DUX4 vectors and engineered guide rnas

Abstract

Disclosed herein are plasmids encoding engineered guide RNAs and compositions comprising the same. Also disclosed herein are AAVs containing engineered guide RNAs. Also disclosed herein are methods of treating diseases or conditions in a subject by administering engineered guide RNAs or pharmaceutical compositions described herein.

Description

Attorney Docket No.199235-774601 DUX4 VECTORS AND ENGINEERED GUIDE RNAS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application No.63 / 663,749 filed on June 25, 2024, and U.S. Provisional Application No.63 / 701,772 filed on October 1, 2024, the disclosures of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST .26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on June 18, 2025, is named 199235-774601_generic_SL.xml and is 215,415 bytes in size. BACKGROUND

[0003] Compositions that mediate RNA editing can be viable therapies for genetic diseases. However, efficacious compositions that can maximize on-target RNA editing while minimizing off-target RNA editing are needed. Moreover, vectors encoding guide RNAs that are capable of facilitating RNA editing are also needed. SUMMARY

[0004] Disclosed herein are recombinant AAVs encapsidating a vector. In some embodiments, the vector can comprise a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 81. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 72. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 121. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 122. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 123. In some embodiments, the sequence of the vector can comprise SEQ ID NO:Attorney Docket No.199235-774601 124. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 125. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 126. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 127. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 128. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 129. In some embodiments, the sequence of the vector can comprise SEQ ID NO: 130. In some embodiments, the vector can encode an engineered guide RNA, and the engineered guide RNA can comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136.

[0005] Disclosed herein are recombinant AAVs encapsidating a vector. In some embodiments, the vector can comprise a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO: 111. In some embodiments, the sequence of the vector comprises SEQ ID NO: 74. In some embodiments, the sequence of the vector comprises SEQ ID NO: 63. In some embodiments, the sequence of the vector comprises SEQ ID NO: 102. In some embodiments, the sequence of the vector comprises SEQ ID NO: 103. In some embodiments, the sequence of the vector comprises SEQ ID NO: 104. In some embodiments, the sequence of the vector comprises SEQ ID NO: 105. In some embodiments, the sequence of the vector comprises SEQ ID NO: 106. In some embodiments, the sequence of the vector comprises SEQ ID NO: 107. In some embodiments, the sequence of the vector comprises SEQ ID NO: 108. In some embodiments, the sequence of the vector comprises SEQ ID NO: 109. In some embodiments, the sequence of the vector comprises SEQ ID NO: 110. In some embodiments, the sequence of the vector comprises SEQ ID NO: 111. In some embodiments, the vector can encode an engineered guide RNA, wherein the engineered guide RNA, upon hybridization to a region of a target DUX4-FL RNA, forms a guide-target RNA scaffold that comprises one or more structural features. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate a knockdown of DUX4 protein encoded by the target DUX4-FL RNA. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNA or protein downstream of DUX4 is encoded by a geneAttorney Docket No.199235-774601 comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, or MBD3L2. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA. In some embodiments, the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene. In some embodiments, the target DUX4-FL RNA comprises a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence. In some embodiments, the RNA editing entity can comprise a human ADAR1, or a human ADAR2. In some embodiments, the one or more structural features can comprise a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof. In some embodiments, the one or more structural features can comprise a bulge. In some embodiments, the bulge can be an asymmetric bulge. In some embodiments, the bulge can be a symmetric bulge. In some embodiments, the one or more structural features can comprise an internal loop. In some embodiments, the internal loop can be a symmetric internal loop. In some embodiments, the internal loop can be an asymmetric internal loop. In some embodiments, the one or more structural features can comprise a hairpin. In some embodiments, the hairpin can be a recruitment hairpin. In some embodiments, the hairpin can be a non-recruitment hairpin. In some embodiments, the vector can encode an engineered guide RNA, and the engineered guide RNA can comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136.

[0006] Also disclosed herein are recombinant AAVs encapsidating a vector. In some embodiments, the vector can comprise a sequence that can encode an engineered guide RNA sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 137 – SEQ ID NO: 142. In some embodiments, the vector can further comprise a sequence encoding a SmOPT and U7 hairpin sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the vector can further comprise a sequence encoding a SmOPT and U5 hairpin sequence withAttorney Docket No.199235-774601 at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73. In some embodiments, the vector can further comprise a terminator sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 65 – SEQ ID NO: 68. In some embodiments, the vector can further comprise an engineered mU7 promoter sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the vector can further comprise an engineered promoter sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the vector can further comprise a ITR-to-ITR sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO: 111. In some embodiments, the recombinant AAV can comprise an AAV1 virion, AAV2 virion, AAV3 virion, AAV4 virion, AAV5 virion, AAV6 virion, AAV7 virion, AAV8 virion, AAV9 virion, AAV10 virion, AAV11 virion, or a derivative, a chimera, or a variant thereof. In some embodiments, the recombinant AAV can comprise a recombinant AAV (rAAV) virion, a hybrid AAV virion, a chimeric AAV virion, a self-complementary AAV (scAAV) virion, or any combination thereof.

[0007] Also disclosed herein are plasmids encoding an engineered guide RNA. In some embodiments, the plasmid can comprise a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 81. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 72. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 121. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 122. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 123. In someAttorney Docket No.199235-774601 embodiments, the sequence of the plasmid can comprise SEQ ID NO: 124. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 125. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 126. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 127. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 128. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 129. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 130. In some embodiments, the engineered guide RNA can comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136.

[0008] Also disclosed herein are plasmids encoding an engineered guide RNA. In some embodiments, the plasmid can comprise a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO: 111. In some embodiments, the sequence of the plasmid can comprise SEQ ID NO: 74. In some embodiments, the plasmid can comprise SEQ ID NO: 45. In some embodiments, the plasmid can comprise SEQ ID NO: 63. In some embodiments, the plasmid can comprise SEQ ID NO: 102. In some embodiments, the plasmid can comprise SEQ ID NO: 103. In some embodiments, the plasmid can comprise SEQ ID NO: 104. In some embodiments, the plasmid can comprise SEQ ID NO: 105. In some embodiments, the plasmid can comprise SEQ ID NO: 106. In some embodiments, the plasmid can comprise SEQ ID NO: 107. In some embodiments, the plasmid can comprise SEQ ID NO: 108. In some embodiments, the plasmid can comprise SEQ ID NO: 108. In some embodiments, the plasmid can comprise SEQ ID NO: 109. In some embodiments, the plasmid can comprise SEQ ID NO: 110. In some embodiments, the plasmid can comprise SEQ ID NO: 111. In some embodiments, the engineered guide RNA, upon hybridization to a region of a target DUX4-FL RNA, can form a guide-target RNA scaffold that comprises one or more structural features. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate a knockdown of DUX4 protein encoded by the target DUX4-FL RNA. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNAAttorney Docket No.199235-774601 or protein downstream of DUX4 is encoded by a gene comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, or MBD3L2. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA. In some embodiments, the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene. In some embodiments, the target DUX4-FL RNA can comprise a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence. In some embodiments, the RNA editing entity can comprise a human ADAR1, or a human ADAR2. In some embodiments, the one or more structural features can comprise a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof. In some embodiments, the one or more structural features can comprise a bulge. In some embodiments, the bulge can be an asymmetric bulge. In some embodiments, the bulge can be a symmetric bulge. In some embodiments, the one or more structural features can comprise an internal loop. In some embodiments, the internal loop can be a symmetric internal loop. In some embodiments, the internal loop can be an asymmetric internal loop. In some embodiments, the one or more structural features can comprise a hairpin. In some embodiments, the hairpin can be a recruitment hairpin. In some embodiments, the hairpin can be a non-recruitment hairpin. In some embodiments, the engineered guide RNA can comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136.

[0009] Also disclosed herein are DNA sequences comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat. In some embodiments, the DNA sequence can comprise SEQ ID NO: 81. In some embodiments, the DNA sequence can comprise SEQ ID NO: 72. In some embodiments, the DNA sequence can comprise SEQ ID NO: 121. In some embodiments, the DNA sequence can comprise SEQ ID NO: 122. In some embodiments, the DNA sequence can comprise SEQ IDAttorney Docket No.199235-774601 NO: 123. In some embodiments, the DNA sequence can comprise SEQ ID NO: 124. In some embodiments, the DNA sequence can comprise SEQ ID NO: 125. In some embodiments, the DNA sequence can comprise SEQ ID NO: 126. In some embodiments, the DNA sequence can comprise SEQ ID NO: 127. In some embodiments, the DNA sequence can comprise SEQ ID NO: 128. In some embodiments, the DNA sequence can comprise SEQ ID NO: 129. In some embodiments, the DNA sequence can comprise SEQ ID NO: 130. In some embodiments, the DNA sequence encodes an engineered guide RNA and the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136..

[0010] Also disclosed herein are DNA sequences comprising a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO: 111. In some embodiments, the DNA sequence can comprise SEQ ID NO: 74. In some embodiments, the DNA sequence can comprise SEQ ID NO: 63. In some embodiments, the DNA sequence can comprise SEQ ID NO: 102. In some embodiments, the DNA sequence can comprise SEQ ID NO: 103. In some embodiments, the DNA sequence can comprise SEQ ID NO: 104. In some embodiments, the DNA sequence can comprise SEQ ID NO: 105. In some embodiments, the DNA sequence can comprise SEQ ID NO: 106. In some embodiments, the DNA sequence can comprise SEQ ID NO: 107. In some embodiments, the DNA sequence can comprise SEQ ID NO: 108. In some embodiments, the DNA sequence can comprise SEQ ID NO: 109. In some embodiments, the DNA sequence can comprise SEQ ID NO: 110. In some embodiments, the DNA sequence can comprise SEQ ID NO: 111. In some embodiments, the DNA sequence can encode an engineered guide RNA, and wherein the engineered guide RNA upon hybridization to a region of a target DUX4-FL RNA, forms a guide-target RNA scaffold that comprises one or more structural features. In some embodiments, the engineered guide RNA can comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO: 136. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate a knockdown of DUX4 protein encoded by the target DUX4-FL RNA. In some embodiments, the engineered guide RNA whenAttorney Docket No.199235-774601 hybridized to the target DUX4-FL RNA can facilitate a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNA or protein downstream of DUX4 is encoded by a gene comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, or MBD3L2. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA. In some embodiments, the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene. In some embodiments, the target DUX4-FL RNA can comprise a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA. In some embodiments, the engineered guide RNA when hybridized to the target DUX4-FL RNA can facilitate RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence. In some embodiments, the RNA editing entity can comprise a human ADAR1, or a human ADAR2. In some embodiments, the one or more structural features can comprise a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof. In some embodiments, the one or more structural features can comprise a bulge. In some embodiments, the bulge can be an asymmetric bulge. In some embodiments, the bulge can be a symmetric bulge. In some embodiments, the one or more structural features can comprise an internal loop. In some embodiments, the internal loop can be a symmetric internal loop. In some embodiments, the internal loop can be an asymmetric internal loop. In some embodiments, the one or more structural features can comprise a hairpin. In some embodiments, the hairpin can be a recruitment hairpin. In some embodiments, the hairpin can be a non-recruitment hairpin.

[0011] Also disclosed herein are pharmaceutical compositions comprising the recombinant AAV encapsidating the vector described previously, the plasmid encoding the engineered guide RNA described previously, or the DNA sequence described previously; and a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition can be in unit dose form.

[0012] Also disclosed herein are methods of administering to a subject an effective amount of the recombinant AAV encapsidating the vector described previously, the plasmid encoding the engineered guide RNA described previously, or the DNA sequence described previously, or the pharmaceutical composition described previously. In some embodiments, the subject can be a mouse, a non-human primate, or a human. In some embodiments, the pharmaceutical composition can be administered is in unit dose form.Attorney Docket No.199235-774601

[0013] Also disclosed herein are methods of treating a facioscapulohumeral muscular dystrophy (FSHD) in a subject comprising administering to a subject an effective amount of the recombinant AAV encapsidating the vector described previously, the plasmid encoding the engineered guide RNA described previously, or the DNA sequence described previously, or the pharmaceutical composition described previously, wherein the administering treats the FSHD in the subject. In some embodiments, the subject can be a mouse, a non-human primate, or a human. In some embodiments, the pharmaceutical composition can be administered in unit dose form.

