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 30% of the sample;
b. contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, and whereby the targeted sequences are cleaved.
2 . The method of claim 1 , further comprising extracting the sequences of interest and the targeted sequences for depletion from the sample.
3 . The method of claim 2 , further comprising fragmenting the extracted sequences.
4 . The method of claim 3 , further comprising adapter ligating the 5′ and 3′ ends of the fragmented extracted sequences.
5 . The method of claim 1 , wherein the cleaved targeted sequences are removed by size-exclusion or with the use of biotin.
6 . The method of claim 1 , further comprising amplifying the sequences of interest.
7 . The method of claim 1 , wherein the method comprises contacting the sample with at least 10 2 unique CRISPR/Cas system protein-gRNA complexes.
8 . 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.
9 . The method of claim 1 , wherein the sample comprises host nucleic acid sequences targeted for depletion and non-host nucleic acid sequences of interest.
10 . The method of claim 9 , wherein the non-host nucleic acid sequences comprise microbial nucleic acid sequences.
11 . The method of claim 10 , wherein the microbial nucleic acid sequences are bacterial, viral or eukaryotic parasitic nucleic acid sequences.
12 . 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.
13 . 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.
14 . The method of claim 2 , wherein the extracted nucleic acids incudes any one of single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, cDNA, synthetic DNA, artificial DNA, and DNA/RNA hybrids.
15 . The method of claim 1 , wherein the sequences of interest comprise less than 10% of the extracted nucleic acids.
16 . The method of claim 1 , wherein the CRISPR/Cas system protein is Cas9.
17 . The method of claim 1 , wherein the CRISPR/Cas system protein is catalytically dead.
18 . The method of claim 17 , wherein the catalytically dead CRISPR/Cas system protein is dCas9.
19 . The method of claim 1 , wherein the CRISPR/Cas system protein is a CRISPR/Cas system protein nickase.
20 . The method of claim 19 , wherein the CRISPR/Cas system protein nickase is a Cas9 nickase.
21 - 70 . (canceled)