CRISPR-based DNA editing has revolutionized the study of the human genome by allowing precise deletion of any human gene to glean insights into its function. But one feature remained challenging—the ability to simultaneously remove multiple genes or gene fragments in the same cell.
My lab is interested in knowing how genes interact and how these interactions break down in cancer and other diseases. To study gene interactions, we needed a tool that would allow us to remove multiple genes at a time from the same cell, which occurs under most disease scenarios.
We’re pretty well experienced in advancing CRISPR technology and were amongst the first labs to apply CRISPR to knock out from cells almost all human genes one at a time and establish which ones are essential for fitness (https://www.theatlantic.com/science/archive/2015/11/a-revolutionary-gene-editing-technique-reveals-cancers-weaknesses/417495/). This was easy to do with the most commonly used CRISPR-Cas9 system where engineered guide RNA molecules (gRNA) deliver the DNA cutting enzyme Cas9 from Streptococcus pyogenes to the target site to excise a single DNA fragment.
For editing at multiple sites, we needed to find another enzyme.
At the same time, my colleague Benjamin Blencowe, renowned for his work on alternative splicing, a process allowing cells to diversify their protein content, was looking for a tool that would allow his team to knock out gene coding segments, or exons, which could not be easily targeted with CRISPR-Cas9.
Our two labs joined forces to find another Cas enzyme that has the right properties and is compatible with CIRPSR. We were lucky to have extremely talented researchers on the team to carry this through: research associate Michael Aregger and senior research associate Kevin Brown from my lab, and research associate Thomas Gonatopoulos-Pournatzis and graduate student Shaghayegh Farhangmehr from the Blencowe lab.
Image from left to right - research associates Thomas Gonatopoulos-Pournatzis, Michael Aregger (standing) and Kevin Brown with graduate student Shaghayegh Farhangmehr form the creative minds behind CHyMErA.
We created a hybrid guide RNA (hgRNA) and combined Cas9 with an enzyme called Cas 12, which cuts both DNA and RNA, meaning it can be employed to generate multiple guide RNAs from a single hgRNA transcript in the same cell for simultaneous editing. We therefore named our tool CHyMErA, for Cas Hybrid for Multiplexed Editing and Screening Applications.
According to Greek mythology, the Chimera was a monstrous fire-breathing hybrid creature of Lycia in Asia Minor, composed of parts from more than one animal. In The Iliad, Homer described the Chimera as “a thing of immortal make, not human, lion-fronted and snake behind, a goat in the middle, and snorting out the breath of the terrible flame of bright fire”. Like the Chimera, our method is multi-functional and can elicit multiple actions simultaneously.
Cas12 also allowed us to target more sites in the genome than with Cas9 alone, as we discovered. For the cutting to occur, the enzyme must first bind the DNA at precise sites, known as PAM sequences, which are scattered across the genome and distinct to those of Cas9, effectively doubling the genome real estate that can be targeted.
We then harnessed CHyMErA in large-scale screens to systematically analyze how genes act together, as well the functions of individual exons.
We first targeted pairs of genes known as paralogs, which have a similar DNA code but remain poorly studied because they were difficult to research. Because paralogs arose by duplication of an ancestral gene, it had been assumed they would largely have similar roles. But their function could not be revealed by the existing single-gene targeting methods typically employed in genetic screens, mostly because the other paralog would compensate for the one that’s missing.
After knocking out ~700 paralog pairs, almost all that exist in the human genome, we worked with computational biologist Chad Myers and his student Henry Ward at the University of Minnesota to confirm that many of these strict gene pairs do indeed perform similar roles in cell survival, whereas others have distinct functions.
Another feature of CHyMErA is that both Cas9 and Cas12a can be deployed to nearby genome sites to cut out gene fragments such as exons. This allowed us to individually delete thousands of exons that have been linked to cancer and brain function but were not amenable to targeting with Cas9 alone. Exons are variably included into genes’ transcripts and can modify the function of the encoded proteins, although how individual exons contribute to cellular processes remains largely unknown. Out of 2,000 exons analyzed by CHyMErA, over 100 were found to be critical for cell survival, enabling future research to now focus on shining light on their potential roles in disease.
Access to a view-only version of the paper can be found with the following Sharedit link (http://em.rdcu.be/ls/click?upn...).
Now that we have CHyMErA working robustly, this technology opens up a realm of new possibilities for combinatorial genetics in mammalian cells to explore the complex genomic landscapes of cancer and other genetic diseases.
I would like to thank Jovana Drinjakovic for help with this blog.
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