Scientists headed by a team at the Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, have developed a CRISPR-based system that can edit multiple target sites in the genome at the same time to help study how different genes may cooperate in health and disease. The new system, dubbed Cas Hybrid for Multiplexed Editing and screening Applications (CHyMerA), utilizes co-expression of both Cas9 and Cas12a CRISPR-associated enzymes, with machine learning-optimized hybrid Cas9-Cas12a guide RNAs, and can be applied to any type of mammalian cell.

“With CHyMErA, you can use the best of the two enzymes,” said Michael Aregger, PhD, a research associate in the lab of molecular genetics professor Jason Moffat, PhD. Aregger played a key role in developing the screening-based applications of CHyMErA. “Cas9 has been improved by the community to have a very high editing efficiency, whereas Cas12a allows multiplexing of guide RNAs and therefore provides a lot more flexibility in finding sites in the genome that we can cut.” The researchers reported on their developments in Nature Biotechnology, in a paper titled, “Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.”

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. “In particular, genome-scale screens employing CRISPR–Cas nucleases have already begun to deliver unprecedented insight into genotype–phenotype relationships,” the authors wrote. But challenges do remain, including the ability to simultaneously remove multiple genes or gene fragments in the same cell. Scientists would need to be able to carry out this kind of genome editing to help understand how different parts of the genome work together in the contexts of both normal physiology and disease. “Identifying GIs [genomic interactions] and the roles of gene segments is crucial in the advancement of knowledge of gene function and how genome alterations contribute to diseases and disorders,” the team noted. “However, systematic mapping of GIs in mammalian cells has been hampered by the lack of efficient and readily scalable targeting systems.”

CRISPR works by sending a DNA-cutting enzyme to desired sites in the genome via guide RNA molecules, engineered to adhere to the target site. While the most widely used DNA-cutting enzyme is Cas9, scientists working to improve and expand the applications of CRISPR technology have also identified other Cas enzymes that have distinct properties.

Unlike CRISPR-Cas9 systems, the new CHyMerA tool developed by researchers in the labs of Moffat and molecular genetics professor Benjamin Blencowe, PhD, combines two different DNA-cutting enzymes, Cas9 and Cas12a, which offers more versatility. Cas12a is an enzyme that can be used to generate multiple guide RNA molecules in the same cell, which is key for simultaneous DNA editing. Thomas Gonatopoulos-Pournatzis, PhD, a research associate in the Blencowe lab, had spent several years trying to develop combinatorial gene editing by testing Cas9 and Cas12a enzymes on their own. He then had the idea to combine these enzymes to generate the CHyMErA system.

“We had been trying a number of approaches to induce genetic fragment deletions and nothing worked as well as CHyMErA,” he said. “I was thrilled when together with Shaghayegh Farhangmehr, a PhD student in the Blencowe lab, we saw the first evidence that CHyMErA was successful in deleting gene segments. We obtained these results on Boxing Day and it was the best Christmas present I could have wished for.”

As a next step, the researchers harnessed CHyMErA in large-scale screens to systematically analyze how genes act together, as well as to identify the functions of individual parts of genes. Blencowe’s team, which studies the regulation and function of gene exons, approached Moffat, whose group had developed extensive experience with CRISPR technology.

In one application of CHyMErA, the researchers targeted pairs of genes known as paralogs, which have a similar DNA code but have remained poorly studied. Because paralogs arose by duplication of an ancestral gene, it had been assumed that 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, primarily because the other paralog would compensate for the one that was missing.

“With CHyMErA, we can take out both paralogs in pairs to see if that ancestral function is important for the cell to survive,” said Kevin Brown, PhD, senior research associate in the Moffat lab and co-lead author on the study together with Aregger and Gonatopoulos-Pournatzis. “We are able to now interrogate a class of genes that was previously missed.”

The team knocked out about 700 paralog pairs, which represent nearly all that exist in the human genome. Their analyses confirmed that many of the gene pairs do perform similar roles in cell survival, although 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 exon fragments. This allowed the team to individually delete thousands of exons that have been linked to cancer and brain function, but which hadn’t previously been 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, more than 100 were found to be critical for cell survival. Future research will be able to focus on trying to understand their potential roles in disease. “Once we identify exons that have a critical role in disease, we can use this information to develop new therapies,” said Gonatopoulos-Pournatzis.

Commenting on their work with CHyMErA, the authors concluded, “These screens demonstrate a previously unappreciated degree of complexity of GIs among paralogous genes, reveal new chemical GIs, and identify numerous alternative exons that impact cell growth …The results in the present study support the efficacy of CHyMErA as a system for addressing these timely challenges … As such, we anticipate its future use in charting GIs and the functions of genome segments, such as the myriad of previously uncharacterized alternative splicing events and noncoding RNA sequences linked to development and disease.”

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