Short multi-repeat RNA targeting gene silencing
Short multi-repeat RNA targeting constructs for manipulating RNA targets and obtaining variable levels of gene silencing, and methods of using the same to silence RNA targets in mammals, insects, plants, and fungus. The constructs comprise two or more distinct guide nucleotide sequences (in the absence of CRISPR nuclease) that are complementary to one or more RNA targets, wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 100 nt or less.
1 . A system comprising a short multi-repeat RNA targeting construct for manipulating an RNA target without introducing a nuclease protein, said construct comprising three or more distinct guide nucleotide sequences that are complementary to the same RNA target, wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragments of 100 nt or less, wherein said single-stranded antisense nucleotide fragments are not self-complementary and do not form double-stranded structures with themselves, said system being free of constructs comprising nuclease-encoding nucleotide sequences or nuclease protein.
2 . The system of claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 15 nt to 100 nt.
3 . The system of claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 80 nt or less.
4 . The system of claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 50 nt or less.
5 . The system of claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 22 nt to 30 nt.
6 . The system of claim 1 , wherein said guide nucleotide sequences are guide RNA (crRNA) or DNA encoding for the guide RNA, which are capable of binding or hybridizing to the RNA target.
7 . The system of claim 1 , wherein the RNA target is a coding sequence selected from mRNA or pre-mRNA, or a non-coding sequence selected from ncRNA, IncRNA, tRNA, or rRNA.
8 . The system of claim 1 , wherein said guide nucleotide sequences each bind to a different region of the same RNA target.
9 . The system of claim 1 , wherein each of said three or more distinct guide nucleotide sequences are:
directly linked on a single expression cassette; or
indirectly linked on a single expression cassette, such that each guide nucleotide sequence is separated by an intervening sequence on said expression cassette.
10 . The system of claim 9 , wherein said single expression cassette is a homogenous multimer wherein each of said three or more distinct guide nucleotide sequences consists of the same sequence and has complementarity with the same region of the same RNA target.
11 . The system of claim 10 , wherein said homogenous multimer comprises up to eight repeats of the same guide nucleotide sequence.
12 . The system of claim 9 , wherein said single expression cassette is a heterogenous multimer wherein each of said three or more distinct guide nucleotide sequences consists of different sequences having complementarity with different regions of the same RNA target.
13 . The system of claim 9 , wherein said expression cassette comprises at least one regulatory element adjacent said three or more distinct guide nucleotide sequences.
14 . A composition for manipulation of RNA targets without introducing a nuclease protein, said composition comprising a system comprising plurality of short multi-repeat RNA targeting constructs according to claim 1 dispersed in a carrier or vehicle, wherein said composition is free of nuclease-encoding nucleotide sequence or nuclease protein.
15 . A method of modifying an RNA target without introducing a nuclease protein, said method comprising delivering to said RNA target a system comprising a short multi-repeat RNA targeting construct according to claim 1 without introducing a nuclease-encoding nucleotide sequence or nuclease protein.
16 . A method of gene silencing without introducing a nuclease protein, the method comprising delivering to a cell, tissue, organ, or organism, a system comprising a short multi-repeat RNA targeting construct according to claim 1 without introducing a nuclease-encoding nucleotide sequence or nuclease protein, wherein at least one of said two or more distinct guide nucleotide sequences is complementary to an RNA target associated with said gene.
17 . A method of manipulating a plant characteristic without introducing a nuclease protein, said method comprising delivering to a plant cell, tissue, or plant a system comprising a short multi-repeat RNA targeting construct according to claim 1 without introducing a nuclease-encoding nucleotide sequence or nuclease protein, wherein at least one of said three or more distinct guide nucleotide sequences is complementary to an RNA target associated with a gene encoding for said plant characteristic.
18 . A seed, seedling, or progeny of a plant produced according to the method of claim 17 , wherein said seed, seedling, or progeny expresses said short multi-repeat RNA targeting construct without a bacterial nuclease protein.
19 . The system of claim 12 , said expression cassette further comprising three or more additional distinct guide nucleotide sequences having complementarity with a different RNA target.