High-plex guide pooling for nucleic acid detection
Provided herein, in certain embodiments, are various methods, reagents, and devices for detection of multiple target nucleic acids in a sample, or multiple segments of a target nucleic acid in a sample, using a programmable nuclease. In certain embodiments, the present disclosure provides compositions of pools of guide nucleic acids, programmable nucleases, and detector nucleic acids and methods of using said compositions for detection of different segments of one target nucleic acid or different target nucleic acids in a sample.
1 . A composition comprising a programmable nuclease, a detector nucleic acid, and a pool of guide nucleic acids comprising greater than 20 distinct guide nucleic acid sequences, wherein:
(i) at least one guide nucleic acid of the pool hybridizes to a segment of a target nucleic acid;
(ii) the detector nucleic acid comprises a detection moiety that produces a detectable signal responsive to cleavage by the programmable nuclease upon formation of a complex comprising the programmable nuclease, the at least one guide nucleic acid, and the segment of the target nucleic acid; and
(iii) a total concentration of the pool of guide nucleic acids is about 400 nM to about 2000 nM (2 μM).
2 . The composition of claim 1 , wherein the pool of guide nucleic acids comprises at least 50 distinct guide nucleic acid sequences, at least 100 distinct guide nucleic acid sequences, at least 500 distinct guide nucleic acid sequences, or at least 1000 distinct guide nucleic acid sequences.
3 . The composition of claim 1 , wherein the pool of guide nucleic acids comprises at least two guide nucleic acids that hybridize to a different segment of the target nucleic acid.
4 . The composition of claim 1 , wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acids that correspond to nucleic acids of the target nucleic acid.
5 . The composition of claim 4 , wherein:
a) the tiled guide nucleic acids are sequential along the target nucleic acid upon hybridization to the target nucleic acid;
b) the tiled guide nucleic acids are non-sequential along the target nucleic acid upon hybridization to the target nucleic acid;
c) the tiled guide nucleic acids are overlapping along the target nucleic acid upon hybridization to the target nucleic acid; or
d) any combination thereof.
6 . The composition of claim 1 , wherein the target nucleic acid is from a pathogen.
7 . The composition of claim 1 , wherein at least two guide nucleic acids of the pool of guide nucleic acids hybridize to segments of distinct target nucleic acids.
8 . The composition of claim 7 , wherein at least two target nucleic acids of the distinct target nucleic acids are from different pathogens.
9 . The composition of claim 6 , wherein the pathogen is a virus, a bacterium, a fungus, a protozoan, or a worm.
10 . The composition of claim 1 , wherein at least two guide nucleic acids of the pool of guide nucleic acids differs from one another by at least one base.
11 . The composition of claim 1 , wherein the total concentration of the pool of guide nucleic acids is about 400 nM, about 1000 nM (1 μM), or about 2000 nM (2 μM).
12 . The composition of claim 1 , wherein each guide nucleic acid of the pool of guide nucleic acids comprises from 20 to 50 bases.
13 . The composition of claim 1 , wherein the programmable nuclease is a Type V CRISPR-Cas enzyme or a Type VI CRISPR-Cas enzyme.
14 . The composition of claim 1 , wherein the target nucleic acid is DNA or RNA.
15 . The composition of claim 1 , wherein the composition further comprises the target nucleic acid.
16 . The composition of claim 15 , wherein the target nucleic acid comprises distinct target nucleic acids.
17 . A method of assaying for a segment of a target nucleic acid in a sample, the method comprising:
contacting the sample to the composition of claim 1 ; and
assaying for the detectable signal.
18 . The method of claim 17 , further comprising reverse transcribing the target nucleic acid, amplifying the target nucleic acid, in vitro transcribing the target nucleic acid, or any combination thereof.
19 . The method of claim 18 , wherein the amplifying is isothermal amplification.