[0014] Also disclosed herein are methods of editing a DUX4-FL RNA transcript in a subject comprising administering to a subject an effective amount of the recombinant AAV encapsidating the vector described previously, the plasmid encoding the engineered guide RNA described previously, or the DNA sequence described previously, or the pharmaceutical composition described previously, wherein after the administering the DUX4-FL RNA transcript is edited in the subject. In some embodiments, the subject can be a mouse, a non- human primate, or a human. In some embodiments, the pharmaceutical composition can be administered in unit dose form. In some embodiments, the editing of the DUX4-FL RNA transcript comprises editing of a polyA signal sequence. In some embodiments, the editing of polyA signal sequence can comprise editing of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of a total DUX4-FL transcripts in a muscle of the subject.

[0015] Also disclosed herein are kits comprising the recombinant AAV encapsidating the recombinant AAV encapsidating the vector described previously, the plasmid encoding the engineered guide RNA described previously, or the DNA sequence described previously, or the pharmaceutical composition described previously and containers. INCORPORATION BY REFERENCE

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGSAttorney Docket No.199235-774601

[0017] Novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which exemplary principles of the present disclosure are utilized, and the accompanying drawings of which:

[0018] FIG.1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8 / 7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2 / 2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1 / 1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5 / 5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2 / 3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side). Figure discloses SEQ ID NOS 143-144, respectively, in order of appearance.

[0019] FIG.2A – FIG.2C shows guide RNA abundance of different AAV cassettes. FIG.2A shows an exemplary tandem and bidirectional ITR-to-ITR AAV vector with cassette 1 and cassette 2 (SEQ ID NO: 63 and SEQ ID NO: 64). FIG.2B shows the Cassette 2 / Cassette 1 abundance for DUX4 guide RNAs and additional guide RNAs in various FSHD patient-derived muscle cell lines. The Y-axis shows the Cassette 2 / Cassette 1 expression and the X-axis shows the tested vector. FIG. 2C shows knockdown of expression of genes downstream of DUX4 (LEUTX, KHDC1L, and ZSCAN4) by DUX4 bidirectional vectors. The Y-axis shows the relative expression of the downstream gene to RPL30, and the X-axis shows the FSHD patient- derived muscle cell line.

[0020] FIG.3A – FIG.3C show cell-free high-throughput screening (HTS) of DUX4 PAS for exemplary engineered gRNAs. FIG. 3A shows the different DUX4 polyadenylation signal (PAS) positions across which the gRNA micro-footprint designs demonstrated robust editing of DUX4 PAS. FIG.3B shows an ADAR-mediated RNA editing plot of the different DUX4 polyadenylation signal (PAS) positions relative to target labeled as fraction edited. Each column represents a position relative to target, cells are shaded based on fraction edited. FIG. 3C shows five different graphs correlating to DUX4 PAS positions (or sites) selected forAttorney Docket No.199235-774601 further optimization. Y-axis depicts ADAR2 fraction edited, and X-axis depicts ADAR1 fraction edited.

[0021] FIG. 4A – FIG. 4B show gRNAs targeting DUX4 impair canonical cleavage and polyadenylation in luciferase cleavage assay performed on HEK293 cells. FIG. 4A shows polyA-seq captured the canonical PAS and revealed minimal to no editing. Figure discloses SEQ ID NO: 145. FIG.4B shows PAS RNA editing was observed in non-canonical transcripts. Y-axis shows percent editing, and X-axis shows position edited after with No_Guide, Guide_1 (SEQ ID NO: 116), Guide_2 (SEQ ID NO: 117), and Guide_3 (SEQ ID NO: 118) (left to right), and X-axis shows position edited .

[0022] FIG. 5A – FIG. 5B shows an inducible DUX4 in vitro model enabled phenotypic gRNA screening as it relates to DUX4-mediated cellular toxicity. FIG.5A shows a schematic view of the inducible DUX4 model, which was doxycycline induced and involved reverse-tTA (rtTA) variant binding to TRE3G. FIG. 5B shows a graphical view of the results of gRNA screening as it relates to DUX4-mediated cellular toxicity for SEQ ID NO: 37 as compared to engineered gRNAs of SEQ ID NO: 131 – SEQ ID NO: 136, and controls, as measured in luminescence units in the inducible DUX4 model. Y-axis shows relative Caspase Activity (Luminescence Units), and X-axis shows treatment group.

[0023] FIG. 6 shows a graphical view of the results of DUX4-mediated cellular toxicity screening in the iHEK DUX4-FL model after treatment with exemplary engineered gRNAs (SEQ ID NO: 36 – SEQ ID NO: 43) as compared to controls. Y-axis shows relative Caspase Activity (Luminescence Units), and X-axis shows treatment group.

[0024] FIG.7A – FIG.7B shows in vivo activity of engineered gRNAs disclosed herein. FIG. 7A shows transduction efficiency (vg / dg), and gRNA expression after treatment with engineered gRNA (SEQ ID NO: 37) as compared to negative control (vehicle). Top graph: Y- axis shows vg / dg normalized to mTfrc, and X-axis shows treatment group; Bottom graph: Y- axis shows gRNA copies per U1 snRNA, and X-axis shows treatment group. FIG.7B shows effects of the exemplary engineered gRNA on DUX4-activated genes Agtr2 (Top) and Wfdc3 (bottom), as compared to vehicle control, and no treatment (No Tdx). Y-axis shows fold change, and X-axis shows treatment group.

[0025] FIG. 8A – FIG. 8C shows RAB7A 3’UTR editing and RAB7A gRNA expression in an in vivo AAV expression assay in NHP skeletal muscle. FIG. 8A shows RAB7A 3’UTR editing in the vastus lateralis muscle of male and female NHP, respectively. Y-axis shows A>G editing at target site (%), and X-axis shows muscle group. FIG.8B shows a graph comparingAttorney Docket No.199235-774601 gRNA expression and % RAB7A 3’UTR editing in the vastus lateralis muscle of male NHPs. Y-axis shows % RAB7A editing, and X-axis guide expression (U1 snRNA-normalized). FIG. 8C shows a graph comparing gRNA expression and % RAB7A 3’UTR editing in the vastus lateralis muscle of female NHPs. Y-axis shows % RAB7A editing, and X-axis guide expression (U1 snRNA-normalized).

[0026] FIG.9A – FIG.9B shows exemplary ITR-to-ITR constructs’ therapeutic potential as evidenced by the % knockdown of DUX4-activated genes, Zscan4 and Khcd1l, in an in vitro AAV expression assay in FSHD muscle cells. FIG.9A shows percent knockdown of Zscan4 in cells treated with SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, or nonTC (non- targeting gRNA control) at a concentration of 5, 5e1, 5e2, 5e3, 5e4, and 5e5 vg / cell. Y-axis shows % knockdown, and X-axis shows dose tested (vg / cell). FIG. 9B shows percent knockdown of Khcd1l in cells treated with SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, or nonTC at a concentration of 5, 5e1, 5e2, 5e3, 5e4, and 5e5 vg / cell. Y-axis shows % knockdown, and X-axis shows dose tested (vg / cell).

[0027] FIG.10 shows relative MYH1 gene expression relative to the untransduced condition (“% untransduced”) in an in vitro AAV expression assay in FSHD cells treated with SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, or nonTC (non-targeting gRNA control) at a concentration of 5, 5e1, 5e2, 5e3, 5e4, and 5e5 vg / cell. Y-axis shows % untransduced, and X- axis shows treatment multiplicity of infection (MOI) (vg / cell).

[0028] FIG.11A – FIG.11B show in vitro activity of engineered gRNAs disclosed herein in FSHD cells. FIG.11A shows gRNA expression in cells treated with SEQ ID NO: 112 or SEQ ID NO: 74 at a concentration of 5, 5e1, 5e2, 5e3, 5e4, and 5e5 vg / cell. Y-axis shows gRNA copies per U1 snRNA, and X-axis shows dose tested (vg / cell). FIG. 11B shows percent knockdown of Zscan4 in cells treated with SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, nonTC at a concentration of 5, 5e1, 5e2, 5e3, 5e4, and 5e5 vg / cell. Y-axis shows % knockdown, and X-axis shows treatment MOI (vg / cell).

[0029] FIG. 12A – FIG.12B shows exemplary ITR-to-ITR constructs’ therapeutic potential as evidenced by the % knockdown of DUX4-activated genes, Wfdc3 and Agtr2, in an in vivo AAV expression assay in ACTA1-MCM;FLExDUX4 female mice. FIG.12A shows percent knockdown of Wfdc3 in mice treated with SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 112, or “nonTC”. Y-axis shows % knockdown as fold change relative to vehicle control, and X-axis shows treatment group. FIG. 12B shows percent knockdown of Agtr2 in mice treated withAttorney Docket No.199235-774601 SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 112, or “nonTC”. Y-axis shows % knockdown as fold change relative to vehicle control, and X-axis shows treatment group.

[0030] FIG.13A – FIG.13B shows in vivo activity of engineered gRNAs disclosed herein in ACTA1-MCM;FLExDUX4 mice. FIG. 13A shows gRNA expression in mice treated with SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 112, or nonTC. Y-axis shows gRNA copies per U1 snRNA, and X-axis shows treatment group. FIG. 12B shows transduction efficiency (vg / dg) in mice treated with SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 112, or nonTC. Y-axis shows vg / dg, and X-axis shows treatment group.

[0031] FIG. 14A – FIG. 14B shows exemplary ITR-to-ITR constructs (various macrofootprints) therapeutic potential as evidenced by the % knockdown of DUX4-activated genes, KHDCL1 and ZSCAN4 in an in vitro AAV expression assay in FSHD cells. FIG.14A shows percent knockdown of KHDCL1 in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding non-targeting gRNA control (“NTGC-01”, SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 101 – SEQ ID NO: 106 at a concentration of 5e2, 5e3,and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to NTGC-01 and X-axis shows treatment group. FIG.14B shows percent knockdown of ZSCAN4 in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 101 – SEQ ID NO: 106 at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to Scramble, and X-axis shows treatment group.

[0032] FIG.15A – FIG.15C shows exemplary ITR-to-ITR constructs (various vector designs) therapeutic potential as evidenced by the % knockdown of DUX4-activated genes, ZSCAN4, KHDCL1 and LEUTX in an in vitro AAV expression assay in FSHD cells. FIG. 15A shows percent knockdown of ZSCAN4 in FSHD cells treated with AAV vectors encapsidating an ITR- to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109, independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to NTGC-01, and X-axis shows treatment group. FIG.15B shows percent knockdown of KHDCL1 in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109 as compared to untransduced , independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to Scramble, andAttorney Docket No.199235-774601 X-axis shows treatment group. FIG.15C shows percent knockdown of LEUTX in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109 as compared to untransduced, independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to NTGC-01 and X-axis shows treatment group.

[0033] FIG. 16 shows a graphical view of the impact of engineered gRNA macro-footprints on gRNA expression in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109 in vitro in FSHD cells.

[0034] FIG. 17A – FIG. 17F shows exemplary ITR-to-ITR constructs’ therapeutic potential as evidenced by the % knockdown of DUX4-activated genes, ZSCAN4, KHDCL1 and LEUTX in an in vitro AAV expression assay in FSHD cells. FIG.17A shows percent knockdown of ZSCAN4 in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109, independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to untransduced, and X-axis shows treatment group. FIG.17B shows percent knockdown of KHDCL1 in FSHD cells treated with AAV vectors encapsidating an ITR-to- ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109, independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to untransduced, and X-axis shows treatment group. FIG. 17C shows percent knockdown of LEUTX in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct encoding NTGC-01 (SEQ ID NO: 113), SEQ ID NO: 112, SEQ ID NO: 74, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 108, SEQ ID NO: 109, independent of vector design, at a concentration of 5e2, 5e3, and 5e4 vg / cell. Y-axis shows % knockdown as fold change relative to untransduced, and X-axis shows treatment group. FIG.17D shows percent knockdown of KHDCL1 in FSHD cells treated with AAV vectors encapsidating ITR-to-ITR constructs (SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110) at a concentration of 5e2, 5e3, and 5e4 vg / cell, as compared to untreated control,Attorney Docket No.199235-774601 non-targeting controls (NTGC-01, NTGC-02, NTGC-03), and non-expressing (NE) control. Y-axis shows relative expression to RPL30 and untransduced 12Abic, and X-axis shows treatment group. FIG.17E shows percent knockdown of ZSCAN4 in FSHD cells treated with AAV vectors encapsidating ITR-to-ITR constructs (SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110) at a concentration of 5e2, 5e3, and 5e4 vg / cell, as compared to untreated control, NTGC-01, NTGC-02, NTGC-03, and NE control. Y-axis shows relative expression to RPL30 and untransduced 12Abic, and X-axis shows treatment group. FIG. 17F shows percent knockdown of LEUTX in FSHD cells treated with AAV vectors encapsidating ITR-to-ITR constructs (SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110) at a concentration of 5e2, 5e3, and 5e4 vg / cell, as compared to untreated control, NTGC-01, NTGC-02, NTGC-03, and NE control. Y-axis shows relative expression to RPL30 and untransduced 12Abic, and X-axis shows treatment group.

