Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing

A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:

• (a) a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD) and
• (b) a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule;
• wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM);
• wherein the guide RNA is:
• (i) a single guide RNA that includes a complementarity region at the 5′ end of the single guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or
• (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA;
• wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell;
• and
• wherein the dCas9-HFD comprises a heterologous functional domain (HFD) that modifies gene expression, histones, or DNA.