Development of dual-gRNA approach with undetectable off-target effect to correct C9ORF72 repeat expansion and C9ORF72 pathology
A group of gRNAs is provided, and the group of gRNAs comprises a first gRNA molecule; and a second gRNA molecule capable of defining a region in a genome sequence with the first gRNA molecule, wherein the region in the genome comprises a target sequence in need of removal.
1 . A group of gRNAs, comprising:
a first gRNA molecule; and
a second gRNA molecule capable of defining a region in a genome sequence with the first gRNA molecule, wherein the region in the genome comprises a target sequence in need of removal, wherein the target sequence comprises GGGGCC repeats of C90RF72 gene and that is located between the exon 1a and the exon 1b, wherein the first gRNA molecule is set between the exon 1a and the GGGGCC repeats, the second gRNA molecule is set downstream of the exon 1b but upstream of initiation codon, wherein the first gRNA molecule comprises the nucleic acid sequence of SEQ ID NO: 1; and the second gRNA molecule comprises the nucleic acid sequence of SEQ ID NO: 2.
2 . The group of gRNAs of claim 1 , wherein the target sequence has at least two GGGGCC repeats.
3 . A construct, comprising a sequence encoding the group of gRNAs of claim 1 .
4 . The construct of claim 3 , further comprising:
a first promoter operably linked to a first nucleic acid molecule encoding the first gRNA molecule; and
a second promoter operably linked to a second nucleic acid molecule encoding the second gRNA molecule.
5 . The construct of claim 4 , wherein each of the first and second promoters comprises at least one selected independently from a group consisting of U6, H1, CMV, EF-1, RSV, and LTR promoters.
6 . The construct of claim 3 , wherein the construct is a virus.
7 . The construct of claim 6 , wherein the construct is a virus selected from the group consisting of a retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV).
8 . A kit comprising the group of gRNAs of claim 1 and an isolated nucleic acid molecule encoding a nuclease, wherein the nuclease can target a specific genome sequence as a genome editing tool, wherein the nuclease comprises CRISPR/Cas9.
9 . A method of modifying a cell in vitro, comprising introducing the group of gRNAs of claim 1 into the cell in need of modification; and introducing a sequence encoding a nuclease, wherein the nuclease can target a specific genome sequence as a genome editing tool, wherein the nuclease can target specific genome sequence as a genome editing tool, and wherein the nuclease comprises CRISPR/Cas9.
10 . The method of claim 9 , wherein said modifying a cell comprises correcting C9ORF72 repeat expansion for cells.
11 . A therapeutic composition for treating C9ORF72 related disease, comprising:
the group of gRNAs of claim 1 and a pharmaceutically acceptable adjuvant.