[0035] FIG.18A – FIG.18C shows exemplary ITR-to-ITR constructs’ therapeutic potential as evidenced by the expression level of DUX4-activated gene, MYH1 in an in vitro AAV expression assay in FSHD cells. FIG. 18A shows expression level of MYH1 in FSHD cells treated with AAV vectors encapsidating an ITR-to-ITR construct of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 112, as compared to NTGC 01-03 (SEQ ID NO: 113 – SEQ ID NO: 115) and untreated control (un). Y-axis shows relative expression to RPL30, and X-axis shows treatment group. FIG.18B shows gRNA expression in 12Abic cells treated with AAV vectors encapsidating an ITR-to-ITR construct of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 112 as compared to NTGC 01-03 (SEQ ID NO: 113 – SEQ ID NO: 115) and untreated control (un). Y-axis shows guide expression (U1snRNA- normalized), and X-axis shows treatment group. FIG.18C shows gRNA expression in 12Ubic cells treated with AAV vectors encapsidating an ITR-to-ITR construct of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110 as compared to NTGC 01-03 (SEQ ID NO: 113 – SEQ ID NO: 115) and untreated control (un). Y-axis shows guide expression (U1snRNA-normalized), and X-axis shows treatment group.Attorney Docket No.199235-774601 DETAILED DESCRIPTION RNA Editing

[0036] RNA editing refers to a process by which RNA is enzymatically modified post synthesis at specific nucleosides. RNA editing can comprise any one of an insertion, deletion, or substitution of a nucleotide(s). Examples of RNA editing include chemical modifications, such as pseudouridylation (the isomerization of uridine residues) and deamination (removal of an amine group from: cytidine to give rise to uridine, or C-to-U editing; or from adenosine to inosine, or A-to-I editing). RNA editing can be used to correct mutations (e.g., correction of a missense mutation) in order to restore protein expression and to introduce mutations or edit coding regions of RNA to affect protein knockdown, mRNA knockdown, or both.

[0037] Described herein are engineered guide RNAs that facilitate RNA editing by an RNA editing entity (e.g., an Adenosine Deaminase Acting on RNA (ADAR)) or biologically active fragments thereof. For example, engineered guide RNAs of the present disclosure can facilitate editing of a polyA signal sequence of a target DUX4 mRNA (for example, an engineered guide RNA of any one SEQ ID NO: 52 – SEQ ID NO: 59). In some instances, ADARs can be enzymes that catalyze the chemical conversion of adenosines to inosines in RNA. Because the properties of inosine mimic those of guanosine (inosine will form two hydrogen bonds with cytosine, for example), inosine can be recognized as guanosine by the translational cellular machinery. “Adenosine-to-inosine (A-to-I) RNA editing”, therefore, effectively changes the primary sequence of RNA targets. In general, ADAR enzymes share a common domain architecture comprising a variable number of amino-terminal dsRNA binding domains (dsRBDs) and a single carboxy-terminal catalytic deaminase domain. Human ADARs possess two or three dsRBDs. Evidence suggests that ADARs can form homodimer as well as heterodimer with other ADARs when bound to double-stranded RNA, however it can be currently inconclusive if dimerization is needed for editing to occur. The engineered guide RNAs disclosed herein can facilitate RNA editing by any of or any combination of the three human ADAR genes that have been identified (ADAR1, ADAR2, ADAR3, or a combination thereof). ADARs have a typical modular domain organization that includes at least two copies of a dsRNA binding domain (dsRBD; ADAR1with three dsRBDs; ADAR2 and ADAR3 each with two dsRBDs) in their N-terminal region followed by a C-terminal deaminase domain.

[0038] The present disclosure, in some embodiments, provides engineered guide RNAs (e.g., SEQ ID NO: 52 – SEQ ID NO: 59) that facilitate edits at particular regions in a target RNA (e.g., mRNA or pre-mRNA). For example, the engineered guide RNAs disclosed herein canAttorney Docket No.199235-774601 target a coding sequence of an RNA. The engineered guide RNAs disclosed herein can target a non-coding sequence of an RNA, for example, a polyadenylation (polyA) signal sequence in the 3’UTR. The engineered guides herein are encoded in ITR-to-ITR regions such as any one of SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 63 – SEQ ID NO: 64, SEQ ID NO: 74 – SEQ ID NO: 80, or SEQ ID NO: 101 – SEQ ID NO: 111. The engineered guides herein are encoded in DNA regions such as any one of SEQ ID NO: 69 – SEQ ID NO: 72, SEQ ID NO: 81 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 120 – SEQ ID NO: 130. The ITR-to-ITR regions can be packaged in a viral vector such as an AAV. In some cases, the ITR-to-ITR region can be packaged in a recombinant AAV.

[0039] The present disclosure provides for engineered guide RNAs that knockdown expression of DUX4-FL mRNA, and hence DUX4 activity. In some cases, an engineered guide RNA described herein can hybridize to a DUX4 target sequence (e.g., DUX4-FL mRNA). In some instances, the DUX4 target sequence can comprise a polyA signal sequence. Alternatively or in addition, the engineered guide RNAs of the present disclosure facilitate knockdown of DUX4-activated genes. Expression knockdown (e.g., knockdown of DUX4-FL mRNA, DUX4 activated genes, or a combination thereof) may result from RNA editing of DUX4-FL mRNA (e.g., editing of the polyA signal sequence) facilitated by an engineered guide RNA, destabilization of the DUX4-FL mRNA (e.g., by masking the polyA signal sequence) facilitated by an engineered guide RNA, or a combination thereof. Facioscapulohumeral muscular dystrophy (FSHD) is a rare neuromuscular disease characterized by progressive skeletal muscle weakness and wasting with significant heterogeneity in phenotypic severity and age of onset. FSHD affects mostly the face (facio), shoulder girdle (scapula), and upper arm (humeral) regions of the body. As the disease progresses, muscles of the upper arms, the legs, and the postural muscles in the back lose mass and strength. Patients often first present with weakness of the face and periscapular muscles, eventually resulting in the inability to raise their arms above shoulder height, make facial expressions, or even close their eyes. In about 20% of the patients with FSHD, paraspinal muscle weakness is debilitating enough to result in patients becoming wheelchair-bound. FSHD is one of the most prevalent adult muscular dystrophies caused by an epigenetic derepression of the subtelomeric D4Z4 microsatellite array on chromosome 4q. This epigenetic derepression leads to hypomethylation in the distal-most D4Z4 unit and misexpression of the DUX4 gene in skeletal muscle. There are two subtypes of FSHD – FSHD1 and FSHD2. FSHD1 accounts for 95% of FSHD cases and is associated with the pathogenic contraction of D4Z4 microsatellite repeats, while FSHD2 accounts for 5% ofAttorney Docket No.199235-774601 the FSHD cases and is contraction-independent but associated with mutations in the chromatin regulator gene SMCHD1. The mutations for both FSHD1 and FSHD2 result in derepression of D4Z4 array and DUX4 mRNA misexpression. Said DUX4 mutations are autosomal dominant in 2 / 3 of FSHD1 patients and are prevalent in 1:8,000-12,000 Americans (~16,000-38,000 patients in the US). DUX4 (double homeobox 4) is a germline transcription factor and its misexpression in muscle activates the expression of a broad set of genes (DUX4-activated genes), many involved in stem and germ cell biology. Some known DUX4-activated genes include MBD3L2, TRIM43, PRAMEF12, ZSCAN, and LEUTX. In some cases, treatment of a DUX4-associated disease such as FSHD can be at least in part due to the knockdown of DUX4. In some cases, treatment of a DUX4-associated disease such as FSHD can be at least in part due to the knockdown of protein, mRNA, or both of a gene downstream of DUX4. In some cases, treatment of a DUX4-associated disease such as FSHD can be at least in part due to the knockdown of protein, mRNA, or both of a DUX4-activated gene. Although physical therapy, pain management, and surgery can alleviate some of the disabilities associated with FSHD, these treatments are not curative, and none of them address the underlying cause of the disease pathology. While healthy subjects generate a non-toxic splice form of DUX4 mRNA that lacks the C-term transactivation domain of DUX4 (referred to as DUX4-S for short), affected subjects produce a toxic splice form of DUX4 mRNA (referred to as DUX4-FL for full length) leading to expression of a toxic form of the DUX4 protein in muscle. Although various pharmaceutical and cell-based intervention approaches are being explored to treat FSHD, these generally offer little to no therapeutic benefit based on results from clinical trials. To develop a more targeted form of treatment, approaches that reduce muscle-specific DUX4-FL expression and DUX4- mediated toxicity have become attractive goals of FSHD therapy. Indeed, genetic treatments that target the root cause of the disease (e.g., DUX4) are expected to lead to a more effective or far-reaching therapeutic effect. The exact amount of DUX4 inhibition required for effective therapy is currently unknown, but data from clinically affected and asymptomatic FSHD patients support the idea that any reduction in DUX4-FL mRNA expression will have a therapeutic benefit. In some embodiments, the present disclosure provides compositions of engineered guide RNAs that target DUX4 and facilitate ADAR-mediated RNA editing of DUX4, specifically, DUX4-FL to mediate DUX4-FL knockdown. In some embodiments, the engineered guide RNAs of the present disclosure target a non-coding sequence in DUX4-FL mRNA. The non-coding sequence can be a polyA signal sequence (ATTAAA) in the pLAM region. The engineered guide RNA can facilitate ADAR-mediated RNA editing of one or moreAttorney Docket No.199235-774601 adenosines in the polyA signal sequence of DUX4. RNA editing of this polyA signal sequence reduces mRNA cleavage and polyadenylation, and genetic excision of the DUX4-FL polyA sequence can result in DUX4-FL mRNA knockdown, DUX4-FL protein knockdown, or both. In some embodiments, engineered guide RNAs of the present disclosure can be designed to target more than one adenosine within the polyA signal sequence in DUX4. In some embodiments, engineered guide RNAs of the present disclosure can be multiplexed to target more than one polyA signal sequence. In some instances, the guide RNAs disclosed herein can bind and mask the polyA signal site and result in destabilization of the DUX4-FL mRNA. In some cases, masking the polyA signal site can comprise binding to and inhibiting translation of DUX4-FL mRNA.

[0040] PolyA Signal Sequence. In some embodiments, the engineered guide RNAs of the present disclosure target one or more adenosines in the polyA signal sequence. In some cases, the polyA signal sequence is referred to herein as the polyA signal site. In some embodiments, an engineered guide RNA facilitates ADAR-mediated RNA editing of the one or more adenosines in the polyA signal sequence, thereby resulting in disruption of RNA processing and degradation of the target mRNA and, thereby, reduction in DUX4 activity. In some embodiments, a target can have one or more polyA signal sequences. In these instances, one or more engineered guide RNAs, varying in their respective sequences, of the present disclosure can be multiplexed to target adenosines in the one or more polyA signal sequences. In both cases, the engineered guide RNAs of the present disclosure facilitated ADAR-mediated RNA editing of adenosines to inosines (read as guanosines by cellular machinery) in the polyA signal sequence, resulting in protein knockdown, mRNA knockdown, or both. In some cases, the engineered guide RNAs of the present disclosure can bind and mask the polyA signal sequence to reduce translation of DUX4-FL. In some embodiments, an engineered guide RNA of the present disclosure (e.g. an engineered guide RNA that comprises a polynucleotide sequence of any one of SEQ ID NO: 52 – SEQ ID NO: 59) can target a polyA signal sequence. In some embodiments, a polynucleotide encoding an engineered guide RNA that targets a polyA signal sequence of DUX4-FL comprises a polynucleotide sequence of any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136.

