Synthetic guide rna, compositions, methods, and uses thereof

The method of any one of the preceding claims, wherein the ligating enzyme is selected from the group consisting of T4 RNA ligase 1, T4 RNA Ligase 2, RtcB Ligase, Thermo-stable 5′ App DNA/RNA Ligase, ElectroLigase, T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, Taq DNA Ligase, SplintR Ligase E. coli DNA Ligase, 9° N DNA Ligase, CircLigase, CircLigase II, DNA Ligase I, DNA Ligase III, and DNA Ligase IV.

The method of any one of the preceding claims, wherein the first and/or second RNA is chemically synthesized.

The method of any one of the preceding claims, wherein the first RNA is a clustered regularly interspersed short palindromic repeats (CRISPR) RNA (crRNA) and the second RNA is a trans-activating RNA (tracrRNA).

The method of any one of the preceding claims wherein a guide RNA (gRNA) is produced.

The method of any one of the preceding claims, wherein the first RNA and/or the second RNA is chemically synthesized.

The method of any one of the preceding claims, wherein ligating the first RNA and the second RNA with a ligating enzyme creates phosphodiester linkages between the first and the second RNA.

The method of any one of the preceding claims, wherein the first RNA and/or second RNA nucleotide is engineered to allow for non-covalent assembly.

The method of any one of the preceding claims, wherein the stem loop has a length of between about 2-50 nucleotides.

The method of any one of the preceding claims, wherein the first RNA and the second RNA comprise at least two RNA nucleotides that have perfect complementarity.

The method of any one of the preceding claims, wherein the first RNA and the second RNA comprise at least five, six, or seven consecutive RNA nucleotides that are complementary at a lower stem formed by the first RNA and the second RNA.

The method of any one of the preceding claims, wherein the first RNA and the second RNA comprise at least four to fourteen consecutive RNA nucleotides that are complementary at an upper stem.

The method of any one of the preceding claims, wherein the first and the second RNA comprise 7 consecutive RNA nucleotides that are complementary at a lower stem.

The method of any one of the preceding claims, wherein the first RNA and/or the second RNA is engineered to create a ligation site for a ligation enzyme.

The method of any of the preceding claims, wherein the stem loop comprises a loop of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides.

The method of any one of the preceding claims, wherein the stem loop comprises a tetraloop.

The method of any one of the preceding claims, wherein ligating the first RNA and the second RNA occurs at a ligation site that is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs from a bulge.

The method of any one of the preceding claims, wherein the first RNA and/or second RNA is enzymatically produced.

The method of any one the preceding claims, wherein the first RNA comprises a 3′ sequence that is capable of base pairing with a portion of the second RNA.

The method of any one of the preceding claims, wherein the first RNA comprises a phosphate at the 5′ terminus.

The method of any one the preceding claims, wherein the second RNA comprises a variable protospacer region.

The method of any one of the preceding claims, wherein about 8-50 nucleotides are complementary and allow for base pairing between the first and the second RNA.

The method of any one of the preceding claims, wherein the first and the second RNA have different nucleotide lengths.

The method of claim any one of the preceding claims, wherein the second RNA has from about 20-70 nucleotides.

The method of any one of the preceding claims, wherein base pairing occurs in a lower stem.

The method of any one of the preceding claims, wherein the gRNA is an extended guide RNA, prime editor guide RNA (pegRNA), or a Cas12 guide RNA such as Cas12a guide RNA, Cas12b guide RNA, Cas12c guide RNA, Cas12d, guide RNA, Cas12e guide RNA, Cas12f guide RNA, Cas12g guide RNA, Cas12h guide RNA, Cas12i guide RNA, Cas12j guide RNA, or Cas12k guide RNA.

The method of any one of the preceding claims, wherein the gRNA comprises one or more of the following:

• a spacer, a lower stem, a bulge, an upper stem, a nexus and a hairpin.

The method of any one of the preceding claims, wherein the first RNA and the second RNA are present at a ratio of about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, or 1:0.5.

The method of any one of the preceding claims, wherein the gRNA is produced at a yield of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.

The method of any one of the preceding claims, wherein the gRNA is produced at 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more improvement in yield as compared to conventional synthetic methods.

A method for targeted transcription activation, targeted transcription repression, targeted epigenome modification, or targeted genome modification, the method comprising introducing into a eukaryotic cell:

• (a) a synthetic guide RNA (gRNA) as defined in any one of the preceding claims;
• (b) at least one CRISPR/Cas protein or a nucleic acid encoding the at least one CRISPR/Cas protein;
• wherein interactions between (a) and (b) and a target sequence in chromosomal DNA leads to targeted transcription activation, targeted transcription repression, targeted epigenome modification, or targeted genome modification.

A method for targeted RNA modification, the method comprising introducing into a eukaryotic cell:

• (a) a synthetic guide RNA (gRNA) as defined in any one of the preceding claims;
• (b) at least one CRISPR/Cas protein or a nucleic acid encoding the at least one CRISPR/Cas protein;
• wherein interactions between (a) and (b) and an RNA expressed by chromosomal DNA leads to a modification of the RNA expressed by the chromosomal DNA.

A composition produced by the method of any one of the proceeding claims comprising a first RNA comprising a phosphate at a 5′ terminus and a second RNA comprising a variable protospacer region, wherein the first and the second RNA are non-covalently bound.

A composition comprising an RNA comprising a nucleotide sequence at least about 80% identical to CGAUACGACAGAAC.

A composition comprising an RNA comprising a nucleotide sequence at least about 80% identical to CGCCG.

A composition comprising an RNA comprising a nucleotide sequence at least about 80% identical to CGGCCGC.

A composition comprising an RNA comprising a nucleotide sequence at least about 80% identical to CGCGC.

A kit comprising a first RNA comprising trans-activating RNA (tracrRNA) sequence, a second RNA comprising a variable protospacer region, and a ligase.