Putting the Brakes on CRISPR

CRISPR-Cas9, more commonly referred to as CRISPR, has been one of the hottest terms in science over the last few years. For goodness sake, Jennifer Lopez is the executive producer of the prospective NBC bio-terror drama CRISPR, which is centered around the gene-editing technique. Starting in 2012, CRISPR began its rise to becoming the newest and most promising gene-editing tool. It has since transformed the way many labs conduct research and has turned into a multi-billion dollar industry. Even though CRISPR has become more mainstream with seemingly limitless applications, recent research has shown we must move forward cautiously and be patient as this technology matures. 

 

CRISPR Therapeutics currently is the largest biotechnology company that specializes in gene editing via CRISPR. After initially opening at $15 per share on October 19, 2016, CRISPR Therapeutics climbed to $74 per share and had a market cap of $3.47 billion on May 30, 2018. The company’s surge was in response to their announcement in April 2018 that they were moving forward with an Investigational New Drug (IND) application with their partner Vertex Pharmaceuticals Inc. The two companies also had plans to start a Phase 1/2 clinical trial to treat adults with sickle cell disease using CRISPR gene-editing technology in both the United States and Europe. On May 30, 2018, CRISPR Therapeutics announced that the FDA put a clinical hold on its IND application pending the resolution of certain questions by the FDA. The announcement offers no insight into the questions or concerns of the FDA, but this news was enough to spook investors. CRISPR Therapeutics’ stock fell 20.2% in June and 19.1% in July. Currently, it is trading about $48 per share and its market cap is valued at $2.295 billion.

Only part of the CRISPR Therapeutics decline can be attributed to the FDA’s announcement. Three separate studies published this summer negatively influenced investors outlook on CRISPR Therapeutics, as well as the two other major biotechs specializing in CRISPR, Editas Medicine and Intellia Therapeutics. The first study, which was published June 11 in Nature Medicine, highlights how double stranded breaks in DNA caused by Cas9, the molecular scissors of the CRISPR-Cas9 system, are toxic to human pluripotent stem cells (hPSCs). hPSCs, cell lines similar to an early embryo that are capable of differentiating into other cell types, depend on the tumor suppressor protein p53 for its toxic response to prevent growth of aberrant cells. Given that p53 mutations are prevalent in hPSCS, there is also a concern that hPSCs engineered using CRISPR-Cas9 could cause cancer. A second study, published in the same issue of Nature Medicine on June 11, used human retinal pigment epithelial cells and reached a conclusion similar to the previous study, CRISPR-Cas9 induces a p53-dependent DNA damage response. In addition, this group also found CRISPR-Cas9 causes cell cycle arrest. Both studies clearly indicate that it is crucial to monitor p53 function when developing cell-based therapies using CRISPR-Cas9.

A third study, published July 16 in Nature Biotechnology, further casts a cloud of uncertainty over CRISPR. This study revealed that CRISPR-Cas9 causes “significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitor and a human differentiated cell line.” Even though these three articles were all released in the last two months and were the primary reason investors in CRISPR-based companies have been more reluctant to invest, two other studies also show negative effects of CRISPR. One shows an adaptive immune response to Cas9 and the other shows major genomic rearrangements from in a mouse model following the use of CRISPR-Cas9 also show negative effects of CRISPR.

Even though it has been a disappointing summer for companies specializing in CRISPR, CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics remain adamant that the future looks bright. For many people, these studies might be more a speed bump rather than a road block.

Peer edited by Justine Grabiec.

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Move over, Mendel

Recently featured in Science, Valentino Gantz and Ethan Bier have developed a novel genome editing method that subverts traditional heritability. Termed the mutagenic chain reaction, this process  can insert new mutations in the genome that automatically spread themselves to neighboring chromosomes. Thus, homozygous mutants are generated after just one generation, instead of the two generations normally required under Mendelian rules of heritability.Gregor_Mendel_frownsauce

To successfully impart mutations, the system uses CRISPR/Cas9 technology. CRISPR/Cas9 consists of two parts: a bacterial protein (Cas9) that cuts DNA and a guide RNA (gRNA), which determines where cutting will occur. When expressed together in cells, Cas9 cuts DNA at a site in the genome specified by the gRNA. The CRISPR system can be used to silence genes or to insert new genetic material into the genome. In this report, the researchers use genes encoding CRISPR components as their insert. This powerful and potentially dangerous improvement led to creation of homozygous mutants in fruit flies after only one generation.

First, genetic material encoding CRISPR components is injected into a fruit fly embryo. After being expressed in the injected flies, the CRISPR components find and cut the DNA at the gRNA-specified site. After the DNA is cut, the chromosome must be repaired through a process called homology-dependent repair. This is a process by which DNA sequences are exchanged between identical or similar DNA molecules to repair the damaged DNA. If there are extra genes included in one of those sequences, they will be inserted as well. As you might guess, the genetic material that was originally injected carries the aforementioned identical DNA sequences, leading to permanent incorporation of the CRISPR components into the genome.

Next, the process begins again. The second target sequence, residing on the second copy of the chromosome, is cleaved, and the identical DNA sequences from the first chromosome are incorporated. The end result is a genome, previously unedited, that possesses two copies of brand new DNA. Traditional models of heritability dictate that homozygous mutants should take at least two generations to be present in the genome. The mutagenic chain reaction can achieve this effect in only one generation.

MCR Figure

The implications of this research are a double-edged sword. The mutagenic chain reaction has tremendous potential in expediting the creation of model organisms. Additionally, scientists could modify mosquitoes and other human disease carriers, reducing their potential to spread disease. However, once an edit is in the wild, its effects, good or ill, are irreversible. Discoveries such as this are exciting, but must be carefully controlled. The authors acknowledge this fact, and went to great lengths to ensure that their engineered flies never made it out of the laboratory. The flies were kept inside three layers of containment, and were only ever handled by one researcher in secure biosafety areas. The authors even suggest having another major molecular biology conference, similar to the 1975 Asilomar conference on recombinant DNA, to set guidelines for future use of genome editing technology.

Technology like CRISPR and the mutagenic chain reaction hold potential to fundamentally change how humans interact with nature, for better and for worse. Already, much knowledge has been gained from CRISPR. For one, we now know better than ever that rules—even 150 year-old ones—are made to be broken.

edited by Rachel Cohen and Erinn Brigham

Gantz and Bier Article: Valentino Gantz and Ethan Bier, “The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations”. DOI: 10.1126/science.aaa5945

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This article was co-published on the TIBBS Bioscience Blog.