[0041] Target Sequence. In some embodiments, an engineered guide disclosed herein is at least partially complementary to a target DUX4 RNA. In some cases, the target DUX4 RNA comprises the sequence of: ACCUGGAUUAGAGUUACAUCUCCUGGAUGAUUAGUUCAGAGAUAUAUUAAAAAttorney Docket No.199235-774601 UGCCCCCUCCCUGUGGAUCCUAUAGAAGAUUUGCAUCUUUUGUGUGAUGAGU GCAGAG (SEQ ID NO: 61). In some cases, the target DNA sequence encoding the target RNA sequence comprises the sequence of ACCTGGATTAGAGTTACATCTCCTGGATGATTAGTTCAGAGATATATTAAAATGC CCCCTCCCTGTGGATCCTATAGAAGATTTGCATCTTTTGTGTGATGAGTGCAGAG (SEQ ID NO: 62). In some cases, the target DUX4 RNA comprises the sequence of GAGUUACAUCUCCUGGAUGAUUAGUUCAGAGAUAUAUUAAAAUGCCCCCUCCC UGUGGAUCCUAUAGAAGAUUUGCAUCUUUUGUGUGAUGAGUGCAGAG (SEQ ID NO: 90). In some cases, the target DNA sequence encoding the target RNA sequence comprises the sequence of GAGTTACATCTCCTGGATGATTAGTTCAGAGATATATTAAAATGCCCCCTCCCTG TGGATCCTATAGAAGATTTGCATCTTTTGTGTGATGAGTGCAGAG (SEQ ID NO: 88). In some cases, the target DUX4 RNA comprises the sequence of ACCUGGAUUAGAGUUACAUCUCCUGGAUGAUUAGUUCAGAGAUAUAUUAAAA UGCCCCCUCCCUGUGGAUCCUAUAGAAGAUUUGCAUCUUUUGUGUGAU (SEQ ID NO: 91). In some cases, the target DNA sequence encoding the target RNA sequence comprises the sequence of ACCTGGATTAGAGTTACATCTCCTGGATGATTAGTTCAGAGATATATTAAAATGC CCCCTCCCTGTGGATCCTATAGAAGATTTGCATCTTTTGTGTGAT (SEQ ID NO: 89).

[0042] PolyA Signal Sequence Masking. Alternatively, or in addition to RNA editing, the engineered guide RNAs of the present disclosure can facilitate knockdown of DUX4 protein expression, expression of proteins or mRNAs downstream of DUX4, or a combination thereof by hybridizing with DUX4-FL mRNA and masking the polyA signal sequence. Masking the polyA signal sequence with an engineered guide RNA can facilitate knockdown by facilitating destabilization of the target DUX4-FL mRNA. In some embodiments, an engineered guide RNA may facilitate knockdown of DUX4 protein expression, expression of proteins or mRNAs downstream of DUX4, or a combination thereof, through polyA signal sequence masking without facilitating editing of the DUX4-FL mRNA. In some embodiments, an engineered guide RNA may facilitate knockdown of DUX4 protein expression, expression of proteins or mRNAs downstream of DUX4, or a combination thereof, through polyA masking and by facilitating editing of the DUX4-FL mRNA

[0043] In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,Attorney Docket No.199235-774601 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61. In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62. In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90. In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88. In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91. In some cases, the target DUX4 DNA sequence encoding the target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89. In some cases, the target DUX4 RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61. In some cases, the target DUX4 RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atAttorney Docket No.199235-774601 least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90. In some cases, the target DUX4 RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA (e.g., DUX4-FL). In some embodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence of SEQ ID NO: 61. In some embodiments, an engineered guide RNA hybridizes to at least 60, 70, or 80 bases of a target RNA sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 61, SEQ ID NO: 90 or SEQ ID NO: 91 and facilitates mRNA knockdown, protein knockdown, or both. Thus, the provided encoded engineered guide RNA (e.g., the guides encoded in the ITR-to-ITR constructs) knockdown DUX4-FL by targeting its polyA signal sequence.

[0044] In some cases, the target DUX4 RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA (e.g., DUX4-FL). In some embodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence of SEQ ID NO: 90. In some embodiments, an engineered guide RNA hybridizes to at least 60, 70, or 80 bases of a target RNA sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90 and facilitates mRNA knockdown, protein knockdown, or both. Thus, the provided encoded engineered guide RNA (e.g., the guides encoded in the ITR-to-ITR constructs) knockdown DUX4-FL by targeting its polyA signal sequence.

[0045] In some cases, the target DUX4 RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity toAttorney Docket No.199235-774601 SEQ ID NO: 91. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA (e.g., DUX4-FL). In some embodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence of SEQ ID NO: 91. In some embodiments, an engineered guide RNA hybridizes to at least 60, 70, or 80 bases of a target RNA sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91 and facilitates mRNA knockdown, protein knockdown, or both. Thus, the provided encoded engineered guide RNA (e.g., the guides encoded in the ITR-to-ITR constructs) knockdown DUX4-FL by targeting its polyA signal sequence.

[0046] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0047] Throughout this application, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0048] As used herein, the term “about” a number can refer to that number plus or minus 10% of that number.

[0049] As disclosed herein, a base paired (bp) region refers to a region of the guide-target RNA scaffold in which bases in the guide RNA (e.g., the bases in the targeting sequence of the guide RNA) are paired with opposing bases in the target polynucleotide. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to the other end of the guide-target RNA scaffold. Base paired regions can extend between two structural features. Base paired regions can extend from one end or proximal to one end of theAttorney Docket No.199235-774601 guide-target RNA scaffold to or proximal to a structural feature. Base paired regions can extend from a structural feature to the other end of the guide-target RNA scaffold. In some embodiments, a base paired region has from 1 to 50, 1 to 75, 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 225, 1 to 250, 1 to 275, 1 to 300, 50 to 75, 50 to 100, 50 to 125, 50 to 150, 50 to 175, 50 to 200, 50 to 225, 50 to 250, 50 to 275, 50 to 300, 60 to 75, 60 to 100, 60 to 125, 60 to 150, 60 to 175, 60 to 200, 60 to 225, 60 to 250, 60 to 275, 60 to 300, 70 to 100, 70 to 125, 70 to 150, 70 to 175, 70 to 200, 70 to 225, 70 to 250, 70 to 275, 70 to 300, 80 to 100, 80 to 125, 80 to 150, 80 to 175, 80 to 200, 80 to 225, 80 to 250, 80 to 275, 80 to 300, 90 to 125, 90 to 150, 90 to 175, 90 to 200, 90 to 225, 90 to 250, 90 to 275, 90 to 300, 100 to 125, 100 to 150, 100 to 175, 100 to 200, 100 to 225, 100 to 250, 100 to 275, 100 to 300, 150 to 200, 150 to 225, 150 to 250, 150 to 275, or 150 to 300 base pairs. In some embodiments, a base paired region has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 base pairs.

[0050] As disclosed herein, a “bulge” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a singleAttorney Docket No.199235-774601 participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair – a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a “mismatch.” Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an “internal loop.” A “symmetrical bulge” refers to a bulge where the same number of nucleotides is present on each side of the bulge. An “asymmetrical bulge” refers to a bulge where a different number of nucleotides are present on each side of the bulge.

[0051] The term “complementary” or “complementarity” refers to the ability of a nucleic acid to form one or more bonds with a corresponding nucleic acid sequence by, for example, hydrogen bonding (e.g., traditional Watson-Crick), covalent bonding, or other similar methods. In Watson-Crick base pairing, a double hydrogen bond forms between nucleobases T and A, whereas a triple hydrogen bond forms between nucleobases C and G. For example, the sequence A-G-T can be complementary to the sequence T-C-A. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” can mean that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein can refer to a degree of complementarity that can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.97%, 98%, 99%, or 100% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions). Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” or “not specific” can refer to a nucleic acid sequence that contains a series of residues that may not be designed to be complementary to or can be only partially complementary to any other nucleic acid sequence.

[0052] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” can be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount ofAttorney Docket No.199235-774601 something present in addition to determining whether it is present or absent depending on the context.

[0053] The term “encode,” as used herein, refers to an ability of a polynucleotide to provide information or instructions in a sequence sufficient to produce a corresponding gene expression product. In a non-limiting example, mRNA can encode a polypeptide during translation, whereas DNA can encode an mRNA molecule during transcription.

[0054] As used herein, the term “engineered guide RNA” can be used interchangeably with “guide RNA” and refers to a designed polynucleotide that is at least partially complementary to a target RNA. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA. Modification of the target RNA includes alteration of RNA splicing, reduction or enhancement of protein translation, target RNA knockdown, target RNA degradation, and / or ADAR mediated RNA editing of the target RNA. In some cases, guide RNAs facilitate ADAR-mediated RNA editing for the purpose of target mRNA knockdown, downstream protein translation reduction or inhibition, downstream protein translation enhancement, correction of mutations (including correction of any G to A mutation, such as missense or nonsense mutations), introduction of mutations (e.g., introduction of an A to I (read as a G by cellular machinery) substitution), or alter the function of any adenosine containing a regulatory motif (e.g., polyadenylation signal, miRNA binding site, etc.). In some cases, a guide RNA can affect a functional outcome (e.g., target RNA modulation, downstream protein translation) via a combination of mechanisms, for example, ADAR-mediated RNA editing and binding and / or degrading target RNA. In some cases, a guide RNA can facilitate introduction of mutations at sites targeted by enzymes in order to modify the affinity of such enzymes for targeting and cleaving such sites. The guide RNAs of this disclosure can contain one or more structural features. A structural feature can be formed from latent structure in latent (unbound) guide RNA upon hybridization of the engineered latent guide RNA to a target RNA. Latent structure refers to a structural feature that forms or substantially forms only upon hybridization of a guide RNA to a target RNA. For example, upon hybridization of the guide RNA to the target RNA, the latent structural feature is formed in the resulting double stranded RNA (also referred herein as guide-target RNA scaffold). In such cases, a structural feature can include, but is not limited to, a mismatch, a wobble base pair, a symmetric internal loop, an asymmetric internal loop, a symmetric bulge, or an asymmetric bulge. In other instances, a structural feature can be a pre-formed structure (e.g., a GluR2 recruitment hairpin, or a hairpin from U7 snRNA).Attorney Docket No.199235-774601

[0055] An “engineered latent guide RNA” refers to an engineered guide RNA that comprises a portion of sequence that, upon hybridization or only upon hybridization to a target RNA, substantially forms at least a portion of a structural feature, other than a single A / C mismatch feature at the target adenosine to be edited.

[0056] As disclosed herein, a structured motif comprises two or more structural features in a guide-target RNA scaffold.

[0057] As used herein, the term “facilitates RNA editing” by an engineered guide RNA refers to the ability of the engineered guide RNA when associated with an RNA editing entity and a target RNA to provide a targeted edit of the target RNA by the RNA edited entity. In some instances, the engineered guide RNA can directly recruit or position / orient the RNA editing entity to the proper location for editing of the target RNA. In other instances, the engineered guide RNA when hybridized to the target RNA forms a guide-target RNA scaffold with one or more structural features as described herein, where the guide-target RNA scaffold with structural features recruits or positions / orients the RNA editing entity to the proper location for editing of the target RNA.

[0058] A “guide-target RNA scaffold,” as disclosed herein, is the resulting double stranded RNA formed upon hybridization of a guide RNA, with latent structure, to a target RNA. A guide-target RNA scaffold has one or more structural features formed within the double stranded RNA duplex upon hybridization. For example, the guide-target RNA scaffold can have one or more structural features selected from a bulge, mismatch, internal loop, hairpin, or wobble base pair.

[0059] The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0060] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50%Attorney Docket No.199235-774601 to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows:

[0061] [(Number of Identical Positions) / (Total Number of Positions in the Test Sequence)] × 100%.

[0062] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match / mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows:Attorney Docket No.199235-774601

[0063] Percent Sequence Identity = (“Percent Identity” output value) × (“Query Coverage” output value).

[0064] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions) / (total number of nucleotides in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 1: AAAAAGGGGG (SEQ ID NO: 1) (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (SEQ ID NO: 2) (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5) / (10)] ×100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 3) (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (SEQ ID NO: 4) (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10) / (20)] ×100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (SEQ ID NO: 4) (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 3) (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10) / (10)] ×100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6.

[0065] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions) / (total number of amino acids in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (SEQ ID NO: 5) (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (SEQ ID NO: 6) (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5) / (10)] ×100% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 7) (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (SEQ ID NO: 5) (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10) / (20)] ×100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11: FFFFFYYYYY (SEQ ID NO: 5) (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 7)Attorney Docket No.199235-774601 (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10) / (10)] ×100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12. As disclosed herein, an “internal loop” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a “bulge” or a “mismatch,” depending on the size of the structural feature. A “symmetrical internal loop” is formed when the same number of nucleotides is present on each side of the internal loop. An “asymmetrical internal loop” is formed when a different number of nucleotides is present on each side of the internal loop.

