Methods of making guide RNA
A nucleic acid construct is provided that encodes two or more or a plurality of spacer sequences separated by restriction endonuclease recognition site. A plurality of such nucleic acid sequences are provided as a library for making guide RNAs for use with CRISPR/Cas systems.
1. A method of making a library of nucleic acids encoding guide RNA spacer sequences wherein the guide RNA spacer sequences are complementary to corresponding target nucleic acid sequences comprising
synthesizing a plurality of nucleic acid sequences each encoding (1) a plurality of spacer sequences including a pair of end spacer sequences, (2) inner restriction endonuclease cut sites, wherein the restriction endonuclease cut sites separate each of the plurality of spacer sequences from each other, (3) a pair of outer restriction endonuclease cut sites flanking the pair of end spacer sequences, and (4) a pair of amplification primer binding sites flanking the outer restriction endonuclease cut sites, and
wherein the outer restriction endonuclease cut sites are different from the inner restriction endonuclease cut sites.
2. The method of claim 1 wherein each nucleic acid sequence encodes 3 to 20 spacer sequences.
3. The method of claim 1 wherein the inner restriction endonuclease cut sites are different from each other.
4. The method of claim 1 wherein the plurality of nucleic acid sequences are synthesized on a support.
5. The method of claim 4 wherein the plurality of nucleic acid sequences are removed from the support.