Anna Cereseto

TnpBs are compact RNA-guided nucleases ideally suited for in vivo delivery. By screening novel TnpBs identified from large metagenomic datasets, ISPmu1 was discovered and its protein and ωRNA were engineered to generate En-ISPmu1, which achieved editing efficiencies exceeding 65% across 12 human loci. Owing to its compact size, En-ISPmu1 was delivered together with a donor DNA in a single AAV vector, yielding up to 12.7% targeted integration in mouse liver. These findings establish En-ISPmu1 as a promising versatile platform for in vivo therapeutic genome editing applications.

Compact Cas nucleases offer advantages over the widely used SpCas9 due to their smaller size, which enables more efficient delivery for in vivo applications. Among these, the phage-encoded CasΦ2 (Cas12j2) is highly promising due to its relaxed PAM requirement (5′-TTN-3′) and compact size (757 aa); however, its translational potential is limited by low editing activity. To enhance the efficacy of CasΦ2, we optimized the previously reported EPICA system, developing EPICA.2, a eukaryotic directed evolution platform to improve nucleases with nearly undetectable activity. EPICA.2 integrates additional yeast evolution rounds to enrich for active variants along with a low background mammalian reporter system that improves detection and selection of enhanced variants. Finally, we set up a long-read sequencing protocol which uses unique molecular identifiers (UMIs) to reduce sequencing errors, enabling accurate identification of the mutation combinations in each evolved variant. Among the most frequent variants, we obtained evoCasΦ2, which contains six activity-boosting mutations with a synergistic effect not predictable by rational engineering. Overall, evoCasΦ2 showed up to 70-fold increased activity in human cells compared to wild-type and outperformed variants generated through rational approaches, highlighting the potential of EPICA.2 as a powerful strategy to evolve genome editing tools with low native activity.

The 1717-1G>A is a prevalent splicing mutation causing cystic fibrosis (CF) for which no pharmacological treatments have been approved. This mutation disrupts a canonical 3′ AG splice acceptor site in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, leading to severe RNA missplicing, which prevents the correct synthesis of the encoded protein. In this study, we developed an adenine base editing (ABE) strategy to efficiently correct the 1717-1G>A mutation. By using the ABE9 base editor with the protospacer adjacent motif–relaxed Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) variant SpRY, we obtained up to 30% editing with limited bystander effects in a human embryonic kidney (HEK) 293–based cellular model. Through systematic optimizations of the ABE system, delivered by electroporation of base editor messenger RNA (mRNA) and single guide RNA (sgRNA), we demonstrated genetic repair of the 1717-1G>A mutation in airway epithelial cells and intestinal organoids derived from people with CF. Functional analysis was performed by measuring short-circuit current in air-liquid interface (ALI) culture and by assessing forskolin-induced swelling (FIS) in intestinal organoids, which revealed restoration of CFTR channel activity. These results highlight SpRY-ABE9 as a potential genome editing strategy to permanently correct the CFTR 1717-1G>A mutation and restore CFTR function.

The biological mechanisms underlying the cooperation between germline genetic variants and somatic mutations during carcinogenesis are rarely elucidated. In this study, characterizing isogenic prostate cancer cell lines, we dissected the interplay between a germline variant at the 7p14.3 locus (rs1376350, G>A) and early recurrent prostate cancer–specific mutation in the speckle-type POZ protein (SPOP) gene across human prostate adenocarcinomas. The transcriptomes of multiple edited models pointed to GLI3 and the Hedgehog signaling pathway in a genotype-specific manner, whereas SPOP mutation and androgen receptor stimulation promote GLI3 accumulation in the full-length, transcriptionally active form. This, in turn, triggers the cell-autonomous production of steroids that prostate cancer relies on, in line with the enhanced responsiveness of SPOP-mutated prostate cancer to androgen deprivation therapy. These data demonstrate that germline variants dictate prostate cancer somatic evolution and suggest opportunities to jointly model germline–somatic relationship to help untangle the complexity of human cancer.Significance:Significant heritability is observed for common cancer types worldwide. The molecular mechanisms by which inherited genetics facilitate cancer initiation might transit through its cooperation with specific somatic events that then dictate the tumor features. Through a germline–somatic tandem leading to steroid biosynthesis, we suggest a paradigm to study cancer initiation.

