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Gene network rewiring by an engineered CRIPSR-Cas12a variant

Periodic Reporting for period 1 - GNRECC (Gene network rewiring by an engineered CRIPSR-Cas12a variant)

Reporting period: 2020-09-01 to 2022-08-31

A major obstacle to the understanding of cellular physiology in both health and disease arise from the ability of genes to execute cellular programs. Therefore, how these elements are interconnected is still an open question which limits our comprehension of complex cell behaviors.

To elucidate and control the gene network underlying cellular functions, this project aims to engineer Cas12a, a member of CRISPR/Cas protein family used for cell transcriptional reprogramming. Although highly potent in single-gene control, the efficacy of Cas12a decreases when directed to multiple genes. Therefore, more potent Cas12a version are needed to facilitate rapid and large-scale engineering of cellular programs.

By designing novel Cas12a enzyme able to be directed towards multiple genomic loci simultaneously, this project will simplify the generation of novel genetic pathways useful for the biotechnological and industrial sector including biopharmaceuticals production and cell-based therapies.
Therefore, we aim at combining statistical inference techniques with both protein and genome engineering to address current challenges that limit the explosion of gene network engineering field. This project aims to generate a potent Cas12a useful for multiplexed orthogonal gene control and employ this optimized enzyme for the validation of an inferred molecular network. To address this issue, the following work packages (WPs) will be executed: rational design of novel Cas12a variants (WP1), validation of novel Cas12a variants (WP2), gene network rewiring by a novel Cas12a variant (WP3).
The work starts in September 2020 during the Covid-19 pandemic and, it was anticipately terminated in February 28th 2021, after 6 months from the starting date.

Therefore, this report cover the entire duration of the 6 month period, between September 2020 and February 2021, in which the work was organized to match both social and working restrictions imposed by the COVID-19 outbreak. In particular, the job was focused in: i) implementing the first work package (WP1) of the project, ii) strengthening the academic profile of the fellow.

i) The research team lead by the Fellow analyzed Cas12a orthologs in order to find aminoacidic residues that, whether mutagenized, might increase Cas12a activity. Using a statistical inference approach, they found that: i) Cas12 residues involved in DNA binding displayed an higher percentage of conservation than other sites, ii) the PI and WEDII2 Cas12 domains have more variability in their aminoacidic content than the BH domain, suggesting that PI and WEDII domains are potential regions in which mutagenesis might improve enzyme activity. Consequently, a library of Cas12a mutants encompassing both the PI and WEDII regions was generated and cloned
Therefore, thanks to Marie-Curie fellowship program, the fellow reach his career objective and find potential regions in which residue mutagenesis might improve Cas12a activity.

ii) The fellow prepared 2 grant application and sent them to distinct private foundation (Associazione Italiana di Ricerca sul Cancro - AIRC, Telethon Foundation). Both of them reached the final evaluation round and, one of the two was deemed worthy of funding from the Associazione Italiana di Ricerca sul Cancro (AIRC). This success was pivotal for the advancement of fellow career. In particular, the financial support of AIRC to the Fellow captured the interest of the Italian Institute for Genomic Medicine – IIGM, a private research institution that supports excellence in biomedical research. IIGM offered a position as a Junior Group Leader to the fellow in order to create a new independent research unit which starts on March 2021.
The planned research presents several innovative aspects which confer to the overall proposal a high level of originality.
These include: 1) the use of statistical inference to guide the design of both novel Cas enzyme variants and gene networks, 2) the assessment of Cas12a residues function in the framework of its protein sequence evolution, 3) the employment of Cas12a for orthogonal gene editing and transcriptional gene activation for rapid and efficient gene network engineering.

Notably, all these aspects were not implemented in their respective research fields. As an example, rational design of CRISPR effectors is mainly based on information derived from structural data. However, this approach is inadequate to generate highly active enzyme, as few structures were solved for this protein enzyme family. Consequently, methods based on the comparison of the hundreds of Cas protein sequence variants represent an inestimable resource to drive optimization of Cas effectors. In parallel, validation of statistical inference is currently based on the perturbation of a single node at time of a gene network. However, successive genetic alterations induce complex adaptive responses that mask the true/real network structure. Consequently, methods which enable simultaneous modification of an entire gene network, will provide a deeper understanding of the interconnection between genes.

Overall, the planned research is highly innovative both in terms of its technical approach as well as in its scientific content paving new ways for the next generation of cellular engineering.
Sequences of novel Cas12a variant