Detection of targeted sequence regions
Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.
1. A method of detecting a nucleic acid, the method comprising:
protecting, in a population of nucleic acids, first and second ends of a target nucleic acid wherein the target nucleic acid is greater than 1000 nucleotides in length by binding first Cas endonuclease and a second Cas endonuclease to the target nucleic acid;
degrading unprotected nucleic acids with an exonuclease while the first and second Cas endonucleases are bound to the target nucleic acid; and
sequencing the target nucleic acid using a single-molecule sequencing platform.
2. The method of claim 1 , wherein:
substantially all of the unprotected nucleic acids are degraded, and
the target nucleic acid is from a genome of a pathogen.
3. The method of claim 1 , wherein the target nucleic acid has a length of at least 10 kb.
4. The method of claim 1 , wherein the first Cas endonuclease comprises a first Cas9 protein complexed with a first guide RNA, and the second Cas endonuclease comprises a second Cas9 protein complexed with a second guide RNA.
5. The method of claim 4 , wherein at least one of the first Cas9 complex and the second Cas9 complex comprises a catalytically inactive Cas9 protein.
6. The method of claim 1 , wherein the target nucleic acid is from a genome of a pathogen.
7. The method of claim 1 , wherein the population of nucleic acids is isolated from an organism and the target nucleic acid comprises a sequence foreign to a genome of the organism.
8. The method of claim 7 , wherein the target nucleic acid is from a genome of a pathogen of the organism.
9. The method of claim 8 , wherein the pathogen is a virus, bacterium, or fungus.
10. The method of claim 9 , wherein the pathogen is a virus, and the target nucleic acid is integrated into a nucleic acid within the genome of the organism.
11. The method of claim 10 , wherein the pathogen is a virus, and the target nucleic acid exists separately from nucleic acids within the genome of the organism.
12. The method of claim 1 , wherein: the population of nucleic acids is isolated from an organism and the target nucleic acid is from a mitochondrial genome of the organism.
13. The method of claim 1 , wherein the population of nucleic acids is isolated from a source selected from the group consisting of a soil sample, a water sample, and a food sample.
14. The method of claim 13 , wherein the target nucleic acid is from a genome of a pathogen.
15. The method of claim 1 , wherein:
the population of nucleic acids is from a sample, and
a plurality of nucleic acids from genomes of organisms in the sample are detected, thereby identifying the metagenome of the sample.
16. The method of claim 1 , wherein:
the population of nucleic acids is from a sample, and
a plurality of nucleic acids from genomes of microbes are detected, thereby identifying the microbiome of the sample.