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 for sequences of interest, comprising:
a. providing a sample comprising sequences of interest and targeted sequences for depletion, wherein the sequences of interest comprise less than 50% of the sample, wherein the sequences of interest and targeted sequences for depletion are adapter-ligated, wherein the adapters are ligated to the 5′ and 3′ ends of the sequences of interest and targeted sequences for depletion, and wherein the sequences of interest and targeted sequences for depletion are DNA;
b. contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to targeted sequences for depletion, and whereby the targeted sequences are cleaved, thereby generating cut targeted sequences for depletion that are adapter-ligated only on one end and leaving uncut the sequences of interest that are adapter-ligated on both the 5′ and 3′ ends; and
c. amplifying the sequences of interest using adapter-specific PCR, thereby enriching the sample for the uncut sequences of interest.
2. The method of claim 1 , wherein the method comprises contacting the sample with at least 10 unique CRISPR/Cas system protein-gRNA complexes.
3. 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.
4. The method of claim 1 , wherein the sample comprises host nucleic acid sequences targeted for depletion and non-host nucleic acid sequences of interest.
5. The method of claim 4 , wherein the non-host nucleic acid sequences comprise microbial nucleic acid sequences.
6. The method of claim 1 , wherein the sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to mitochondrial DNA.
7. The method of claim 1 , wherein the sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to DNA corresponding to ribosomal RNA sequences, sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences.
8. The method of claim 1 , wherein the sequences of interest comprise less than 10% of the extracted nucleic acids.
9. 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.
10. The method of claim 4 , wherein:
step (a) comprises providing host nucleic acids targeted for depletion that are adapter-ligated and non-host nucleic acids of interest that 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; wherein step (b) comprises contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to targeted sites in the host nucleic acids, thereby generating host-nucleic acids adapter ligated only on one end and non-host nucleic acids adapter ligated on both the 5′ and 3′ ends.
11. The method of claim 10 , wherein the method comprises contacting the sample with at least 10 unique CRISPR/Cas system protein-gRNA complexes.
12. The method of claim 10 , wherein the non-host nucleic acids comprise less than 50% of the total nucleic acids in the sample.
13. The method of claim 10 , wherein the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample.
14. The method of claim 10 , wherein the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
15. A method for serially depleting targeted nucleic acids in a sample comprising:
a. providing a sample comprising sequences of interest and targeted sequences for depletion, wherein the targeted sequences for depletion comprise sequences from a first genome and a second genome, and wherein the sequences of interest and targeted sequences for depletion are adapter-ligated on their 5′ and 3′ ends, and wherein the sequences of interest and targeted sequences for depletion are DNA;
b. 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 first genome of the sequences targeted for depletion, whereby a portion of the first genome nucleic acids are cleaved, thereby generating cut first genome nucleic acid sequences that are adapter-ligated only on one end and leaving uncut the sequences of interest 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 second genome of the sequences targeted for depletion, whereby a portion of the second genome nucleic acids are cleaved, thereby generating cut second genome nucleic acid sequences that are adapter-ligated only on one end and leaving uncut the sequences of interest 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 sequences of interest.
16. The method of claim 15 , wherein the method comprises contacting the sample with at least 10 unique CRISPR/Cas system protein-gRNA complexes configured to hybridize to targeted sequences in the targeted sequences for depletion.
17. The method of claim 15 , wherein the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
18. The method of claim 15 , wherein the sequences of interest comprise less than 5% of the total nucleic acids in the sample.