Mutant myocilin disease model and uses thereof

A non-human animal cell comprising in its genome a humanized endogenous MYOC locus in which a region of the endogenous MYOC locus has been deleted and replaced with a corresponding human MYOC sequence,

• wherein the region of the endogenous MYOC locus comprises both a MYOC exonic sequence and a MYOC intronic sequence and the corresponding human MYOC sequence comprises both a MYOC exonic sequence and a MYOC intronic sequence, and
• wherein the non-human animal cell further comprises in its genome a genomically integrated expression cassette, wherein the genomically integrated expression cassette comprises:
• (a) a nucleic acid encoding a chimeric Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains;
• and
• (b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains, and
• wherein the non-human animal cell further comprises one or more guide RNAs that were administered or one or more expression cassettes that encode the one or more guide RNAs, each guide RNA comprising one or more adaptor-binding elements to which the chimeric adaptor protein can specifically bind,
• wherein each of the one or more guide RNAs is capable of forming a complex with the Cas protein and guiding it to a target sequence within a target gene, and
• wherein at least one of the one or more guide RNAs targets the humanized endogenous MYOC locus.

A method of increasing MYOC expression in a non-human animal,

• wherein the non-human animal comprises in its genome a humanized endogenous MYOC locus in which a region of the endogenous MYOC locus has been deleted and replaced with a corresponding human MYOC sequence,
• wherein the region of the endogenous MYOC locus comprises both a MYOC exonic sequence and a MYOC intronic sequence and the corresponding human MYOC sequence comprises both a MYOC exonic sequence and a MYOC intronic sequence,
• wherein the non-human animal further comprises in its genome a genomically integrated expression cassette, wherein the genomically integrated expression cassette comprises:
• (a) a nucleic acid encoding a chimeric Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains;
• and
• (b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains,
• wherein the method comprises administering to the non-human animal one or more guide RNAs or one or more expression cassettes that encode the one or more guide RNAs, each guide RNA comprising one or more adaptor-binding elements to which the chimeric adaptor protein can specifically bind,
• wherein each of the one or more guide RNAs is capable of forming a complex with the Cas protein and guiding it to a target sequence within a target gene, and
• wherein at least one of the one or more guide RNAs targets the humanized endogenous MYOC locus.

A method of increasing intraocular pressure in a non-human animal,

• wherein the non-human animal comprises in its genome a humanized endogenous MYOC locus in which a region of the endogenous MYOC locus has been deleted and replaced with a corresponding human MYOC sequence,
• wherein the region of the endogenous MYOC locus comprises both a MYOC exonic sequence and a MYOC intronic sequence and the corresponding human MYOC sequence comprises both a MYOC exonic sequence and a MYOC intronic sequence,
• wherein the non-human animal further comprises in its genome a genomically integrated expression cassette, wherein the genomically integrated expression cassette comprises:
• (a) a nucleic acid encoding a chimeric Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains;
• and
• (b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains,
• wherein the method comprises administering to the non-human animal one or more guide RNAs or one or more expression cassettes that encode the one or more guide RNAs,
• each guide RNA comprising one or more adaptor-binding elements to which the chimeric adaptor protein can specifically bind,
• wherein each of the one or more guide RNAs is capable of forming a complex with the Cas protein and guiding it to a target sequence within a target gene, and
• wherein at least one of the one or more guide RNAs targets the humanized endogenous MYOC locus.

A method of modeling glaucoma in a non-human animal,

• wherein the non-human animal comprises in its genome a humanized endogenous MYOC locus in which a region of the endogenous MYOC locus has been deleted and replaced with a corresponding human MYOC sequence,
• wherein the region of the endogenous MYOC locus comprises both a MYOC exonic sequence and a MYOC intronic sequence and the corresponding human MYOC sequence comprises both a MYOC exonic sequence and a MYOC intronic sequence,
• wherein the non-human animal further comprises in its genome a genomically integrated expression cassette, wherein the genomically integrated expression cassette comprises:
• (a) a nucleic acid encoding a chimeric Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains;
• and
• (b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains,
• wherein the method comprises administering to the non-human animal one or more guide RNAs or one or more expression cassettes that encode the one or more guide RNAs,
• each guide RNA comprising one or more adaptor-binding elements to which the chimeric adaptor protein can specifically bind,
• wherein each of the one or more guide RNAs is capable of forming a complex with the Cas protein and guiding it to a target sequence within a target gene, and
• wherein at least one of the one or more guide RNAs targets the humanized endogenous MYOC locus.