Targeted enrichment by endonuclease protection

A method for preparing an adapter ligated target nucleic acid fragment from a sample comprising a nucleic acid molecule, wherein the target nucleic acid fragment comprises a sequence of interest, wherein the method comprises the steps of:

• a) providing the sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest;
• b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating the target nucleic acid fragment comprising the sequence of interest and at least one non-target nucleic acid fragment;
• c) contacting the cleaved nucleic acid molecules obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment;
• d) optionally purifying the target nucleic acid fragment comprising the sequence of interest from the digest obtained in step c;
• and
• e) ligating adapters to the target nucleic acid fragment.

A method for sequencing a target nucleic acid fragment from a sample comprising a nucleic acid molecule, wherein the target nucleic acid fragment comprises a sequence of interest, wherein the method comprises the steps of:

• a) providing the sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest;
• b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating the target nucleic acid fragment comprising the sequence of interest and at least one non-target nucleic acid fragment;
• c) contacting the cleaved nucleic acid molecules obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment;
• d) optionally purifying the target nucleic acid fragment comprising the sequence of interest from the digest obtained in step c;
• e) optionally ligating adapters to the target nucleic acid fragment;
• and
• f) sequencing the at least one target nucleic acid fragment.