Enrichment of mutated cell free nucleic acids for cancer detection

Abstract

Provided herein are methods of enriching mutated cell free nucleic acids for detection and diagnosis of cancer. Also provided are methods using a CRISPR-Cas system to target and deplete unwanted more abundant cell free nucleic acid sequences thereby enriching for less abundant sequences.

1 - 40 . (canceled) 
     
     
         41 . A method for analyzing the genome of a cancer patient, comprising:
 providing an endonuclease system that comprises a plurality of crRNAs, or derivatives thereof, and a plurality of Cas proteins, or variants thereof;
 wherein each crRNA comprises a targeting sequence; and 
 wherein each Cas protein is capable of binding to a PAM site on a target nucleic acid; 
   contacting a patient sample, obtained from the cancer patient and comprising a plurality of target nucleic acids, with the endonuclease system to obtain a pool of target nucleic acid fragments associated with the patient sample,
 wherein each target nucleic acid fragment of the pool of target nucleic acid fragments is a DNA fragment of cell-free DNA (cfDNA); 
   sequencing the pool of target nucleic acid fragments to obtain first sequencing data from the cancer patient; and   comparing the first sequencing data from the cancer patient with second sequencing data from a reference genome fragmented by the endonuclease system to detect structural rearrangements and mutations in the genome of the cancer patient.   
     
     
         42 . The method of  claim 41 , wherein comparing the sequencing data comprises:
 comparing a first fragmentation pattern of the pool of target nucleic acids fragments associated with the patient sample with a second fragmentation pattern of a pool of target nucleic acids fragments from the reference genome.   
     
     
         43 . The method of  claim 42 , further comprising:
 contacting a control sample, obtained from a control subject and comprising a plurality of target nucleic acids, with the endonuclease system to obtain the pool of target nucleic acid fragments from the reference genome and associated with the control sample; and   sequencing the pool of target nucleic acid fragments associated with the control sample to obtain the second sequencing data from the reference genome.   
     
     
         44 . The method of  claim 43 , wherein the endonuclease system cleaves target nucleic acids in the control sample at a predetermined interval. 
     
     
         45 . The method of  claim 44 , wherein the predetermined interval is about 300 bp. 
     
     
         46 . The method of  claim 41 , wherein the plurality of Cas proteins comprises Cas9, or a variant thereof, a Cas9 ortholog, or a variant thereof, or Cpf1, or a variant thereof. 
     
     
         47 . The method of  claim 46 , wherein the Cas9, or variant thereof, is derived from Streptococcus pyogenes, and wherein the Cpf1, or variant thereof, is derived from  Francisell novicida  U112. 
     
     
         48 . The method of  claim 41 , wherein the patient sample comprises a blood sample, a serum sample, a plasma sample, a urine sample, or a cerebrospinal fluid sample. 
     
     
         49 . The method of  claim 41 , wherein a sequence in the plurality of target nucleic acids in the patient sample comprises a mutant allele sequence selected from a group consisting of: AKT1, BRAF, EGFR, KRAS, MAP2K1, NRAS, PI3KCA and PTEN. 
     
     
         50 . The method of  claim 41 , wherein a sequence in the plurality of target nucleic acids in the patient sample comprises a point mutation of at least one base pair. 
     
     
         51 . The method of  claim 41 , wherein the cfDNA comprises circulating tumor DNA (ctDNA) that includes a mutation selected from a group consisting of: a single nucleotide mutation, an insertion, and a deletion. 
     
     
         52 . The method of  claim 41 , wherein a target nucleic acid fragment of the pool of target nucleic acid fragments has either a blunt end or a staggered end. 
     
     
         53 . The method of  claim 41 , wherein a sequence in the plurality of target nucleic acids in the patient sample comprises a mutant allele sequence selected from a group consisting of: AKT1-E17K, BRAF-V600E, BRAF-L597V, BRAF-G469A, BRAF-G466V, EGFR-E709 T710delins, EGFR-G719S, EGFR-G719C, EGFR-G719A, EGFR-Exon19del, EGFR-T790M, EGFR-L858R, EGFR-L861Q, KRAS-Q61H, KRAS-Q61L, KRAS-Q61R, KRAS-Q61K, KRAS-G13A, KRAS-G13D, KRAS-G13C, KRAS-G13R, KRAS-G13D, KRAS-G13C, KRAS-G13R, KRAS-G13S, KRAS-G12V, KRAS-G12A, KRAS-G12D, KRAS-G12D, KRAS-G12C, KRAS-G12R, KRAS-G12S, MAP2K1-Q56P, NRAS-Q61H, NRAS-Q61L, NRAS-Q61R, NRAS-Q61K, NRAS-G12A, NRAS-G12D, NRAS-G12C, NRAS-G12R, NRAS-G12S, PI3KCA-E542K, PI3KCA-E545Q, PI3KCA-E545K, PI3KCA-H1047R, PI3KCA-H1047L, and PTEN-R233*. 
     
     
         54 . The method of  claim 41 , wherein a sequence in the plurality of target nucleic acids in the patient sample comprises a mutation identified in  FIG.  7   .