[0066] Latent structure refers to a structural feature that substantially forms only upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed, and the latent structure provided in the guide RNA is, thus, unmasked. The formation and structure of a latent structural feature upon binding to the target RNA depends on the guide RNA sequence. For example, formation and structure of the latent structural feature may depend on a pattern of complementary and mismatched residues in the guide RNA sequence relative to the target RNA. The guide RNA sequence may be engineered to have a latent structural feature that forms upon binding to the target RNA. “Messenger RNA” or “mRNA” are RNA molecules comprising a sequence that encodes a polypeptide or protein. In general, RNA can be transcribed from DNA. In some cases, precursor mRNA containing non-protein coding regions in the sequence can be transcribed from DNA and then processed to remove all or a portion of the non-coding regions (introns) to produce mature mRNA. As used herein, the term “pre-mRNA” can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions.

[0067] As disclosed herein, a “mismatch” refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNAAttorney Docket No.199235-774601 scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a “bulge” or an “internal loop,” depending on the size of the structural feature.

[0068] As used herein, the term “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5’ to the 3’ end. The term “RNA” is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), lncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA), RNAi (inhibitory RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA). The term DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. A sequence of a polynucleotide may be provided interchangeably as an RNA sequence (containing U) or a DNA sequence (containing T). A sequence provided as an RNA sequence is intended to also cover the corresponding DNA sequence and the reverse complement RNA sequence or DNA sequence. A sequence provided as a DNA sequence is intended to also cover the corresponding RNA sequence and the reverse complement RNA sequence or DNA sequence.

[0069] The term “protein”, “peptide” and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence. As used herein the term “amino acid” can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term “fusion protein” can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function. In this regard, the term “linker” can refer to a protein fragment that can be used to link these domains together – optionally to preserve the conformation of the fused protein domains, prevent unfavorable interactions between the fused protein domains which can compromise their respective functions, or both.Attorney Docket No.199235-774601

[0070] The term “structured motif” refers to a combination of two or more structural features in a guide-target RNA scaffold.

[0071] The terms “subject,” “individual,” or “patient” can be used interchangeably herein. A “subject” refers to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0072] As used herein, the term “targeting sequence” can be used interchangeable with “targeting domain” or “targeting region” and refers to a polynucleotide sequence within an engineered guide RNA sequence that is at least partially complementary to a target polynucleotide. The target polynucleotide (e.g., a target RNA or a target DNA) may be a region of a polynucleotide of interest, such as a gene or a messenger RNA. As used herein, a “complementary” sequence refers to a sequence that is a reverse complement relative to a second sequence. A targeting sequence of an engineered guide RNA allows the engineered guide RNA to hybridize to a target polynucleotide (e.g., a target RNA) through base pairing, such as Watson Crick base pairing. A targeting sequence can be located at either the N-terminus or C-terminus of the engineered guide RNA, or both, or the targeting sequence can be within the engineered guide RNA. The targeting sequence can be of any length sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence is at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or up to about 200 nucleotides in length. In an embodiment, an engineered polynucleotide comprises a targeting sequence that is about 25 to 200, 50 to 150, 75 to 100, 80 to 110, 90 to 120, 95 to 115, 60 to 200, 60 to 180, 60 to 160, 60 to 140, 70 to 200, 70 to 180, 70 to 160, 70Attorney Docket No.199235-774601 to 140, 80 to 200, 80 to 190, 80 to 170, 80 to 160, 80 to 150, 80 to 140, 80 to 130, 80 to 120, 90 to 200, 90 to 190, 90 to 180, 90 to 170, 90 to 160, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 100 to 200, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 110 to 200, 110 to 190, 110 to 180, 110 to 170, 110 to 160, 110 to 150, 110 to 140, 110 to 120, 120 to 200, 120 to 190, 120 to 180, 120 to 170, 120 to 160, 120 to 150, 120 to 140, 130 to 200, 130 to 190, 130 to 180, 130 to 170, 130 to 160, 130 to 150, 140 to 200, 140 to 190, 140 to 180, 140 to 170, 140 to 160, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 160 to 200, 160 to 190 or 160 to 180 nucleotides in length.

[0073] A targeting sequence comprises at least partial sequence complementarity to a target polynucleotide. The targeting sequence may have a degree of sequence complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to the target polynucleotide. In some cases, the targeting sequence comprises less than 100% complementarity to the target polynucleotide sequence. For example, the targeting sequence may have a single base mismatch relative to the target polynucleotide when bound to the target polynucleotide. In other cases, the targeting sequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 30, 40 or up to about 50 base mismatches relative to the target polynucleotide when bound to the target polynucleotide. In some aspects, nucleotide mismatches can be associated with structural features provided herein. In some aspects, a targeting sequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or up to about 15 nucleotides that differ in complementarity from a wildtype polynucleotide of a subject target polynucleotide.

[0074] A targeting sequence comprises nucleotide residues having complementarity to a target polynucleotide. The targeting sequence may have a number of residues with complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. The complementary residues may be contiguous or non-contiguous. In some cases, the targeting sequence comprises at least 50 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 250 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases,Attorney Docket No.199235-774601 the targeting sequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises more than 50 nucleotides total and has at least 50 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 250 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases, the at least 50 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 150 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 200 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 250 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 300 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one orAttorney Docket No.199235-774601 more bulges, or one or more loops, or any combination thereof. For example, a targeting sequence comprises a total of 54 nucleotides wherein, sequentially, 25 nucleotides are complementary to the target polynucleotide, 4 nucleotides form a bulge, and 25 nucleotides are complementary to the target polynucleotide. As another example, a targeting sequence comprises a total of 118 nucleotides wherein, sequentially, 25 nucleotides are complementary to the target polynucleotide, 4 nucleotides form a bulge, 25 nucleotides are complementary to the target polynucleotide, 14 nucleotides form a loop, and 50 nucleotides are complementary to the target polynucleotide.

[0075] The term “in vivo” refers to an event that takes place in a subject’s body.

[0076] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay may not be performed on a subject. Rather, it can be performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample can be an “in vitro” assay.

[0077] The term “in vitro” refers to an event that takes place within in a container for holding laboratory reagent such that it can be separated from the biological source from which the material can be obtained. In vitro assays can encompass cell-based assays in which living or dead cells can be employed. In vitro assays can also encompass a cell-free assay in which no intact cells can be employed.

[0078] The term “wobble base pair” refers to two bases that weakly pair. For example, a wobble base pair can refer to a G paired with a U.

[0079] The term “substantially forms” as described herein, when referring to a particular secondary structure, refers to formation of at least 80% of the structure under physiological conditions (e.g., physiological pH, physiological temperature, physiological salt concentration, etc.).

[0080] As used herein, the terms “treatment” or “treating” can be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can refer to eradication or amelioration of one or more symptoms of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement can be observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease orAttorney Docket No.199235-774601 condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made. Engineered Guide RNAs

[0081] Disclosed herein are engineered guide RNAs (e.g. SEQ ID NO: 52 – SEQ ID NO: 59) and engineered polynucleotides encoding the same (e.g. SEQ ID NO: 36 – SEQ ID NO: 43) for site-specific, selective editing of a target RNA (for example, a polyA signal sequence of DUX4) via an RNA editing entity or a biologically active fragment thereof. In some embodiments, the engineered guide RNA of the present disclosure can be encoded by a polynucleotide comprising a polynucleotide sequence of any one of SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 63 – SEQ ID NO: 64, SEQ ID NO: 69 – SEQ ID NO: 72, SEQ ID NO: 74 – SEQ ID NO: 80, SEQ ID NO: 81 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, SEQ ID NO: 101 – SEQ ID NO: 111, or SEQ ID NO: 120 – SEQ ID NO: 130. In some embodiments, the engineered guide RNAs of the present disclosure target one or more adenosines in the RNA sequence of SEQ ID NO: 61 or a sequence that is at least 80% identical to SEQ ID NO: 61. In some embodiments, the engineered guide RNAs of the present disclosure target one or more adenosines in the RNA sequence of SEQ ID NO: 90 or a sequence that is at least 80% identical to SEQ ID NO: 90. In some embodiments, the engineered guide RNAs of the present disclosure target one or more adenosines in the RNA sequence of SEQ ID NO: 91 or a sequence that is at least 80% identical to SEQ ID NO: 91.

[0082] In some embodiments, engineered guide RNAs of the present disclosure that target DUX4 (e.g., DUX4-FL) comprise a micro-footprint sequence and / or a macro-footprint sequence that each comprise latent structures, such that when the engineered guide RNA is hybridized to the target RNA, the latent structures manifest. A latent structure, when manifested, produces at least one structural feature selected from the group consisting of: a bulge, an internal loop, a mismatch, a hairpin, and any combination thereof. In some embodiments, the engineered guide RNA of the disclosure, upon hybridization of the engineered guide RNA and the sequence of the target RNA form a guide-target RNA scaffold, comprising (i) a region that comprises at least one structural feature; and (ii) a macro-footprint, such as a first internal loop (also referred to as a “left bell” or “LB”) and a second internal loop (also referred to as a “right bell” or “RB”) that flank opposing ends of the region of the guide-Attorney Docket No.199235-774601 target RNA scaffold, where the engineered guide RNA facilitates an increase in the amount of the targeted edit of the adenosine of the target RNA via the adenosine deaminase enzyme RNA editing entity, relative to an otherwise comparable engineered guide RNA lacking the first internal loop and the second internal loop. As described herein, a first internal loop and a second internal loop can be described with respect to their position relative to an A / C mismatch in the target RNA scaffold, where the A in the A / C mismatch is the target adenosine of the DUX4 target RNA.

[0083] As described herein, a “micro-footprint” sequence refers to a sequence with latent structures that, when manifested, facilitate editing of the adenosine of a target RNA via an adenosine deaminase enzyme. A micro-footprint can serve to guide an RNA editing enzyme and direct its activity towards the target adenosine to be edited. In some embodiments, included within the micro-footprint sequence is a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes the adenosine to be edited by the adenosine deaminase and does not base pair with the adenosine to be edited. This nucleotide is referred to herein as the “mismatched position” or “mismatch” and can be a cytosine. Micro- footprint sequences as described herein have upon hybridization of the engineered guide RNA and target RNA, at least one structural feature selected from the group consisting of: a bulge, an internal loop, a mismatch, a hairpin, and any combination thereof. Engineered guide RNAs with superior micro-footprint sequences can be selected based on their ability to facilitate editing of a specific target RNA (such as DUX4 mRNA).

[0084] In some embodiments, guide RNAs of the present disclosure (e.g., SEQ ID NO: 52 – SEQ ID NO: 59) can further comprise a macro-footprint. In some embodiments, the macro- footprint comprises a barbell macro-footprint. A macro-footprint can serve to guide an RNA editing entity (e.g., ADAR) and direct its activity towards a micro-footprint. A “barbell” as described herein refers to a pair of internal loop latent structures that manifest upon hybridization of the guide RNA to the target RNA. In some embodiments, each internal loop is positioned towards the 5′ end or the 3′ end of the guide-target RNA scaffold formed upon hybridization of the guide RNA and the target RNA. In some embodiments, each internal loop flanks opposing sides of the micro-footprint sequence. Insertion of a barbell macro-footprint sequence flanking opposing sides of the micro-footprint sequence, upon hybridization of the guide RNA to the DUX4 target RNA, results in formation of barbell internal loops on opposing sides of the micro-footprint, which in turn comprises at least one structural feature that facilitates editing of the DUX4 target RNA.Attorney Docket No.199235-774601

[0085] Provided herein are engineered guide RNAs (such as latent guide RNA that comprise a micro-footprint sequence and / or a macro-footprint sequence) and polynucleotides encoding the same; as well as compositions comprising said engineered guide RNAs or said polynucleotides. As used herein, the term “engineered” in reference to a guide RNA or polynucleotide encoding the same refers to a non-naturally occurring guide RNA or polynucleotide encoding the same. For example, the present disclosure provides for engineered polynucleotides encoding for engineered guide RNAs. In some embodiments, the engineered guide comprises RNA. In some embodiments, the engineered guide comprises DNA. In some examples, the engineered guide comprises modified RNA bases or unmodified RNA bases. In some embodiments, the engineered guide comprises modified DNA bases or unmodified DNA bases. In some examples, the engineered guide comprises both DNA and RNA bases.

[0086] An engineered guide RNA as described herein comprises a targeting domain with complementarity to a target RNA described herein. As such, a guide RNA can be engineered to site-specifically / selectively target and hybridize to a particular target RNA, thus facilitating editing of specific nucleotide in the target RNA via an RNA editing entity or a biologically active fragment thereof. The targeting domain can include a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or biologically active fragment thereof and does not base pair, or does not fully base pair, with the base to be edited. This mismatch can help to localize editing of the RNA editing entity to the desired base of the target RNA. However, in some instances there can be some, and in some cases significant, off target editing in addition to the desired edit.

