Genome engineering with CRISPR/Cas

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for their work to pioneer genome engineering with CRISPR/Cas. This acronym derives from Clustered Regularly Interspaced Short Palindromic Repeats and their CRISPR-associated genes (Cas). CRISPR loci, which are found in many species of bacteria and most archae, have been collectively described as an RNA-based “immune system” due to their ability to recognize and destroy foreign phage and plasmid DNA. Although the acronym was first coined in a 2002 paper1, CRISPR has only recently been exploited as a research tool.

scientific pioneers of CRISPR/Cas
The four scientific pioneers of CRISPR/Cas. From left to right: George Church, Jennifer Doudna, Feng Zhang, and Emmanuelle Charpentier. Image Source: How the battle lines over CRISPR were drawn.

What is the CRISPR/Cas system?

There are at least three distinct types of CRISPR system. A typical “type II” CRISPR locus consists of several protein-coding Cas genes adjacent to an array of direct repeat and spacer sequences. The direct repeats are usually palindromic and conserved, in contrast to the much more variable spacers; these repeat-spacer sequences are transcribed as one unit and then processed into short CRISPR-RNAs (crRNAs). A 2007 Science article demonstrated that a bacterial population could acquire resistance to phage infection by incorporating DNA fragments from the invading phage genome into a CRISPR locus, in the form of new spacer sequences.2 The newly acquired spacers are then transcribed and processed into crRNAs, associate with a trans-activating RNA (tracRNA) and Cas protein, and are eventually guided to a homologous DNA sequence to catalyze a double-stranded break.

Genome engineering with CRISPR/Cas

The CRISPR system can be flexibly “reprogrammed” by designing custom chimeric RNAs (chiRNA), which serve the function of both crRNA and tracrRNA in one molecule. By co-expressing a “designer” chiRNA with a Cas protein, a targeted and specific DNA break can be created in the genome; after providing an exogenous DNA template to help repair the break, customized knock-ins or knock-outs can be generated. To date, knock-outs have been created in a variety of organisms including rats, flies, and human cells.

genome engineering using CRISPR/Cas
Image source: Introduction to the CRISPR/Cas9 system
  1. A single guide RNA (sgRNA) consists of a crRNA sequence specific to a DNA target, and a tracrRNA that interacts with the Cas9 protein
  2. The sgRNA binds to a specially engineered Cas9 protein
  3. The sgRNA-Cas9 complex causes target-specific dsDNA cleavage at a gene of interest
  4. The non-homologous end joining (NHEJ) DNA repair pathway is inaccurate and results in random insertions/deletions that knock out the gene

What are the potential applications of CRISPR/Cas?

CRISPR/Cas technology has attracted scientific attention as well as commercial interest, and its applications seem almost limitless. CRISPR Therapeutics, a recently formed company dedicated to translating the technology into genetic disease therapies, has multiple CRISPR-based therapies in clinical development.

But a technology as transformative as this one does not develop without controversy, both ethical and financial. In 2019 Chinese scientist He Jiankui was sentenced to three years in prison, and banned for life from reproductive medicine, for his role in creating CRISPR-modified human babies. There are also battles over intellectual property rights to CRISPR/Cas: In 2014 the Broad Institute and MIT were awarded a patent which covers the use of CRISPR genome-editing technology in eukaryotes. Feng Zhang, who is listed as Inventor on the patent, and his lab at MIT were the first to publish on CRISPR’s functionality in human cells.3 More recently the Broad Institute was awarded even more complete patent rights over the use of CRISPS/Cas technology in eukaryotic cells.

Only time will tell if genome engineering with CRISPR/Cas produces the amazing breakthroughs that scientists, and the general public, are eagerly awaiting.


If you enjoyed this summary of genome engineering using CRISPR/Cas, please check out my post on how Drosophila melanogaster genes are named.


References

  1. Jansen R, et al. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565-75. DOI: 10.1046/j.1365-2958.2002.02839.x
  2. Barrangou R, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315(5819):1709-1712. DOI:10.1046/j.1365-2958.2002.02839.x
  3. Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-823. DOI: 10.1126/science.1231143

Leave a comment

Your email address will not be published.