Altered guide rnas for modulating cas9 activity and methods of use

A synthetic nucleic acid construct, comprising:

• (a) a crRNA comprising a 3′ region and a 5′ region, wherein the 3′ region comprises at least about 10 consecutive nucleotides of a CRISPR repeat, and/or a functional fragment of the CRISPR repeat, and the 5′ region comprises at least about 20 consecutive nucleotides of a spacer sequence located immediately upstream of the repeat;
• (b) a tracrRNA comprising a 5′ and a 3′ region, wherein at least a portion of the 5′ region of the tracrRNA is complementary to the 3′ region (i.e., CRISPR repeat) of the crRNA,
• wherein, when the 5′ region of the tracrRNA that is complementary to the 3′ region of the crRNA hybridizes to the 3′ region of the crRNA, the synthetic nucleic acid construct forms secondary structures from 5′ to 3′ of:
• (i) a stem, the stem comprising a duplex between the 5′ end of the tracrRNA and the repeat of the crRNA, and optionally, the stem further comprising a kink or a bulge;
• (ii) a nexus hairpin;
• (iii) at least one terminal hairpin;
• and
• (c) optionally, a linker that when present links the 3′end of the crRNA to the 5′ end of the tracrRNA (i.e., linking the 3′ end and 5′ end of the stem),
• wherein the construct comprises an insertion, substitution, and/or deletion of about one to about five nucleotides or base pairs in the nexus hairpin as compared to a wild type Type II crRNA:tracrRNA duplex;
• and/or
• an insertion, substitution and/or deletion of about one to about nine nucleotides and/or base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) in the stem as compared to a wild type Type II crRNA:tracrRNA duplex.