[0087] Hybridization of the target RNA and the targeting domain of the guide RNA produces specific secondary structures in the guide-target RNA scaffold that manifest upon hybridization, which are referred to herein as “latent structures.” Latent structures when manifested become structural features described herein, including mismatches, bulges, internal loops, and hairpins. Without wishing to be bound by theory, the presence of structural features described herein that are produced upon hybridization of the guide RNA with the target RNA configure the guide RNA to facilitate a specific, or selective, targeted edit of the target RNA via the RNA editing entity or biologically active fragment thereof. Further, the structural features in combination with the mismatch described above generally facilitate an increased amount of editing of a target adenosine, fewer off target edits, or both, as compared to a construct comprising the mismatch alone or a construct having perfect complementarity to aAttorney Docket No.199235-774601 target RNA. Accordingly, rational design of latent structures in engineered guide RNAs of the present disclosure to produce specific structural features in a guide-target RNA scaffold can be a powerful tool to promote editing of the target RNA with high specificity, selectivity, and robust activity.

[0088] Provided herein are engineered guides and polynucleotides encoding the same; as well as compositions comprising said engineered guide RNAs or said polynucleotides. As used herein, the term “engineered” in reference to a guide RNA or polynucleotide encoding the same refers to a non-naturally occurring guide RNA or polynucleotide encoding the same. For example, the present disclosure provides for engineered polynucleotides encoding engineered guide RNAs. In some embodiments, the engineered guide comprises RNA. In some embodiments, the engineered guide comprises DNA. In some examples, the engineered guide comprises modified RNA bases or unmodified RNA bases. In some embodiments, the engineered guide comprises modified DNA bases or unmodified DNA bases. In some examples, the engineered guide comprises both DNA and RNA bases.

[0089] In some examples, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target RNA molecule, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA molecule, wherein the guide-target RNA scaffold comprises at least one structural feature, and wherein the guide- target RNA scaffold recruits an RNA editing entity and facilitates a chemical modification of a base of a nucleotide in the target RNA molecule by the RNA editing entity.

[0090] In some examples, a target RNA of an engineered guide RNA of the present disclosure can be a pre-mRNA or mRNA. In some embodiments, the engineered guide RNA of the present disclosure hybridizes to a sequence of the target RNA. In some embodiments, part of the engineered guide RNA (e.g., a targeting domain) hybridizes to the sequence of the target RNA. The part of the engineered guide RNA that hybridizes to the target RNA is of sufficient complementary to the sequence of the target RNA for hybridization to occur. A. Targeting Domain

[0091] Engineered guide RNAs disclosed herein can be engineered in any way suitable for RNA editing. In some examples, an engineered guide RNA generally comprises at least a targeting sequence that allows it to hybridize to a region of a target RNA molecule (e.g. a polyA signal sequence of DUX4-FL). A targeting sequence can also be referred to as a “targeting domain” or a “targeting region”.Attorney Docket No.199235-774601

[0092] In some cases, a targeting domain of an engineered guide allows the engineered guide to target an RNA sequence through base pairing, such as Watson Crick base pairing. In some examples, the targeting sequence can be located at either the N-terminus or C-terminus of the engineered guide. In some cases, the targeting sequence can be located at both termini. The targeting sequence can be of any length. In some cases, the targeting sequence can be at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or up to about 200 nucleotides in length. In some cases, the targeting sequence can be no greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 200 nucleotides in length. In some examples, an engineered guide comprises a targeting sequence that can be from about 60 to about 500, from about 60 to about 200, from about 75 to about 100, from about 80 to about 200, from about 90 to about 120, or from about 95 to about 115 nucleotides in length. In some examples, an engineered guide RNA comprises a targeting sequence that can be about 100 nucleotides in length.

[0093] In some cases, a targeting domain comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to a target RNA. In some cases, a targeting sequence comprises less than 100% complementarity to a target RNA sequence. For example, a targeting sequence and a region of a target RNA that can be bound by the targeting sequence can have a single base mismatch.

[0094] The targeting sequence can have sufficient complementarity to a target RNA to allow for hybridization of the targeting sequence to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 50 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisenseAttorney Docket No.199235-774601 complementarity of about 60 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 70 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 80 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 90 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 100 nucleotides or more to the target RNA. In some embodiments, antisense complementarity refers to non-contiguous stretches of sequence. In some embodiments, antisense complementarity refers to contiguous stretches of sequence.

[0095] In some cases, an engineered guide RNA targeting DUX4 can comprise multiple targeting sequences. In some instances, one or more target sequence domains in the engineered guide RNA can bind to one or more regions of a target DUX4 mRNA. For example, a first targeting sequence can be configured to be at least partially complementary to a first region of a target RNA (e.g., a polyA signal sequence of DUX4), while a second targeting sequence can be configured to be at least partially complementary to a second region of a target RNA. In some instances, multiple target sequences can be operatively linked to provide continuous hybridization of multiple regions of a target RNA. In some instances, multiple target sequences can provide non-continuous hybridization of multiple regions of a target RNA. A “non- continuous” overlap or hybridization refers to hybridization of a first region of a target DUX4 mRNA by a first targeting sequence, along with hybridization of a second region of a target DUX4 mRNA by a second targeting sequence, where the first region and the second region of the target DUX4 RNA are discontinuous (e.g., where there is intervening sequence between the first and the second region of the target RNA). Use of an engineered guide RNA as described herein configured for non-continuous hybridization can provide a number of benefits. For instance, such a guide can potentially target pre-mRNA during transcription (or shortly thereafter), which can then facilitate chemical modification using a deaminase (e.g., ADAR) co-transcriptionally and thus increase the overall efficiency of the chemical modification. Further, the use of oligo tethers to provide non-continuous hybridization while skipping intervening sequence can result in shorter, more specific guide RNA with less off-target editing.

[0096] In some instances, an engineered guide RNA configured for non-continuous hybridization to a target DUX4 RNA (e.g., an engineered guide RNA comprising a targeting sequence with an oligo tether) can be configured to bind distinct regions or a target DUX4 RNAAttorney Docket No.199235-774601 separated by intervening sequence. In some instances, the intervening sequence can be at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, or 10000 nucleotides. In some instances, the targeting sequence and oligo tether can target distinct non-continuous regions of the same intron, exon or noncoding region. In some instances, the targeting sequence and oligo tether can target distinct non-continuous regions of adjacent exons, introns or noncoding regions. In some instances, the targeting sequence and oligo tether can target distinct non- continuous regions of distal exons, introns, or noncoding regions.

[0097] In some embodiments, a polynucleotide encoding a guide RNA disclosed herein can comprise a targeting sequence, such as the sequences described in Table 1. Table 1 provides different guide RNA sequences (DNA and RNA sequences) and the latent structural features associated with guide RNA sequences. For each sequence, the structural features formed in the double stranded RNA substrate upon hybridization of the guide RNA to the target DUX4 RNA, are shown in the last column of Table 1. For reference, each structural feature formed within a guide-target RNA scaffold (target RNA sequence hybridized to an engineered guide RNA) is annotated as follows: a) the position of the structural feature with respect to the target A (position 0 – the first A (bolded and underline) in the polyA signal sequence AUUAAA) of the target RNA sequence, with a negative value indicating upstream (5’) of the target A and a positive value indicating downstream (3’) of the target A;Attorney Docket No.199235-774601 b) the number of bases in the target RNA sequence and the number of bases in the engineered guide RNA that together form the structural feature – for example, 6 / 6 indicates that six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; c) the name of the structural feature (e.g., symmetric bulge, symmetric internal loop, asymmetric bulge, asymmetric internal loop, mismatch, or wobble base pair), and d) the sequences of bases on the target RNA side and the engineered guide RNA side that participate in forming the structural feature.

[0098] For example, with reference to SEQ ID NO: 52, “-5_6-6_internal_loop- symmetric_UCAGAG-GCAGCU, 0_1-1_mismatch_A-C, 44_6-6_internal_loop- symmetric_UUUUGU-CUACUC” is read as a structural feature formed in a guide-target RNA scaffold (target DUX4 RNA sequence hybridized to an engineered guide RNA of SEQ ID NO: 36), where a structural feature starts 5 nucleotides upstream (5’) (the -5 position) from the target A (0 position) of the target RNA sequence; six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; the structural feature is an internal symmetric loop; and a sequence of UCAGAG from the target RNA side and a sequence of GCAGCU from the engineered guide RNA side participate in forming the internal symmetric loop. A structural feature starts at the target A (0 position) of the target RNA sequence; 1 base from the target RNA and 1 base from the engineered guide RNA form the structural feature; the structural feature is a mismatch; and the sequence of A from the target RNA side and a sequence of C from the engineered guide RNA side participate in forming the mismatch. A structural feature starts 44 nucleotides downstream (3’) (the +44 position) from the target A (0 position) of the target RNA sequence; six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; the structural feature is an internal symmetric loop; and a sequence of UUUUGU from the target RNA side and a sequence of CUACUC from the engineered guide RNA side participate in forming the internal symmetric loop. Table 1 – DUX4 Targeting SequencesAttorney Docket No.199235-774601Attorney Docket No.199235-774601Attorney Docket No.199235-774601Attorney Docket No.199235-774601

[0099] In some embodiments, a polynucleotide encoding a guide RNA disclosed herein can comprise a targeting sequence, such as any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136. In some cases, the targeting sequence targets the polyA signal sequence of DUX4. In some cases, SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136 can be positioned in a vector in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’).

[0100] In some embodiments, an engineered guide RNA disclosed herein can comprise a targeting sequence, such as any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142. In some cases, the targeting sequence targets the polyA signal sequence of DUX4. In some embodiments, a composition can comprise an engineered guide RNA comprising any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142. In some embodiments, a composition can comprise an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136. In some embodiments, a composition can comprise a polynucleotide encoding one or more engineered guide RNAs comprising any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 131 – SEQ ID NO: 136.Attorney Docket No.199235-774601

[0101] In some embodiments, hybridization of a targeting domain of an engineered guide RNA to a target DUX4 mRNA results in mRNA knockdown, protein knockdown, or both. For example, hybridization of a targeting domain of an engineered guide RNA to a sequence of a target DUX4 mRNA containing a polyA signal sequence can result in degradation and / or silencing of the target DUX4 mRNA. This degradation and / or silencing can occur due to, for example, where the hybridization of the targeting domain to the target DUX4 mRNA results in ADAR-mediated editing of an adenosine of the polyA signal sequence, thus converting an “A” in the polyA signal sequence to a “G” and silencing expression of DUX4 mRNA. Alternatively or in addition, degradation and / or silencing can occur due to masking of a polyA signal sequence of a target DUX4 mRNA by the engineered guide RNA. In some cases, masking of the polyA signal sequence can facilitate destabilization of DUX4-FL mRNA. In some embodiments, hybridization of a targeting domain of an engineered guide RNA to a sequence of a target DUX4 mRNA containing a polyA signal sequence can result in expression knockdown of a gene downstream of DUX4 (e.g., SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, Wfdc3, Agtr2, MBD3L2, or combinations thereof). For example, hybridization of a targeting domain of an engineered guide RNA to a sequence of a target DUX4 RNA containing a polyA signal sequence can result in knockdown of a mRNA, a protein, or both encoded by SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, Wfdc3, Agtr2, or MBD3L2. B. Engineered Guide RNAs Having a Recruiting Domain

[0102] In some examples, a subject engineered guide RNA comprises a recruiting domain that recruits an RNA editing entity (e.g., ADAR), where in some instances, the recruiting domain is formed and present in the absence of binding to the target RNA. A “recruiting domain” can be referred to herein as a “recruiting sequence” or a “recruiting region”. In some examples, a subject engineered guide can facilitate editing of a base of a nucleotide in a target sequence of a target RNA that results in modulating the expression of a polypeptide encoded by the target RNA. Said modulation can be increased expression of the polypeptide or decreased expression of the polypeptide. In some cases, an engineered guide can be configured to facilitate an editing of a base of a nucleotide or polynucleotide of a region of an RNA by an RNA editing entity (e.g., ADAR). In order to facilitate editing, an engineered guide RNA of the disclosure can recruit an RNA editing entity (e.g., ADAR). Various RNA editing entity recruiting domains can be utilized. In some examples, a recruiting domain comprises:Attorney Docket No.199235-774601 Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), an Alu sequence, or, in the case of recruiting APOBEC, an APOBEC recruiting domain.

[0103] In some examples, more than one recruiting domain can be included in an engineered guide of the disclosure. In examples where a recruiting domain can be present, the recruiting domain can be utilized to position the RNA editing entity to effectively react with a subject target RNA after the targeting sequence hybridizes to a target sequence of a target RNA (e.g., SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142). In some cases, a recruiting domain can allow for transient binding of the RNA editing entity to the engineered guide. In some examples, the recruiting domain allows for permanent binding of the RNA editing entity to the engineered guide. A recruiting domain can be of any length. In some cases, a recruiting domain can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, up to about 80 nucleotides in length. In some cases, a recruiting domain can be no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 80 nucleotides in length. In some cases, a recruiting domain can be about 45 nucleotides in length. In some cases, at least a portion of a recruiting domain comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting domain comprises about 45 nucleotides to about 60 nucleotides.

[0104] In some embodiments, a recruiting domain comprises a GluR2 sequence or functional fragment thereof. In some cases, a GluR2 sequence can be recognized by an RNA editing entity, such as an ADAR or biologically active fragment thereof. In some embodiments, a GluR2 sequence can be a non-naturally occurring sequence. In some cases, a GluR2 sequence can be modified, for example for enhanced recruitment. In some embodiments, a GluR2 sequence can comprise a portion of a naturally occurring GluR2 sequence and a synthetic sequence.

[0105] In some examples, a recruiting domain comprises a GluR2 sequence, or a sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity and / or length to: GUGGAAUAGUAUAACAAUAUGCUAAAUGUUGUUAUAGUAUCCCAC (SEQ ID NO: 8). In some cases, a recruiting domain can comprise at least about 80% sequence identity to at least about 10, 15, 20, 25, or 30 nucleotides of SEQ ID NO: 8. In some examples, aAttorney Docket No.199235-774601 recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity and / or length to SEQ ID NO: 8.

[0106] Additional RNA editing entity recruiting domains are also contemplated. In an embodiment, a recruiting domain comprises an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) domain. In some cases, an APOBEC domain can comprise a non-naturally occurring sequence or naturally occurring sequence. In some embodiments, an APOBEC-domain-encoding sequence can comprise a modified portion. In some cases, an APOBEC-domain-encoding sequence can comprise a portion of a naturally occurring APOBEC-domain-encoding-sequence. In another embodiment, a recruiting domain can be from an Alu domain.

[0107] Any number of recruiting domains can be found in an engineered guide of the present disclosure. In some examples, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting domains can be included in an engineered guide. Recruiting domains can be located at any position of engineered guide RNAs. In some cases, a recruiting domain can be on an N- terminus, middle, or C-terminus of an engineered guide RNA. A recruiting domain can be upstream or downstream of a targeting sequence. In some cases, a recruiting domain flanks a targeting sequence of a subject guide. A recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting domain comprising both ribo- and deoxyribonucleotides can in some cases not be excluded. C. Engineered Guide RNAs with Latent Structure

[0108] In some examples, an engineered guide disclosed herein useful for facilitating editing of a target RNA by an RNA editing entity can be an engineered latent guide RNA. An “engineered latent guide RNA” refers to an engineered guide RNA that comprises latent structure. “Latent structure” refers to a structural feature that substantially forms upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked.

[0109] A double stranded RNA (dsRNA) substrate is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA (for example, the polyA signalAttorney Docket No.199235-774601 sequence of DUX4). The resulting dsRNA substrate is also referred to herein as a “guide-target RNA scaffold.”

[0110] FIG.1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8 / 7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2 / 2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1 / 1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5 / 5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2 / 3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side). Unless otherwise noted, the number of participating nucleotides in a given structural feature is indicated as the nucleotides on the target RNA side over nucleotides on the guide RNA side. Also shown in this legend is a key to the positional annotation of each figure. For example, the target nucleotide to be edited is designated as the 0 position. Downstream (3’) of the target nucleotide to be edited, each nucleotide is counted in increments of +1. Upstream (5’) of the target nucleotide to be edited, each nucleotide is counted in increments of -1. Thus, the example 2 / 2 symmetric bulge in this legend is at the +12 to +13 position in the guide-target RNA scaffold. Similarly, the 2 / 3 asymmetric bulge in this legend is at the -36 to-37 position in the guide-target RNA scaffold. As used herein, positional annotation is provided with respect to the target nucleotide to be edited and on the target RNA side of the guide-target RNA scaffold. As used herein, if a single position is annotated, the structural feature extends from that position away from position 0 (target nucleotide to be edited). For example, if a latent guide RNA is annotated herein as forming a 2 / 3 asymmetric bulge at position -36, then the 2 / 3 asymmetric bulge forms from -36 position to the -37 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold. As another example, if a latent guide RNA is annotated herein as forming a 2 / 2 symmetric bulge at position +12, then the 2 / 2 symmetric bulge forms from the +12 to the +13 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold.

[0111] In some examples, the engineered guides disclosed herein lack a recruiting region and recruitment of the RNA editing entity can be effectuated by structural features of the guide- target RNA scaffold formed by hybridization of the engineered guide RNA and the target RNA.Attorney Docket No.199235-774601 In some examples, the engineered guide, when present in an aqueous solution and not bound to the target RNA molecule, does not comprise structural features that recruit the RNA editing entity (e.g., ADAR). The engineered guide RNA, upon hybridization to a target RNA, form with the target RNA molecule, one or more structural features that recruits an RNA editing entity (e.g., ADAR).

[0112] In cases where a recruiting sequence can be absent, an engineered guide RNA can be still capable of associating with a subject RNA editing entity (e.g., ADAR) to facilitate editing of a target RNA and / or modulate expression of a polypeptide encoded by a subject target RNA. This can be achieved through structural features formed in the guide-target RNA scaffold formed upon hybridization of the engineered guide RNA and the target RNA. Structural features can comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge, symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, or any combination thereof.

[0113] Described herein are structural features which can be present in a guide-target RNA scaffold of the present disclosure. Examples of features include a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a non-recruiting hairpin). Engineered guide RNAs of the present disclosure can have from 1 to 50 features. Engineered guide RNAs of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features. In some embodiments, structural features (e.g., mismatches, bulges, internal loops) can be formed from latent structure in an engineered latent guide RNA upon hybridization of the engineered latent guide RNA to a target RNA and, thus, formation of a guide-target RNA scaffold. In some embodiments, structural features are not formed from latent structures and are, instead, pre-formed structures (e.g., a GluR2 recruitment hairpin or a hairpin from U7 snRNA).

[0114] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. As disclosed herein, a mismatch refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceedsAttorney Docket No.199235-774601 1, the resulting structure is no longer considered a mismatch, but rather, is considered a bulge or an internal loop, depending on the size of the structural feature. In some embodiments, a mismatch is an A / C mismatch. An A / C mismatch can comprise a C in an engineered guide RNA of the present disclosure opposite an A in a target RNA. An A / C mismatch can comprise an A in an engineered guide RNA of the present disclosure opposite a C in a target RNA. A G / G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA.

[0115] In some embodiments, a mismatch positioned 5’ of the edit site can facilitate base- flipping of the target A to be edited. A mismatch can also help confer sequence specificity. Thus, a mismatch can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0116] In another aspect, a structural feature comprises a wobble base. A wobble base pair refers to two bases that weakly base pair. For example, a wobble base pair of the present disclosure can refer to a G paired with a U. Thus, a wobble base pair can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0117] In some cases, a structural feature can be a hairpin. As disclosed herein, a hairpin includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion. A hairpin can have from 10 to 500 nucleotides in length of the entire duplex structure. The loop portion of a hairpin can be from 3 to 15 nucleotides long. A hairpin can be present in any of the engineered guide RNAs disclosed herein. The engineered guide RNAs disclosed herein can have from 1 to 10 hairpins. In some embodiments, the engineered guide RNAs disclosed herein have 1 hairpin. In some embodiments, the engineered guide RNAs disclosed herein have 2 hairpins. As disclosed herein, a hairpin can include a recruitment hairpin or a non-recruitment hairpin. A hairpin can be located anywhere within the engineered guide RNAs of the present disclosure. In some embodiments, one or more hairpins is proximal to or present at the 3’ end of an engineered guide RNA of the present disclosure, proximal to or at the 5’ end of an engineered guide RNA of the present disclosure, proximal to or within the targeting domain (e.g., the targeting sequence) of the engineered guide RNAs of the present disclosure, or any combination thereof.Attorney Docket No.199235-774601

[0118] A recruitment hairpin, as disclosed herein, can recruit at least in part an RNA editing entity, such as ADAR. In some cases, a recruitment hairpin can be formed and present in the absence of binding to a target RNA. In some embodiments, a recruitment hairpin is a GluR2 domain or portion thereof. In some embodiments, a recruitment hairpin is an Alu domain or portion thereof. A recruitment hairpin, as defined herein, can include a naturally occurring ADAR substrate or truncations thereof. Thus, a recruitment hairpin such as GluR2 is a pre- formed structural feature that may be present in constructs comprising an engineered guide RNA, not a structural feature formed by latent structure provided in an engineered latent guide RNA.

[0119] In some aspects, a structural feature comprises a non-recruitment hairpin. A non- recruitment hairpin, as disclosed herein, does not have a primary function of recruiting an RNA editing entity. A non-recruitment hairpin, in some instances, does not recruit an RNA editing entity. In some instances, a non-recruitment hairpin has a dissociation constant for binding to an RNA editing entity under physiological conditions that is insufficient for binding. For example, a non-recruitment hairpin has a dissociation constant for binding an RNA editing entity at 25 ºC that is greater than about 1 mM, 10 mM, 100 mM, or 1 M, as determined in an in vitro assay. A non-recruitment hairpin can exhibit functionality that improves localization of the engineered guide RNA to the target RNA. In some embodiments, the non-recruitment hairpin improves nuclear retention. In some embodiments, the non-recruitment hairpin comprises a hairpin from U7 snRNA. Thus, a non-recruitment hairpin such as a hairpin from U7 snRNA is a pre-formed structural feature that can be present in constructs comprising engineered guide RNA constructs, not a structural feature formed by latent structure provided in an engineered latent guide RNA.

[0120] A hairpin of the present disclosure can be of any length. In an aspect, a hairpin can be from about 10-500 or more nucleotides. In some cases, a hairpin can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,Attorney Docket No.199235-774601 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more nucleotides. In other cases, a hairpin can also comprise 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to 190, 10 to 200, 10 to 210, 10 to 220, 10 to 230, 10 to 240, 10 to 250, 10 to 260, 10 to 270, 10 to 280, 10 to 290, 10 to 300, 10 to 310, 10 to 320, 10 to 330, 10 to 340, 10 to 350, 10 to 360, 10 to 370, 10 to 380, 10 to 390, 10 to 400, 10 to 410, 10 to 420, 10 to 430, 10 to 440, 10 to 450, 10 to 460, 10 to 470, 10 to 480, 10 to 490, or 10 to 500 nucleotides.

[0121] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. As disclosed herein, a bulge refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand. A bulge can change the secondary or tertiary structure of the guide-target RNA scaffold. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNAAttorney Docket No.199235-774601 scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair – a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a mismatch. Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an internal loop. In some embodiments, the guide- target RNA scaffold of the present disclosure has 2 bulges. In some embodiments, the guide- target RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guide- target RNA scaffold of the present disclosure has 4 bulges. Thus, a bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0122] In some embodiments, the presence of a bulge in a guide-target RNA scaffold can position or can help to position ADAR to selectively edit the target A in the target RNA and reduce off-target editing of non-target A(s) in the target RNA. In some embodiments, the presence of a bulge in a guide-target RNA scaffold can recruit or help recruit additional amounts of ADAR. Bulges in guide-target RNA scaffolds disclosed herein can recruit other proteins, such as other RNA editing entities. In some embodiments, a bulge positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited. A bulge can also help confer sequence specificity for the A of the target RNA to be edited, relative to other A(s) present in the target RNA. For example, a bulge can help direct ADAR editing by constraining it in an orientation that yields selective editing of the target A.

[0123] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. A bulge can be a symmetrical bulge or an asymmetrical bulge. A bulge can be a symmetrical bulge or an asymmetrical bulge. A symmetrical bulge is formed when the same number of nucleotides is present on each side of the bulge. For example, a symmetrical bulge in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-Attorney Docket No.199235-774601 target RNA scaffold target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0124] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An asymmetrical bulge is formed when a different number of nucleotides is present on each side of the bulge. For example, an asymmetrical bulge in a guide-target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 1 nucleotide on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide- target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosureAttorney Docket No.199235-774601 can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the target RNA side of the guide- target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0125] In some embodiments, an asymmetric bulge can be a 1 / 0 asymmetric bulge. In some embodiments, a 1 / 0 asymmetric bulge can be a U deletion. A “U deletion” refers to a 1 / 0 asymmetric bulge in which a U nucleotide of an engineered guide RNA that would be situated opposite a non-target A of a target RNA in the guide-target RNA scaffold is deleted from the engineered guide RNA. In some instances, a 1 / 0 asymmetric bulge comprising a U deletionAttorney Docket No.199235-774601 can reduce editing of the non-target A, relative to a comparable guide RNA lacking the U deletion.

