Compositions and methods for precise editing of human dystrophin

An expression cassette comprising, from 5′ to 3′:

• a) a first inverted terminal repeat (ITR);
• b) a first promoter sequence operably linked to a nucleic acid sequence encoding a guide RNA wherein the guide RNA comprises:
• i. a first region comprising a protein binding sequence;
• and
• ii. a second region comprising a spacer sequence that is complementary to a target sequence of DMD, wherein the spacer sequence comprises a nucleic acid sequence selected from SEQ ID NOs:
• 100-282 and 928-1069;
• c) a second promoter sequence operably linked to a nucleic acid sequence encoding an effector protein;
• d) a poly(A) signal;
• and
• e) a second ITR.

A system for deleting a region of DMD, the system comprising:

• a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell;
• b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP;
• c) a first guide RNA (gRNA) or a nucleic acid encoding the first gRNA, wherein the first gRNA comprises
• i. a first scaffold sequence, and
• ii. a first spacer sequence that hybridizes to a first target sequence on a first strand of the DMD gene,
• wherein the first scaffold sequence is located 5′ of the first spacer sequence, and
• wherein the effector protein and the first gRNA form a first RNP complex that cleaves the DMD gene to form a first single stranded DNA (ssDNA) flap from the first strand of the DMD gene;
• d) a second gRNA or a nucleic acid encoding the second gRNA, wherein the second gRNA comprises
• i. a second scaffold sequence, and
• ii. a second spacer sequence that hybridizes to a second target sequence on a second strand of the DMD gene,
• wherein the second scaffold sequence is located 5′ of the second spacer sequence, and
• wherein the effector protein and the second gRNA form a second RNP complex that cleaves the DMD gene to form a second ssDNA flap from the second strand of the DMD gene;
• e) a first template RNA (retRNA) or a nucleic acid encoding the first retRNA, wherein the first retRNA comprises
• i. a first primer binding sequence (PBS) that hybridizes to at least a portion of the first ssDNA flap, and
• ii. a first template sequence;
• and
• f) a second retRNA or a nucleic acid encoding the second retRNA, wherein the second retRNA comprises
• i. a second PBS that hybridizes to at least a portion of the second ssDNA flap, and
• ii. a second template sequence.

A system for modifying a target strand (TS) of a DMD, the system comprising:

• a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell;
• b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP;
• c) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA comprises
• i. a first region comprising a scaffold sequence, and
• ii. a second region comprising a spacer sequence that hybridizes to a target sequence of the TS of the DMD gene,
• wherein the first region is located 5′ of the second region;
• and
• wherein the effector protein and the gRNA form a RNP complex that produces a double stranded break;
• d) a TS template RNA (retRNA) or a nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises
• i. a TS primer binding sequence (PBS) that hybridizes to the TS of the DMD gene, and
• ii. a TS template sequence.