence of this rare congenital malady is about 200,000 cases per year within the United States.

Ethical Considerations
In 2017, the National Academy of Sciences stated that “editing to prevent transmission of genetically inherited diseases may become a realistic possibility” yet emphasized that “clinical trials using heritable genome editing should be permitted only within a robust and effective regulatory framework” (National Academy of Sciences 2017). In July 2018, the UK Nuffield Council on Bioethics issued a qualified statement that “heritable genome editing interventions to influence the characteristics of future generations could be ethically acceptable in some circumstances” (Nuffield Council on Bioethics 2018). According to a 2018 poll by the Pew Research Center, 72% of adult respondents in the United States indicated gene editing would be acceptable to treat a serious disease or condition a baby would have at birth, but 80% of the same participants rejected this technology to make a baby more intelligent (Pew Research Center 2018).

As with many new technologies, there are regulatory hurdles, research challenges, agriculture-related opportunities, and consumer concerns as well as, in this case, bioethical considerations. In 2016, the U.S. Department of Agriculture noted that since the CRISPR/Cas9 white button mushroom did not contain any foreign DNA integrated into the mushroom genome, additional agency approval was not required. However, the agency required a notification or permit for importation, interstate movement, or environmental release of the mushroom with the new non-browning trait (USDA 2016).

Research Priorities
Approximately nine months later, the U.S. Food and Drug Administration reassured the agency’s science-based approach to genome editing technologies. The agency’s current gene-editing research includes four major areas: 1) treating HIV, cancer, or rare diseases; 2) controlling pathogenic organisms and vectors; 3) improving health and welfare of food animals; and 4) altering specific traits of food plants or fungi (Califf and Nalubola 2017).

In December 2015, the National Academy of Sciences and the National Academy of Medicine’s Human Gene-Editing Initiative, co-hosted with the Chinese Academy of Sciences and the United Kingdom’s Royal Society, convened the first international summit in Washington, D.C., on human gene editing. The participating experts, who discussed scientific, ethical, legal, social, and governance issues associated with human gene editing, reinforced the importance of safety and efficacy of the new tools for clinical applications. They also recognized the potential of off-target effects, which could inactivate essential genes, activate cancer-causing genes, or cause chromosomal rearrangements. The panelists and participants came to the following conclusions: 1) additional research, being sensitive to ethical, legal, and social issues, is required, especially when considering altering germline cells and human embryos; 2) with respect to clinical applications, there is need to understand the risks, such as inaccurate editing, and the potential benefits of each proposed genetic modification; and 3) germline editing poses many important issues, including the risks of inaccurate editing (such as off-target mutations), the difficulty of predicting harmful effects of genetic changes, the obligation to consider implications for both the individual and the future generations who will carry the genetic alterations, the fact that, once introduced into the human population, genetic alterations would be difficult to remove and would not remain within any single community or country, the possibility that permanent genetic ‘enhancements’ to subsets of the population could exacerbate social inequities or be used coercively, and the moral and ethical considerations in purposefully altering human character and longevity using this technology.

Risks and Benefits
The next international summit on human genome editing will convene in Hong Kong in November 2018. The summit organizers note that since 2015, research on the human genome has escalated while gene editing continues to raise questions on the risks and benefits of such technology and its potential lasting health and clinical effects; the potential ethical and cultural issues and perspectives; the challenges associated with legal, regulatory, and policy development; and the critical importance of effective public communication and engagement.

CRISPR/Cas9 and related proteins represent astonishing opportunities in human health and the food supply, but there is much we do not know and must learn about this technology. The global scientific community is learning rapidly, but the optimism must be tempered by scientific humility and imagination regarding unanticipated consequences.

Roger Clemens, DrPH, CFS, Contributing Editor
Adjunct Professor, Univ. of Southern California’s School of Pharmacy, Los Angeles, Calif.
[email protected]

Peter Pressman, MD, is director, The Daedalus Foundation ([email protected]).