Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising

• delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises:
• (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
• I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises
• (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,
• (b) a tracr mate sequence, and
• (c) a tracr sequence, and
• II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,
• wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
• wherein components I and II are located on the same or different vectors of the system,
• wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
• or
• (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
• I. a first regulatory element operably linked to
• (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and
• (b) at least one or more tracr mate sequences,
• II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
• III. a third regulatory element operably linked to a tracr sequence,
• wherein components I, II and III are located on the same or different vectors of the system,
• wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence.

A method of treating or inhibiting a condition caused by a defect in a target sequence in a genomic locus of interest in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing treatment comprising:

• delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system, comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target sequence is manipulated by the composition when expressed, wherein the composition comprises:
• (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
• I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises
• (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,
• (b) a tracr mate sequence, and
• (c) a tracr sequence, and
• II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,
• wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
• wherein components I and II are located on the same or different vectors of the system,
• wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
• or
• (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
• I. a first regulatory element operably linked to
• (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and
• (b) at least one or more tracr mate sequences,
• II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
• III. a third regulatory element operably linked to a tracr sequence,
• wherein components I, II and III are located on the same or different vectors of the system,
• wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence.