After the announcement of the medicine prize and physics prize, the 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the development of the CRISPR/Cas9 method of genetic editing. We discussed the origins, development, and applications of the CRISPR/Cas9 method earlier this year, but in light of this recent award, I thought we could delve a little deeper into the discovery and implications of this technology. Given how significant and ubiquitous the CRISPR/Cas9 technology has become, it was only a matter of time before it garnered a Nobel prize, but there was some disagreement concerning who would be included in that prize. CRISPR/Cas9, like most significant scientific discoveries, was the result of many scientists’ hard work and dedication. But the Nobel prize can only be awarded to a maximum of three individuals each year, so the committee had to decide who made the most significant contributions to the development of CRISPR/Cas9.
There is very little argument that Charpentier and Duodna made a profound impact on the field. In fact, their synthesis of the hybrid sgRNA was really the catalyst that made targeted genetic engineering through CRISPR/Cas9 possible. But there was some skepticism as to whether the Nobel prize committee would properly recognize them for this contribution, given the committee’s history of female exclusion. Before the 2020 award, only five other women had won the Nobel prize in Chemistry (and the award had never been won by more than one woman simultaneously). But the announcement of this award bodes well for the future of female recognition in science, particularly as we see more and more women entering science careers.
The discovery of CRISPR/Cas9 began in the 80s and 90s with the discovery of conserved stretches of palindromic repeats interspersed by highly variable spacers in strains of Bacteria and Archaea. These regions of DNA, which were found to be highly common in prokaryotes (single cell organisms that have free standing DNA, not contained by a nucleus), were tentatively named clustered regularly interspaced short palindromic repeats (CRISPR). Due to the strength with which these regions were conserved across strains and through successive generations, it was clear that CRISPR DNA must serve some vital function. An additional clue to this function came when another conserved region was identified adjacent to the the CRISPR DNA, the CRISPR-associated (cas) genes. Subsequent analysis of these genes revealed that they encoded a family of Cas proteins with helicase (involved in unwinding DNA) and nuclease (involved in cleaving DNA) domains. These discoveries hinted that the CRISPR/Cas system might be involved somehow in gene expression or the breakdown of DNA.
Despite these initial clues, the true function of the CRISPR/Cas system wasn’t revealed until 2005 with the discovery of protospacers. Protospacers are sequences of DNA found in bacteriophages (viruses that attack bacteria) that match a spacer sequence in the CRISPR DNA. From this discovery, scientists deduced that the CRISPR/Cas system must function as some form of adaptive immunity to viral infection. The mechanism of CRISPR/Cas adaptive immunity was first practically demonstrated in 2007 by an industrial microbiologist working on making the bacteria found in dairy products more resistant to infection. Subsequent research revealed that the CRISPR DNA is transcribed into RNA (pre-crRNA), which is then cleaved into smaller RNA molecules (crRNA). These crRNA molecules are picked up by Cas proteins that use them to target cleavage of any matching sequences.
The CRISPR/Cas mechanism is a highly targeted system of adaptive immunity; but if the Cas protein cuts any DNA that matches the crRNA, then how does it avoid cutting up the CRISPR DNA? The key to this specificity turns out to be related to short nucleotide sequences next to the protospacer in the bacteriophage genome. These protospacer adjacent motifs (PAMs) are not copied over into CRISPR DNA during spacer integration so they act as a secondary form of identification to verify foreign DNA before it is cut.
By 2011, it became clear that some aspects of the CRISPR/Cas system could potentially be adapted for use in genetic editing. There are many different Cas proteins involved in CRISPR adaptive immunity and many different types of CRISPR mechanisms. One of the most studied CRISPR mechanisms is the CRISPR/Cas9 system found in the human pathogen Streptococcus pyogenes (responsible for diseases such as strep throat, scarlet fever, and cellulitis). The CRISPR/Cas9 mechanism is simpler and more accurate than other CRISPR systems, making it an ideal candidate for genetic engineering applications. But there was still much that needed to be learned about CRISPR/Cas9 before it could be hijacked for this kind of purpose.
The key discoveries that made CRISPR/Cas9 possible came out of Emmanuelle Charpentier’s and Jennifer A. Doudna’s labs. First, in 2011, Charpentier demonstrated how pre-crRNA was processed into mature crRNA. She found that a region of DNA, 210 base pairs upstream of the CRISPR DNA, is transcribed simultaneously. This RNA transcript (tracrRNA) complements the palindromic repeat regions tailing off the spacers in the crRNA to form a duplex.
Following this initial discovery, Charpentier collaborated with Doudna to investigate how the interaction between crRNA and tracrRNA stimulates Cas9 to cleave a DNA target. Previous experiments indicated that crRNA on its own could not activate Cas9, but the mechanism by which tracrRNA promoted Cas9 target cleavage was still unclear. Through their collaboration, Charpentier and Doudna were able to determine that tracrRNA triggers pre-crRNA processing and activates cleavage of the target by Cas9 dependent on its recognition of an appropriate PAM sequence. They found that cleavage by Cas9 always occurs around 3 base pairs upstream of the target’s PAM sequence (which is always the same in a given bacterial strain). They identified a crucial “seed region” surrounding the PAM sequence in the target strand that was required for recognition and cleavage.
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Based on their understanding of the CRISPR/Cas9 system, Charpentier and Doudna were able to engineer a chimeric RNA molecule, single-guide RNA (sgRNA), from the fusion of tracrRNA with crRNA. They demonstrated how sgRNA can be preprogammed to cut a specific DNA target (provided that the target has a nearby PAM sequence). Charpentier and Doudna’s discovery was a major leap forward for the field of genetic engineering that enabled unprecedented accuracy and precision with gene inactivation, modification, and replacement. In the years since these discoveries, CRISPR/Cas9 has been adapted to serve a gamut of applications in research and development, agriculture, medicine, and more.
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