Guide rna with chemical modifications

A chemically-synthesized single molecule CRISPR guide RNA (gRNA) comprising:

• a 5′-end and a 3′-end;
• one or more chemically modified nucleotides within five nucleotides from said 5′-end or said 3′-end, or within five nucleotides from each of said 5′-end and said 3′-end, wherein each of the one or more chemically modified nucleotides is independently selected from 2′-O-methyl-3′-phosphorothioate (MS), 2′-O-methyl-3′-phosphonoacetate (MP), and 2′-O-methyl-3′-thiophosphonoacetate (MSP);
• a guide sequence that is 18-22 nucleotides long and is capable of hybridizing to a target sequence in a target polynucleotide;
• and
• a scaffold region that can interact with a Cas9 protein to form a gRNA:Cas9 complex, wherein said complex is capable of cleaving both strands of the target polynucleotide when the Cas9 protein has double-strand nuclease activity.