Methods and compositions for generating CRISPR/Cas guide RNAs

A method of generating a library of DNA molecules encoding Cas9 single guide RNAs (sgRNAs) or Cas9 targeter RNAs, the method comprising:

• (a) contacting a target DNA molecule with a first DNA endonuclease that specifically binds to and cleaves within a recognition sequence that includes a Cas9 PAM sequence, to generate a plurality of cleavage fragments;
• (b) attaching a first DNA adapter to the plurality of cleavage fragments to generate a plurality of adapter-attached cleavage fragments, each having an adapter segment and a cleavage fragment segment;
• (c) contacting the plurality of adapter-attached cleavage fragments with a second DNA endonuclease that specifically binds to a recognition sequence present in the adapter segment and cleaves within the cleavage fragment segment to generate a plurality of adapter-attached Cas9 guide sequences each having an adapter segment and a guide sequence segment;
• (d) contacting the plurality of adapter-attached Cas9 guide sequences with a third DNA endonuclease that specifically binds to a recognition sequence present in the adapter segment and cleaves at one or more sites to remove all or a portion of the adapter segment, thereby generating a plurality of Cas9 guide sequences;
• and
• (e) attaching DNA encoding a constant region of a Cas9 sgRNA or a Cas9 targeter RNA to the plurality of Cas9 guide sequences to generate a library of DNA molecules encoding Cas9 sgRNAs or Cas9 targeter RNAs.