CRISPR-Cas systems include several Cas proteins, each with their own enzymatic features and targeting abilities, holding great potential for genome engineering and gene therapy applications. Screenings of metagenomic datasets are continuously providing new additions to the ever-growing CRISPR toolbox. Reliable computational methods for decoding PAM are the key for uncovering unexplored Cas variety. Recently, we have developed a highly-performing PAM prediction pipeline and identified a repertoire of about 33,000 unreported Cas9s. Here, we combined such large variety of orthologs with a prediction-based PAM-specific approach for allele-specific gene editing, and we isolated small Cas9s tailored for ALS-associated SOD1 mutations. CoCas9 from Collinsella showed a good efficiency and specificity profile in both the case studies we investigated, i.e. c.281G>C (p.Gly94Ala) and c.112G>C (p.Gly38Arg) mutations, proving the importance of novel Cas9 discovery to complement existing nucleases and facilitate the advance of CRISPR-based personalized therapies. Nonetheless, not all bacterial orthologs exhibit high functionality when transferred to human cells. Therefore, we evaluated the suitability of the VEGAS SINV-based directed evolution platform to evolve poorly active Cas into catalytically enhanced enzymes. We implemented a selection strategy that made the expression of viral structural genes (SSG) dependent on Cas activity, and we named it CRISPR-SPLICEX. To achieve that coupling, we built an artificial stop/frameshift cassette flanked by introns and inserted it in the coding sequence of a EGFP reporter. We demonstrated that insertion of the cassette abrogated gene function, and that Cas editing at its splice sites could efficiently alter the splicing process to induce cassette skipping and restoration of the gene activity. We moved the cassette into the SSG coding sequence, and we generated a Cas-inducible packaging construct. Simulation of the genetic circuit proved that the Cas-inducible packaging construct could support SINV propagation only in the presence of functional Cas proteins able to rescue the SSG expression. Given the flexibility of the artificial stop/frameshift cassette to accommodate tailored PAMs, the CRISPR-SPLICEX strategy could, in principle, be applied to evolve functional activities of any Cas ortholog.

Novel Cas orthologs from natural reservoirs and design of a directed evolution approach in mammalian cells to enhance their editing potential / Marelli, Federica. - (2024 Apr 16), pp. 1-62. [10.15168/11572_406869]

Novel Cas orthologs from natural reservoirs and design of a directed evolution approach in mammalian cells to enhance their editing potential

Marelli, Federica
2024-04-16

Abstract

CRISPR-Cas systems include several Cas proteins, each with their own enzymatic features and targeting abilities, holding great potential for genome engineering and gene therapy applications. Screenings of metagenomic datasets are continuously providing new additions to the ever-growing CRISPR toolbox. Reliable computational methods for decoding PAM are the key for uncovering unexplored Cas variety. Recently, we have developed a highly-performing PAM prediction pipeline and identified a repertoire of about 33,000 unreported Cas9s. Here, we combined such large variety of orthologs with a prediction-based PAM-specific approach for allele-specific gene editing, and we isolated small Cas9s tailored for ALS-associated SOD1 mutations. CoCas9 from Collinsella showed a good efficiency and specificity profile in both the case studies we investigated, i.e. c.281G>C (p.Gly94Ala) and c.112G>C (p.Gly38Arg) mutations, proving the importance of novel Cas9 discovery to complement existing nucleases and facilitate the advance of CRISPR-based personalized therapies. Nonetheless, not all bacterial orthologs exhibit high functionality when transferred to human cells. Therefore, we evaluated the suitability of the VEGAS SINV-based directed evolution platform to evolve poorly active Cas into catalytically enhanced enzymes. We implemented a selection strategy that made the expression of viral structural genes (SSG) dependent on Cas activity, and we named it CRISPR-SPLICEX. To achieve that coupling, we built an artificial stop/frameshift cassette flanked by introns and inserted it in the coding sequence of a EGFP reporter. We demonstrated that insertion of the cassette abrogated gene function, and that Cas editing at its splice sites could efficiently alter the splicing process to induce cassette skipping and restoration of the gene activity. We moved the cassette into the SSG coding sequence, and we generated a Cas-inducible packaging construct. Simulation of the genetic circuit proved that the Cas-inducible packaging construct could support SINV propagation only in the presence of functional Cas proteins able to rescue the SSG expression. Given the flexibility of the artificial stop/frameshift cassette to accommodate tailored PAMs, the CRISPR-SPLICEX strategy could, in principle, be applied to evolve functional activities of any Cas ortholog.
16-apr-2024
XXXV
2023-2024
CIBIO (29/10/12-)
Biomolecular Sciences
Cereseto, Anna
no
Inglese
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/406869
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