COMMENTARY

5 Things to Know About CRISPR

Ricki Lewis, PhD

Disclosures

July 20, 2018

We've been hearing about CRISPR since 2012, and excitement has been mounting about this molecular tool that can add, remove, or exchange a DNA sequence within the genome of a living organism. Meanwhile, basic research is continuing to troubleshoot the fledgling field, such as the recent finding that the intervention might introduce a risk for cancer. But how exactly does it work, and what is its potential for transforming medicine? Here are five things clinicians should know.

1. CRISPR was inspired by a nucleic acid–based mechanism that bacteria deploy to recognize and remove viruses from their genomes.

CRISPR stands for "clustered regularly interspaced short palindromic repeats," which are repeated short DNA sequences that are palindromes (reading the same in both directions) interspersed with short, nonrepeat "spacers." CRISPRs originally were discovered in archaea and bacteria, where the spacers were bits of DNA from infecting viruses.

The spacers are transcribed into short CRISPR RNAs that attract a cutting enzyme, such as Cas9 (CRISPR-associated protein 9). The complex of CRISPR-Cas9 then searches the DNA for matching spacer sequences and, using natural DNA repair, cuts them out by breaking the double helix across both strands. In this way, a bacterial cell can recognize bits of viral DNA in future encounters and promptly remove them, akin to an animal's immune system.

Cas9 was the first DNA-cutting enzyme used with CRISPRs. Others, with differing targets, include Cas13 and CPF1.

2. Retooled for biotechnology, CRISPR uses synthesized "guide RNAs" to selectively target a specific DNA sequence.

The guide RNAs, corresponding to the targeted viral sequences embedded in a bacterial genome, attract Cas9 or another enzyme to the location in the genome where a researcher wants to intervene, like a drone. Therein lies the technology's versatility: Guide RNAs can be designed to add, replace, or remove a specific DNA sequence.

Strategies to treat sickle cell disease illustrate CRISPR's eclecticism and investigators' creativity. In one approach, for instance, researchers cultured induced pluripotent stem (iPS) cells from patients' skin fibroblasts and then used CRISPR-Cas9 to correct one copy of the single-base mutation that causes the disease. The red blood cells that descend from the altered iPS cells produce adult beta globin and are not sickled. In another approach, using hematopoietic stem cells, researchers used CRISPR-Cas9 to cut small deletions in a gene, BCL11A, which regulates the molecular switch from fetal to adult hemoglobin. The descendant red blood cells produce fetal hemoglobin, restoring the blood's oxygen-carrying capacity.

3. CRISPR gene editing is more precise than the traditional concept of gene therapy.

Gene therapy pioneered in the 1990s introduced copies of wild-type (functional) genes that are deleted or mutant in a particular disease using viral vectors, liposomes, or directly through electroporation. It added genes; it didn't replace them as CRISPR can.

Although CRISPR uses some of the same viral vectors as gene therapy, the cargo is more targeted. In traditional gene therapy, the introduced gene either lies outside the chromosomes in a ring of DNA called an episome, or integrates into a chromosome randomly or at any of several binding sites. (A gene therapy that unexpectedly disrupted an oncogene caused leukemia in boys who had been treated successfully for an inherited immune deficiency in 2002, derailing the field for 2 years.) CRISPR instead deposits its cargo where the guide RNA takes it: to a pre-set, exact location in the genome. This precision is why CRISPR is called "gene editing" rather than "gene therapy."

4. Patients may have an inflated idea of CRISPR's current applications.

Patients who saw the recent 60 Minutes TV segment about CRISPR might conclude that we are on the brink of a tantalizing array of new cures. But a quick check of the database ClinicalTrials.gov reveals only a handful of trials involving CRISPR, and half of those alter patients' cells ex vivo, which must then be delivered to their bodies.

The first clinical trial for a traditional gene therapy was in 1990, yet the first FDA approval for a gene therapy to treat a Mendelian condition came nearly 28 years later, in December 2017. That was for Luxturna, to treat a form of retinal blindness. CRISPR is far behind, but the excitement and energy may drive an accelerated regulatory trajectory.

Meanwhile, preclinical work is progressing for several single-gene conditions, including Leber congenital amaurosis 10, Usher syndrome, beta-thalassemia, Duchenne muscular dystrophy, cystic fibrosis, transthyretin amyloidosis, alpha-1 antitrypsin deficiency, and primary hyperoxaluria type 1, as well as CAR-T approaches against cancers (see Editas Medicine, Intellia Therapeutics, and CRISPR Therapeutics).

5. CRISPR's applications transcend medicine and may spark controversy.

The roots of CRISPR trace back to 1980s-era investigations of homologous genetic recombination and DNA repair, but as a biotechnology ,CRISPR has evolved at a dizzying pace since 2012, and not without drama. Its continuing story now includes a bitter patent dispute, controversy over potential "designer babies" and germline alterations, and fears of controlling pests to the point of species extinction.

Recent reports offer a smorgasbord of CRISPR applications:

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