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US 2019/0161752 A1Published application
Enrichment of mutated cell free nucleic acids for cancer detection
AssigneeGrail, LLC
InventorsGordon Cann, Alex Aravanis, Arash Jamshidi, Rick Klausner, Richard Rava
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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.
Claims40
A method for enriching a plurality of target nucleic acids in a sample, the method comprising:
- providing an endonuclease system;
- wherein each of the plurality of target nucleic acids comprises a first variant and a second variant,
- wherein the endonuclease system comprises a plurality of clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs), or derivatives thereof, each crRNA comprising a targeting sequence, and a plurality of CRISPR-associated (Cas) proteins, or variants thereof, each Cas protein capable of binding to a protospacer adjacent motif (PAM) site on a target nucleic acid,
- wherein the first variant of each target nucleic acid comprises a PAM site adjacent to a region complementary to a crRNA targeting sequence, and wherein the second variant does not comprise the PAM site or does not comprise the region complementary to the crRNA targeting sequence adjacent to the PAM site, and
- contacting the sample with the endonuclease system, thereby depleting the first variant and enriching the second variant of each of the plurality of target nucleic acids in the sample.
2 more elements
A method for analyzing the genome of a cancer patient, comprising:
- providing an endonuclease system,
- wherein the endonuclease system comprises a plurality of crRNAs, or derivatives thereof, each crRNA comprising a targeting sequence, and a plurality of Cas proteins, or variants thereof, each Cas protein capable of binding to a PAM site on a target nucleic acid;
- contacting a sample obtained from the cancer patient comprising a plurality of target nucleic acids with the endonuclease system to obtain a pool of target nucleic acid fragments;
- sequencing the pool of target nucleic acid fragments to obtain sequencing data from the cancer patient, and
- comparing the sequencing data from the cancer patient with sequencing data from a reference genome fragmented by the endonuclease system to detect structural rearrangements and mutations in the genome of the cancer patient.
2 more elements
Dependencies are inferred from the claim text.