Snapshot: What is CRISPR?

A common nuisance for bacteria is the bacteriophage: a virus that uses the internal machinery of a bacteria to replicate its own genetic material. Bacteriophages do this by latching onto bacteria and injecting their DNA into the cell. As the cell grows and divides, the bacteriophage’s hope is that their genetic material is replicated alongside the bacteria’s own genome. Unfortunately for bacteriophages, many bacteria have evolved a method to fight off their attacks. After recognizing a viral infection, the bacteria integrate portions of the injected viral DNA into their own genome. The area where these viral DNA segments end up is known as the CRISPR sequence (short for clustered regularly interspaced short palindromic repeat). The viral DNA segments that were integrated into the CRISPR sequence are then replicated and attached to a bacterial protein called Cas9 (CRISPR-associated protein 9). These CRISPR-Cas9 pairs patrol the cell, acting as the bacteria’s antiviral immune system. If the same viral infection happens again, the DNA in one of the CRISP-Cas9 pairs will match part of the injected viral DNA and bind to it. Once bound, Cas9 cuts the viral DNA, which is then destroyed.

a DNA molelcule that has a fragment cut out of it. Scientific drawing and scribble are faint in the background
Artist’s cartoon of DNA that has been cut by CRISPR. Image courtesy of the NIH.

Recently, scientists have found a way to harness this system for manipulating genes (a process broadly called genetic engineering). By making an artificial CRISPR sequence, attaching that sequence to Cas9, then introducing the man-made CRISPR-Cas9 into a cell, it becomes possible to make a targeted cut in any gene. Making a CRISPR-Cas9 pair that targets one specific gene is as simple as making a CRISPR sequence that matches that gene.

Unlike in bacteria, most organisms repair rather than simply destroy cut DNA. This leaves the targeted genetic sequence available for further manipulation, including the introduction of a short mutation or even the insertion of a whole new DNA sequence. In essence, using the CRISPR-Cas9 system, scientists are now able to edit genes in a simple, targeted way.

CRISPR-Cas9 has become quite popular as a genetic tool in research settings: as of now, the genomes of anything from worms and fruit flies to mice and monkeys have been altered using this technique. While its use in humans is still in its early stages – the first patient treated using CRISPR began therapy earlier this year – is plausible that CRISPR-Cas9 could prove useful in altering the genomes of patients with genetic disorders (like, for instance, the SCAs). For patients, this might sound like a miracle cure. However, it is important to note that several concerns remain as to the ethics of human genetic engineering – the concept of “designer babies” being one of them.

If you’re interested in reading more about the conversation around CRISPR and bioethics, check out the articles by NPR and the National Human Genome Research Institute.

Snapshot written by Logan Morrison and edited by Dr. Maxime W. Rousseaux.

Snapshot: What is Gene Therapy?

Gene therapy is using nucleic acids to treat a genetic disorder.  These nucleic acids can be designed in a variety of ways to achieve the same therapeutic outcome. Gene therapy tools can be used to correct a mutant gene by one of three ways:

  1. Expressing a healthy copy of a gene
  2. Silencing or inactivating the mutant gene transcript
  3. Using genome editing tools to repair or turn-off the mutated gene.
computer desk laptop stethoscope
Photo of a stethoscope by Negative Space on Pexels.com

How is gene therapy used?

Monogenic disorders, like some spinocerebellar ataxias (SCAs), are excellent targets for gene therapy approaches. Gene therapies are currently being used throughout ataxia research for studying disease mechanisms and for preclinical therapeutic application.

Overview of how gene therapy works. First, Package the healthy gene, RNAi, or gene editing tools into the AAV (can also deliver as naked DNA or in a nanoparticle). Second, Inject the packaged AAV into the tissue of interest. Third, AAV will enter the cell and release the genetic material. The cell will become healthy by either 1) expressing the normal gene, 2) repressing the mutant RNA, or by 3) correcting the mutant gene.
Overview of gene therapy, designed by Stephanie Coffin using Biorender.

One gene therapy approach for rescuing SCA1 phenotypes involves overexpressing a healthy gene, ataxin-1-like, which competes with the mutant ATXN1 protein for complex formation. This work, conducted by Keiser and colleagues in 2016, showed phenotypic rescue in a mouse model of SCA1.

There are two common technologies for silencing or inactivating disease genes: RNA interference (RNAi) or antisense oligonucleotides (ASOs). RNAi strategies utilize small RNA molecules to knock down the expression of target mutant RNA transcripts, while ASOs are DNA molecules used to knock down or correct mutant RNA transcripts. Both therapeutic approaches are being pursued in SCAs. For example, Carmo and colleagues in 2013 showed that using RNAi against the SCA3 disease gene, ATXN3, could longitudinally decrease mutant ATXN3 levels. See the SCAsource snapshot on ASOs for further information about their use in SCAs.

The most common genome editing tool is the CRISPR/Cas9 system, which uses an RNA guide to direct the Cas9 nuclease to the region of the genome to be edited. One can then knockout that gene or correct the mutant gene. It is early days for this technology as a potential therapeutic option due to the challenges of delivery and the risk of off-target editing.

How is gene therapy delivered?

One of the most difficult aspects of gene therapy is how to deliver these various molecules to the cells of interest. One of the most common delivery methods is through viral delivery.  The “drug” nucleic acid is transferred into the disease cells by a vector, which is a virus that has been modified to remove viral components. The most common viral vectors for gene therapies currently are adeno-associated viruses (AAVs). Other delivery methods include non-viral vectors such as naked DNA and nanoparticles.

How long-lasting is gene therapy?

Viral delivery of gene therapy products provides a longitudinal expression of the nucleic acid, while naked DNA and nanoparticles express the nucleic acid drug transiently, thus typically requiring ongoing treatment.

If you would like to learn more about gene therapy, take a look at these resources by the National Institutes of Health and KidsHealth.

Snapshot written by Stephanie Coffin and edited by Dr.Hayley McLoughlin.