The supplementary figure file includes Figures S1–S5. Descriptions of each figure are as follows: - Figure S1 shows the approach for editing the 7p14.3 locus in PC3 cells. - Figure S2 shows differential gene expression in bulk and in single clones of PC3 edited cells - Figure S3 shows the impact of single nucleotide editing on Gli3 targets Gli1 and PTCH1, and how Hh inhibitor cyclopamine prevents the accumulation of Gli3 targets. - Figure S4 shows the impact of SPOP status on CYP11A1 expression in LNCaP and 22Rv1 cells. - Figure S5 shows how SPOP status affects PR accumulation in LNCaP and PC3 cells, and how single nucleotide editing influences SRD5A1 and 3BHSD2 expression.

The supplementary table file includes Supplementary Tables S1-S12. Descriptions of each table are as follows: - Supplementary Table 1: Study oligo sequences - Supplementary Table 2: Number of Off Targets calculated with Cas-OFFinder for the final set of guides. - Supplementary Table 3: Description of three sets of deletion editing experiements in prostate cells - Supplementary Table 4: List of study DEGs - all comparisons - Supplementary Table 5: Results of AG vs GG differential analysis (PC3 cells, single clones) - Supplementary Table 6: Results of AA vs GG differential analysis (PC3 cells, single clones) - Supplementary Table 7: Human tissue RNA-seq differential analysis for SPOPmut vs SPOPwt (AA vs AG and AG vs GG clone data DEG set) - Supplementary Table 8: GO analysis of AG vs GG DEGs - Supplementary Table 9: Sample names conversion and annotation - Supplementary Table 10: RNA sequencing statistics - Supplementary Table 11: List of antibodies used in this study - Supplementary Table 12: LC-MS/MS Steroid detection

The biological mechanisms underlying the cooperation between germline genetic variants and somatic mutations during carcinogenesis are rarely elucidated. In this study, characterizing isogenic prostate cancer cell lines, we dissected the interplay between a germline variant at the 7p14.3 locus (rs1376350, G>A) and early recurrent prostate cancer–specific mutation in the speckle-type POZ protein (SPOP) gene across human prostate adenocarcinomas. The transcriptomes of multiple edited models pointed to GLI3 and the Hedgehog signaling pathway in a genotype-specific manner, whereas SPOP mutation and androgen receptor stimulation promote GLI3 accumulation in the full-length, transcriptionally active form. This, in turn, triggers the cell-autonomous production of steroids that prostate cancer relies on, in line with the enhanced responsiveness of SPOP-mutated prostate cancer to androgen deprivation therapy. These data demonstrate that germline variants dictate prostate cancer somatic evolution and suggest opportunities to jointly model germline–somatic relationship to help untangle the complexity of human cancer. Significance: Significant heritability is observed for common cancer types worldwide. The molecular mechanisms by which inherited genetics facilitate cancer initiation might transit through its cooperation with specific somatic events that then dictate the tumor features. Through a germline–somatic tandem leading to steroid biosynthesis, we suggest a paradigm to study cancer initiation.

The Cornelia de Lange syndrome (CdLS) is a rare genetic disease, which is characterized by a cohesinopathy. Mutations of the NIPBL gene are observed in 65% of CdLS patients. A novel iPSC (induced Pluripotent Stem Cell) line was reprogrammed from the leukocytes of a CdLS patient carrying a missense mutation of the NIPBL gene. A mutation-corrected isogenic iPSC-line and two iPSC-lines generated from the healthy parents were used as controls. The iPSC lines were differentiated along the hepatocyte-lineage. Comparative immunofluorescence, RNA-seq and ATAC-seq analyses were performed on undifferentiated and differentiated iPSCs. In addition, chromatin organization was studied by ChIP-Seq analysis on the patient derived iPSCs as well as the respective controls. Relative to the mutation-corrected and the healthy-parents iPSCs, the patient-derived counterparts are defective in terms of differentiation along the hepatocyte-lineage. One-third of the genes selectively up-regulated in CdLS-derived iPSCs and hepatic cells are non-protein-coding genes. By converse, most of the selectively down-regulated genes code for transcription factors and proteins regulating neural differentiation. Some of the transcriptionally silenced loci, such as the DPP6 gene on chromosome 7q36.2 and the ZNF gene cluster on chromosome 19p12, are located in closed-chromatin regions. Relative to the corresponding controls, the global transcriptomic differences observed in CdLS undifferentiated iPSCs are associated with altered chromatin accessibility, which was confirmed by ChIP-Seq analysis. Thus, the deficits in the differentiation along the hepatocyte lineage observed in our CdLS patient is likely to be due to a transcriptional dysregulation resulting from a cohesin-dependent alteration of chromatin accessibility.