Abstract: The present invention relates to the medical field of single-gene disorders caused by functional loss or gain of an allele. The innovative approach developed being based on editing the human genome at the level of the Kozak sequence by means of CRISPR-Cas programmable nucleases. Particularly, the present invention relates to variant Kozak sequences and related in vitro or in vivo methods for obtaining such variant Kozak sequences for therapeutic applications in the treatment of single-gene diseases caused by monoallelic losses or gains. These in vitro and in vivo methods include CRISPR-Cas homology-directed repair, CRISPR-Cas prime editing, CRISPR-Cas base editing or genome editing with other programmable RNA-guided nucleases, and the introduction of specific nucleotide conversions in the Kozak sequence of genes causative of diseases. These nucleotide conversions enhance or inhibit the translation of the mRNA produced by the gene, compensating for the functional loss or gain of one allele in the diseases.

Abstract: Type II Cas proteins referred to as ENQP Type II Cas proteins; gRNAs for Type II Cas proteins; systems comprising Type II Cas proteins and gRNAs; nucleic acids encoding the Type II Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.

Abstract: Type II Cas proteins, for example Type II Cas proteins referred to as AIK Type II Cas proteins, BNK Type II Cas proteins, HPLH Type II Cas proteins, and ANAB Type II Cas proteins; gRNAs for Type II Cas proteins; systems comprising Type II Cas proteins and gRNAs; nucleic acids encoding the Type II Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.

Abstract: To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity, the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease, evoCas9, was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.

Abstract: Engineered Cas12a guide RNA (gRNA) molecules useful, for example, for correcting aberrant RNA splicing resulting from mutations in a genomic DNA sequence and for preventing exon inclusion in mature mRNA.

Abstract: The present invention describes vesicles carrying gRNA(s) and/or CRISPR-associated RNA guided-nuclease ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through fusogenic envelope proteins.

Abstract: To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity, the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease, evoCas9, was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.

Abstract: The present invention describes a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for the Streptococcus pyogenes Cas9 (SpCas9), a first Cas9 self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, SLiCES was integrated into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, the present hit and go system increases safety margins for genome editing.