CRISPR effector system based diagnostics
The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.
1. A system for detecting the presence of a nucleic acid target sequence in an in vitro sample, comprising:
reagents for amplifying the target sequence;
a Cas13;
at least one guide polynucleotide comprising a guide sequence that hybridizes with the target sequence, and designed to form a complex with the Cas13; and
an RNA-based masking construct comprising a non-target sequence; and
wherein the Cas13 exhibits collateral RNase activity and cleaves the non-target sequence of the RNA-based masking construct once activated by the target sequence.
2. The system of claim 1 , wherein the target sequence is a target DNA sequence and the system further comprises an RNA polymerase and a primer designed to bind the target DNA sequence and further comprises an RNA polymerase promoter.
3. The system of claim 1 , wherein the Cas13 comprises one or more HEPN domains.
4. The system of claim 3 , wherein the HEPN domain comprise a RxxxxH motif sequence.
5. The system of claim 4 , wherein the RxxxxH motif comprises a R[N/H/K]X 1 X 2 X 3 H sequence, wherein X 1 is R, S, D, E, Q, N, G, or Y, and X 2 is independently I, S, T, V, or L, and X 3 is independently L, F, N, Y, V, I, S, D, E, or A.
6. The system of claim 1 , wherein the Cas13 is a Cas13a, a Cas13b, or a Cas13c.
7. The system of claim 1 , wherein the reagents for amplifying the one or more target sequences are isothermal amplification reagents.
8. The system of claim 7 , wherein the isothermal amplification reagents are nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification (HDA), or nicking enzyme amplification.
9. A system for detecting the presence of one or more target polypeptides in an in vitro sample comprising:
an RNA-based masking construct comprising a non-target sequence; and
one or more detection aptamers, each designed to bind to one of the one or more target polypeptides, and each detection aptamer comprising a masked RNA polymerase promoter binding site or a masked primer binding site and a trigger sequence template, encoding a trigger sequence;
a Cas13
at least one guide polynucleotide comprising a guide sequence that hybridizes with the trigger sequence encoded by the trigger sequence template; and
wherein the Cas13 exhibits collateral RNase activity and cleaves the non-target sequence of the RNA-based masking construct once activated by the trigger sequence.
10. The system of claim 9 , further comprising nucleic acid amplification reagents to amplify the trigger sequence.
11. The system of claim 10 , wherein the nucleic acid amplification reagents to amplify the trigger sequence are isothermal amplification reagents.
12. The system of claim 11 , wherein the isothermal amplification reagents are nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nicking enzyme amplification.
13. The system of claim 9 , wherein the Cas13 comprises one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains.
14. The system of claim 13 , wherein the HEPN domain comprise a RxxxxH motif sequence.
15. The system of claim 14 , wherein the RxxxxH motif comprises a R[N/H/K]X 1 X 2 X 3 H sequence, wherein X 1 is R, S, D, E, Q, N, G, or Y, and X2 is independently I, S, T, V, or L, and X3 is independently L, F, N, Y, V, I, S, D, E, or A.
16. The system of claim 9 , wherein the Cas13 is a Cas13a, a Cas13b, or a Cas13c.