Method of using cut&run or cut&tag to validate crispr-cas targeting

A method to validate CRISPR-Cas targeting comprising the following steps:

• (a) expressing a catalytically inactive Cas protein (dCas) in target cells, wherein the dCas protein optionally comprises a protein tag,
• (b) optionally hypotonic lysis of the cells of step (a) to release nuclei,
• (c) immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads,
• (d) incubating the product of step (c) with an anti-CRISPR-dCas antibody or an antibody against the tag of the protein tag of step (a),
• (e) incubating the product of step (d) with a secondary antibody against the anti-CRISPR-dCas antibody or the anti-tag antibody,
• (f) incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein loaded with DNA primers duplexes for high-throughput sequencing,
• (g) adding a Ca2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes,
• (h) adding a chelator-containing buffer to stop the reaction of step (f),
• (i) pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant,
• (j) extracting DNA and RNA, respectively, from the chromatin fragments of step (i), and
• (k) sequencing DNA and RNA, respectively.