As the first company-sponsored trial of Clustered Regularly Interspaced Short Palindromic Repeats in humans nears its start date, the debate about the gene editing technology's promise to treat or even cure disease persists.

Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, has made headlines over the past seven years, ranging from the treatment of muscular dystrophy in dogs to a scientist injecting himself with a CRISPR enzyme on live television.

The technology requires a derived protein or enzyme system to do its work, which involves cutting and then revising, removing or replacing DNA sequences.

The Cas9 protein most commonly used in CRISPR gene editing was discovered by Jennifer Doudna of the University of California, Berkeley, and the University of Vienna's Emmanuelle Charpentier in 2011 when they successfully used CRISPR in cell-free systems. The enzyme was leveraged for eukaryotic cells — those with a defined nucleus and plasma membrane — by Massachusetts Institute of Technology scientist Feng Zhang in his work for a venture between MIT and Harvard University, known as the Broad Institute.

All the major CRISPR companies license either Doudna and Charpentier's work or Zhang's work, and the three scientists sit on the boards of several of these companies, even co-founding some. Zhang co-founded Editas Medicine Inc. and Beam Therapeutics Inc., while Charpentier co-founded CRISPR Therapeutics AG, which is slated to run human trials this year. Doudna co-founded Intellia Therapeutics Inc., Caribou Biosciences Inc. and newcomer Mammoth Biosciences Inc. Zhang is also an adviser to Pairwise Plants, which offers gene editing for agriculture.

How CRISPR works

The technology deploys RNA to guide the Cas9 enzyme to a specific target DNA sequence, which the enzyme then cuts. From there, the cell can be altered and repaired.

Editas CFO Andrew Hack likened the CRISPR-Cas enzymes to hardware, while the guide RNA is the software.

"You only need to change the guide RNA piece to make the protein go to a new location," he said.

According to Intellia senior vice president of innovative sciences Tom Barnes, there is a "rich collection" of Cas9s evolved from different bacteria.

Editas, Intellia and CRISPR Therapeutics all began with CRISPR-Cas9 from a bacterium known as Streptococcus pyogenes, according to Editas' Hack.

Cas9 from Streptococcus pyogenes has "repeatedly" been found to be the most effective and the most versatile Cas9, CRISPR Therapeutics CEO Sam Kulkarni said.

Editas also uses a smaller Cas9 from another bacterium, Staphylococcus aureus. According to Hack, Editas is the only one among the major human therapeutics CRISPR companies that has explored Cpf1, or Cas12a, in addition to Cas9.

The Cpf1 and Cas9 enzymes target different genome sites, Hack said. He added that it is common to fuse the two RNA pieces required for Cas9 into one long guide RNA, often over 100 nucleotides in length. Cpf1 requires only one guide RNA, approximately 40 nucleotides in length.

"The shorter guide has advantages from a manufacturing perspective," Hack said. "It's tempting to want to compare the features and benefits of each, and I'd hesitate to characterize one as safer or better than the other."

Pairwise Plants, which has partnered with Bayer AG's newly acquired Monsanto Co. for gene editing in crops, also uses Cpf1. CEO Tom Adams described Cpf1 as more akin to an eraser, as opposed to the scissor-like Cas9.

"We can chemically modify the base, ACTG, and take, for example, a C and a G and do a deamination and replace with a different base," Adams said, referring to the removal of an amino group. The aim is to make affordable, healthy food that is "convenient and sustainable," as well as "more snackable."

Mammoth Biosciences, a recent startup backed by tech superstars such as Apple Inc.'s Tim Cook and former Google executive and GRAIL Inc. Vice Chairman Jeff Huber, is using Cas12 and Cas13 as a DNA detector and an RNA detector, respectively, for its "CRISPR search engine" platform, according to CEO Trevor Martin.

Exploring human uses for CRISPR

CRISPR is being explored for treating inherited conditions such as hemophilia, sickle cell disease, beta thalassemia and cystic fibrosis, as well as certain cancers. CRISPR Therapeutics, Intellia and Editas are all exploring these areas. Editas and CRISPR Therapeutics are also looking into reversing the underlying mutations of Duchenne muscular dystrophy, or DMD. Editas is additionally leveraging CRISPR for the treatment of rare eye diseases.

That follows successes in animal studies, notably research published in Science on Aug. 30 describing how CRISPR was used to restore muscular function in four beagles with DMD.

Mammoth Biosciences is an example of using CRISPR as a life science tool. The startup is looking for partners in various industries to dole out its search function capabilities, beginning with disease detection, and potentially moving into agriculture and food safety as well, Mammoth Biosciences' Martin said.

But CRISPR has never been tested in humans by a company.

That is poised to change. CRISPR Therapeutics, partnered with Vertex Pharmaceuticals Inc., plans to launch human trials in Germany for beta thalassemia. While the FDA put a clinical hold on the treatment's investigational new drug application in May for sickle cell disease, both companies have said the phase 1/2 trial's initiation date in the second half of 2018 remains unchanged.

Editas had previously planned to enter human trials for a rare eye disorder in 2017, but the program was delayed due to third-party manufacturing issues. The program, which targets an inherited retinal degenerative disorder called Leber congenital amaurosis 10, is being jointly developed with Allergan PLC.

Editas plans to file an investigational new drug application for the treatment in October, Hack said, as manufacturing issues have been resolved.

Although for many, the prospect of a transformative CRISPR medicine is a far-off speck over the horizon, Hack said he expects product candidates from multiple companies to move into clinical trials over the next couple of years.

Some scientists have warned of the gene editing tool's potential to perform harmful cuts that may unexpectedly damage DNA. One such finding was published in the journal Nature Biotechnology in July.

Intellia's Barnes said the scrutiny is "something that comes with the territory."

"CRISPR is a major force in the life sciences, so famous that you can gain notoriety and publish papers by flagging potential issues with CRISPR," Barnes said. "We're not offended by it, but we systematically and diligently field questions that come in and reaffirm why we think the issues raised don't apply to us."