Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
Provided herein are methods and compositions for depleting targeted nucleic acid sequences from a sample, enriching for sequences of interest from a sample, and/or partitioning of sequences from a sample. The methods and compositions are applicable to biological, clinical, forensic, and environmental samples.
1. A method of enriching a sample comprising:
a. providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the host nucleic acids and non-host nucleic acids are adapter-ligated, wherein the adapters are ligated to the 5′ and 3′ ends of the host nucleic acids and the non-host nucleic acids, and wherein the host nucleic acids and non-host nucleic acids are DNA;
b. contacting the sample with a plurality of CRISPR/Cas system protein-guide RNA (gRNA) complexes, wherein the gRNAs are complementary to targeted sites in the host nucleic acids, thereby generating cut host nucleic acid fragments that are adapter-ligated only on one end and leaving uncut the non-host nucleic acids that are adapter-ligated on both the 5′ and 3 ′ ends; and
c. amplifying the product of step (b) using adapter-specific PCR, thereby enriching the sample for the uncut non-host nucleic acids.
2. The method of claim 1 , wherein the method comprises contacting the sample with at least 10 2 unique CRISPR/Cas system protein-gRNA complexes.
3. The method of claim 1 , wherein the contacting of step (b) generates host DNA adapter-ligated on the 5′ end but not the 3′ end and non-host DNA adapter-ligated on both the 5′ and 3′ ends.
4. The method of claim 1 , wherein the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2 or Cm5.
5. The method of claim 4 , wherein the CRISPR/Cas system protein is Cas9 or Cpf1.
6. The method of claim 1 , wherein the CRISPR/Cas system protein is Cas9 or Cpf1 nickase.
7. The method of claim 1 , wherein the CRISPR/Cas system protein is thermostable.
8. The method of claim 1 , wherein the host is selected from the group consisting of a human, cow, horse, sheep, pig, avian organism, monkey, dog, cat, gerbil, bird, mouse, and rat.
9. The method of claim 1 , wherein the non-host is selected from the group consisting of a eukaryote, virus, bacterium, fungus, prokaryotic organism and protozoa.
10. The method of claim 1 , wherein the adapter-ligated host nucleic acids and non-host nucleic acids range from 50-1000 bp.
11. The method of claim 1 , wherein the non-host nucleic acids comprise less than 50% of the total nucleic acids in the sample.
12. The method of claim 1 , wherein the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample.
13. The method of claim 1 , further comprising treating the product of step (b) with an enzyme that has exonuclease activity.
14. The method of claim 13 , wherein the enzyme is Exonuclease III or BAL-31.
15. The method of claim 1 , wherein the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
16. A method for serially depleting targeted nucleic acids in a sample comprising:
a. providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the non-host nucleic acids comprise nucleic acids from at least one known non-host organism and nucleic acids from at least one unknown non-host organism, wherein the host nucleic acids and non-host nucleic acids are adapter-ligated to the 5′ and 3′ ends of the host nucleic acids and the non-host nucleic acids, and wherein the host nucleic acids and non-host nucleic acids are DNA;
b. contacting the sample with a plurality of CRISPR/Cas system protein-guide RNA (gRNA) complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the host nucleic acids, whereby a portion of the host nucleic acids are cleaved, thereby generating cut host nucleic acid fragments that are adapter-ligated only on one end and leaving uncut the non-host nucleic acids that are adapter-ligated on both the 5′ and 3′ ends;
c. contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the at least one known non-host nucleic acids, whereby a portion of the at least one known non-host nucleic acids are cleaved, thereby generating cut known non-host nucleic acid fragments that are that are adapter ligated only on one end and leaving uncut the nucleic acids from the at least one unknown non-host organism that are adapter-ligated on both the 5′ and 3′ ends; and
d. amplifying the product of step (c) using adapter-specific PCR, thereby isolating the nucleic acids from the unknown non-host organism.
17. The method of claim 16 , wherein the method comprises contacting the sample with at least 10 2 unique CRISPR/Cas system protein-gRNA complexes configured to hybridize to targeted sequences in the host nucleic acids.
18. The method of claim 16 , wherein the method comprises contacting the sample with at least 10 2 unique CRISPR/Cas system protein-gRNA complexes configured to hybridize to targeted sequences in the at least one known non-host nucleic acids.
19. The method of claim 16 , wherein the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
20. The method of claim 16 , wherein the nucleic acids from an unknown non-host organism comprise less than 5% of the total nucleic acids in the sample.
21. The method of claim 16 , wherein the host is a human.
22. The method of claim 1 , wherein the method comprises contacting the sample with at least 10 4 unique CRISPR/Cas system protein-gRNA complexes.
23. The method of claim 16 , wherein the method comprises contacting the sample with at least 10 4 unique CRISPR/Cas system protein-gRNA complexes.
24. The method of claim 1 , wherein the host nucleic acids and non-host nucleic acids comprise genomic DNA.
25. The method of claim 1 , wherein the host nucleic acids and non-host nucleic acids comprise cDNA.
26. The method of claim 16 , wherein the host nucleic acids and non-host nucleic acids comprise genomic DNA.
27. The method of claim 16 , wherein the host nucleic acids and non-host nucleic acids comprise cDNA.