Variant-specific exogenous dna template-free correction of pathogenic variants

A method of introducing a genetic change in a genome of a cell with an exogenous DNA template-free system at a target site comprising:

• obtaining a nucleic acid comprising a variant-specific sgRNA specific for a target genomic sequence comprising 88% or higher homology with one or more paralogs or pseudogenes;
• wherein the target genomic sequence has one or more nucleotides that differ(s) when compared to a homologous DNA sequence in the one or more paralogs or pseudogenes (allele-specificity);
• and
• wherein a homologous region in the one or more paralogs or pseudogenes has a desired nucleotide sequence for transfer to the target genomic sequence after a double strand break (DSB) by Cas-based genome editing;
• introducing into the cell a variant-specific sgRNA that directs gene editing by gene conversion and nonallelic homologous recombination (NAHR) to cause a unidirectional transfer of genomic DNA from the homologous DNA sequences to the target genomic sequence, wherein gene conversion or NAHR involves a double stranded break;
• and
• contacting the genome of the cell with the variant-specific sgRNA and a Cas-based genome editing system, thereby introducing a genetic conversion without an exogenous DNA template.

A method of making a variant-specific single guide RNA for exogenous DNA template-free gene editing comprising:

• (i) identifying a target genomic sequence of the genome comprising 88% or higher homology with one or more paralogs or pseudogenes;
• wherein the target genomic sequence has one or more nucleotides that differ(s) when compared to a homologous DNA sequence in the one or more paralogs or pseudogenes (allele-specificity);
• and
• wherein a homologous region in the one or more paralogs or pseudogenes has a desired nucleotide sequence for transfer to the target genomic sequence after a double strand break (DSB) by Cas-based genome editing;
• (ii) analyzing the nucleotide sequence and cut site with a computational model to identify a sequence for the variant-specific single guide RNA;
• (ii) synthesizing variant-specific single guide RNA, wherein the variant-specific sgRNA in a presence of Cas-based genome editing enzymes edits the target genomic sequence.

A method of treating a genetic disease in a subject caused by a genetic error in a genome of one or more cells of the subject by introducing a genetic change in the genome of a cell with an exogenous DNA template-free genome editing system at a target site comprising:

• introducing into the cell a variant-specific sgRNA that directs gene editing by gene conversion and nonallelic homologous recombination (NAHR) to cause a unidirectional transfer of genomic DNA from a homologous DNA sequences to a target genomic sequence, wherein the gene conversion or NAHR involves a double-stranded break,
• wherein the variant specific sgRNA is specific for a target genomic sequence comprising 88% or higher homology with one or more paralogs or pseudogenes;
• wherein the target genomic sequence has one or more nucleotides that differ(s) when compared to a homologous DNA sequence in the one or more paralogs or pseudogenes (allele-specificity);
• and
• wherein a homologous region in the one or more paralogs or pseudogenes has a desired nucleotide sequence for transfer to the target genomic sequence after a double strand break (DSB) by Cas-based genome editing;
• and
• contacting the genome of the cell with the variant-specific sgRNA and a Cas-based genome editing system, thereby introducing a genetic conversion without an exogenous DNA template.

A method comprising a computational model for selecting a single guide RNA sequence for use with a Cas-based genome editing system that introduces a genetic change in a genome by gene conversion and nonallelic homologous recombination (NAHR), the method comprising:

• using a processor to identify a polynucleotide sequence for a variant-specific sgRNA specific for a target genomic sequence comprising 88% or higher homology with one or more paralogs or pseudogenes;
• wherein the target genomic sequence has one or more nucleotides that differ(s) when compared to a homologous DNA sequence in the one or more paralogs or pseudogenes (allele-specificity);
• and
• wherein a homologous region in the one or more paralogs or pseudogenes has a desired nucleotide sequence for transfer to the target genomic sequence after a double strand break (DSB) by Cas-based genome editing;
• and
• synthesizing the variant-specific sgRNA.

A method of introducing a genetic change in a genome of a cell with an exogenous DNA template-free Cas-based genome editing system comprising:

• (i) selecting a single guide RNA (sgRNA) for use with a Cas-based genome editing system capable of introducing a genetic change into a nucleotide sequence of a target genomic location;
• (ii) identifying a target genomic sequence of the genome comprising 88% or higher homology with one or more paralogs or pseudogenes;
• wherein the target genomic sequence has one or more nucleotides that differ(s) when compared to a homologous DNA sequence in the one or more paralogs or pseudogenes (allele-specificity);
• and
• wherein a homologous region in the one or more paralogs or pseudogenes has a desired nucleotide sequence for transfer to the target genomic sequence after a double strand break (DSB) by Cas-based genome editing;
• (iii) introducing into the cell a variant-specific sgRNA that directs gene editing by gene conversion and nonallelic homologous recombination (NAHR) to cause a unidirectional transfer of genomic DNA from the homologous DNA sequences to the target genomic sequence, wherein the gene conversion or NAHR involves a double-stranded break;
• and
• (iv) contacting the genome of the cell with the variant-specific sgRNA and a Cas-based genome editing system, thereby introducing a genetic conversion without an exogenous DNA template.