[0126] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. In some cases, a structural feature can be an internal loop. As disclosed herein, an internal loop refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide- target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a bulge or a mismatch, depending on the size of the structural feature. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. Internal loops present in the vicinity of the edit site can help with base flipping of the target A in the target RNA to be edited.

[0127] One side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, can be formed by from 5 to 150 nucleotides. One side of the internal loop can be formed by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 120, 135, 140, 145, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides, or any number of nucleotides there between. One side of the internal loop can be formed by 5 nucleotides. One side of the internal loop can be formed by 10 nucleotides. One side of the internal loop can be formed by 15 nucleotides. One side of the internal loop can be formed by 20 nucleotides. One side of the internal loop can be formed by 25 nucleotides. One side of the internal loop can be formed by 30 nucleotides. One side of the internal loop can be formed by 35 nucleotides. One side of the internal loop can be formed by 40 nucleotides. One side of the internal loop can be formed by 45 nucleotides. One side of the internal loop can be formed by 50 nucleotides. One side of the internal loop can be formed by 55 nucleotides. One side of the internal loop can be formed by 60 nucleotides. One side of the internal loop can be formed by 65 nucleotides. One side of the internal loop can be formed by 70 nucleotides. One side of the internal loop can be formed by 75 nucleotides. One side of the internal loop can be formed by 80 nucleotides. One side of the internal loop can be formed by 85 nucleotides. One side of the internal loop can be formed by 90 nucleotides. One side of the internal loop can be formed byAttorney Docket No.199235-774601 95 nucleotides. One side of the internal loop can be formed by 100 nucleotides. One side of the internal loop can be formed by 110 nucleotides. One side of the internal loop can be formed by 120 nucleotides. One side of the internal loop can be formed by 130 nucleotides. One side of the internal loop can be formed by 140 nucleotides. One side of the internal loop can be formed by 150 nucleotides. One side of the internal loop can be formed by 200 nucleotides. One side of the internal loop can be formed by 250 nucleotides. One side of the internal loop can be formed by 300 nucleotides. One side of the internal loop can be formed by 350 nucleotides. One side of the internal loop can be formed by 400 nucleotides. One side of the internal loop can be formed by 450 nucleotides. One side of the internal loop can be formed by 500 nucleotides. One side of the internal loop can be formed by 600 nucleotides. One side of the internal loop can be formed by 700 nucleotides. One side of the internal loop can be formed by 800 nucleotides. One side of the internal loop can be formed by 900 nucleotides. One side of the internal loop can be formed by 1000 nucleotides. Thus, an internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0128] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. A symmetrical internal loop is formed when the same number of nucleotides is present on each side of the internal loop. For example, a symmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold target and 8 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 9 nucleotidesAttorney Docket No.199235-774601 on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 15 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 15 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 20 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 20 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 30 nucleotides on the engineered polynucleotide side of the guide- target RNA scaffold target and 30 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 40 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 40 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 50 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 60 nucleotides on the engineered polynucleotide side of the guide- target RNA scaffold target and 60 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 70 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 70 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 80 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 80 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 90 nucleotides on the engineered polynucleotide side of the guide- target RNA scaffold target and 90 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 100 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 110 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 110 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the presentAttorney Docket No.199235-774601 disclosure can be formed by 120 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 120 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 130 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 130 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 140 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 140 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 150 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 200 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 250 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 250 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 300 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 350 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 350 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 400 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 450 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 450 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 500 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 600 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 600 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered polynucleotide side of theAttorney Docket No.199235-774601 guide-target RNA scaffold target and 700 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 800 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 800 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 900 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 900 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold target and 1000 nucleotides on the target RNA side of the guide- target RNA scaffold. Thus, a symmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0129] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. An asymmetrical internal loop is formed when a different number of nucleotides is present on each side of the internal loop. For example, an asymmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.

[0130] An asymmetrical internal loop of the present disclosure can be formed by from 5 to 150 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold and from 5 to 150 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by from 5 to 1000 nucleotides on the engineered polynucleotide side of the guide-target RNA scaffold and from 5 to 1000 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 6 nucleotides on the engineered guide RNA side of the guide-Attorney Docket No.199235-774601 target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-Attorney Docket No.199235-774601 target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can beAttorney Docket No.199235-774601 formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide- target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the presentAttorney Docket No.199235-774601 disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can beAttorney Docket No.199235-774601 formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can beAttorney Docket No.199235-774601 formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the presentAttorney Docket No.199235-774601 disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can beAttorney Docket No.199235-774601 formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0131] As disclosed herein, a “base paired (bp) region” refers to a region of the guide-target RNA scaffold in which bases in the guide RNA are paired with opposing bases in the target RNA. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to the other end of the guide-target RNA scaffold. Base paired regions can extend between two structural features. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to a structural feature. Base paired regions can extend from a structural feature to the other end of the guide- target RNA scaffold. In some embodiments, a base paired region has from 1 base pair (bp) toAttorney Docket No.199235-774601 100 base pairs, from 1 bp to 90 bp, from 1 bp to 80 bp, from 1 bp to 70 bp, from 1 bp to 60 bp, from 1 bp to 50 bp, from 1 bp to 45 bp, from 1 bp to 40 bp, from 1 bp to 35 bp, from 1 bp to 30 bp, from 1 bp to 25 bp, from 1 bp to 20 bp, from 1 bp to 15 bp, from 1 bp to 10 bp, from 1 bp to 5 bp, from 5 bp to 10 bp, from 5 bp to 20 bp, from 10 bp to 20 bp, from 10 bp to 50 bp, from 5 bp to 50 bp, at least 1 bp, at least 2 bp, at least 3 bp, at least 4 bp, at least 5 bp, at least 6 bp, at least 7 bp, at least 8 bp, at least 9 bp, at least 10 bp, at least 12 bp, at least 14 bp, at least 16 bp, at least 18 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40 bp, at least 45 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp.

[0132] The present disclosure provides engineered guide RNAs (SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142) that target a sequence of an DUX4 target RNA (for example, the polyA signal sequence of DUX4-FL).

[0133] In some embodiments, an engineered guide RNA of the present disclosure that targets the polyA signal sequence of DUX4 comprises one or more structural features.

[0134] In some embodiments, a first 6 / 6 symmetric internal loop is at position -5, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 1-1 mismatch at position 0 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 44 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 52.

[0135] In some embodiments, a first 6 / 6 symmetric internal loop is at position -6, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 1-1 mismatch at position 0 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 27 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 53.

[0136] In some embodiments, a first 6 / 6 symmetric internal loop is at position -6, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a wobble base at position -35 relative to the target adenosine at position 0, a wobble base at position -32 relative to the target adenosine at position 0, a wobble base at position -27 relative to the targetAttorney Docket No.199235-774601 adenosine at position 0, a wobble base at position -20 relative to the target adenosine at position 0, a wobble base at position -18 relative to the target adenosine at position 0, a wobble base at position -17 relative to the target adenosine at position 0, a wobble base at position -15 relative to the target adenosine at position 0, a wobble base at position 8 relative to the target adenosine at position 0, a wobble base at position 20 relative to the target adenosine at position 0, a wobble base at position 22 relative to the target adenosine at position 0, a wobble base at position 37 relative to the target adenosine at position 0, a wobble base at position 45 relative to the target adenosine at position 0, a wobble base at position 49 relative to the target adenosine at position 0, a wobble base at position 55 relative to the target adenosine at position 0, a wobble base at position 59 relative to the target adenosine at position 0, a 1-1 mismatch at position 0 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 27 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 54.

[0137] In some embodiments, a first 6 / 6 symmetric internal loop is at position -22, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a wobble base at position -21 relative to the target adenosine at position 0, a wobble base at position -14 relative to the target adenosine at position 0, a 1-0 asymmetric bulge at position -13 relative to the target adenosine at position 0, a 1-3 asymmetric bulge at position -7 relative to the target adenosine at position 0, a 4-4 symmetric bulge at position -3 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 36 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 55.

[0138] In some embodiments, a 6 / 6 symmetric internal loop is at position -22, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a wobble base at position -35 relative to the target adenosine at position 0, a wobble base at position -33 relative to the target adenosine at position 0, a wobble base at position -31 relative to the target adenosine at position 0, a wobble base at position -21 relative to the target adenosine at position 0, a wobble base at position -14 relative to the target adenosine at position 0, a wobble base at position 18 relative to the target adenosine at position 0, a wobble base at position 20 relativeAttorney Docket No.199235-774601 to the target adenosine at position 0, a wobble base at position 22 relative to the target adenosine at position 0, a wobble base at position 24 relative to the target adenosine at position 0, a wobble base at position 29 relative to the target adenosine at position 0, a wobble base at position 31 relative to the target adenosine at position 0, a wobble base at position 34 relative to the target adenosine at position 0, a 1 / 1 mismatch at position 41 relative to the target adenosine at position 0, a wobble base at position 45 relative to the target adenosine at position 0, a wobble base at position 48 relative to the target adenosine at position 0, a wobble base at position 51 relative to the target adenosine at position 0, a wobble base at position 55 relative to the target adenosine at position 0, a wobble base at position 58 relative to the target adenosine at position 0, a wobble base at position 62 relative to the target adenosine at position 0, a wobble base at position 64 relative to the target adenosine at position 0, a 1-0 asymmetric bulge at position -13 relative to the target adenosine at position 0, a 1-3 asymmetric bulge at position - 7 relative to the target adenosine at position 0, a 4 / 4 symmetric bulge at position -3 relative to the target adenosine at position 0, a 4 / 4 symmetric internal loop at position 36 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 56.

[0139] In some embodiments, a first 6 / 6 symmetric internal loop is at position -8, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 1-1 mismatch at position 3 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 35 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 57.

[0140] In some embodiments, a first 6 / 6 symmetric internal loop is at position -8, relative to the target adenosine at position 0. In some embodiments, the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 1-1 mismatch at position 0 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 44 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 58.

[0141] In some embodiments, a first 6 / 6 symmetric internal loop is at position -11, relative to the target adenosine at position 0. In some embodiments, the one or more structural featuresAttorney Docket No.199235-774601 further comprises at least one structural feature selected from the group consisting of: a 1-1 mismatch at position 4 relative to the target adenosine at position 0, a second 6 / 6 symmetric internal loop at position 42 relative to the target adenosine at position 0, and any combination thereof. In some embodiments, the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 59. D. Guides with Macro-Footprints

[0142] Guide RNAs of the present disclosure can further comprise a macro-footprint. In some embodiments, the macro-footprint comprises a barbell macro-footprint. A micro-footprint can serve to guide an RNA editing enzyme and direct its activity towards the target adenosine to be edited. A “barbell” as described herein refers to a pair of internal loop latent structures that manifest upon hybridization of the guide RNA to the target RNA. In some embodiments, each internal loop is positioned towards the 5′ end or the 3′ end of the guide-target RNA scaffold formed upon hybridization of the guide RNA and the target RNA. In some embodiments, each internal loop flanks opposing sides of the micro-footprint sequence. Insertion of a barbell macro-footprint sequence flanking opposing sides of the micro-footprint sequence, upon hybridization of the guide RNA to the target RNA, results in formation of barbell internal loops on opposing sides of the micro-footprint. In some cases, barbell internal loops can comprise at least one structural feature that facilitates editing of a specific target RNA.

[0143] As described herein, a “micro-footprint” sequence refers to a sequence with latent structures that, when manifested, facilitate editing of the adenosine of a target RNA via an adenosine deaminase enzyme. A macro-footprint can serve to guide an or focus RNA editing entity (e.g., ADAR) and direct its activity towards a micro-footprint. In some embodiments, included within the micro-footprint sequence is a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, said nucleotide is opposite the adenosine to be edited by the ADAR enzyme and does not base pair with the adenosine to be edited. This nucleotide is referred to herein as the “mismatched position” or “mismatch” and can be a cytosine. Micro-footprint sequences as described herein have upon hybridization of the engineered guide RNA and target RNA, at least one structural feature selected from the group consisting of: a bulge, an internal loop, a mismatch, a hairpin, and any combination thereof. Engineered guide RNAs with superior micro-footprint sequences can be selected based on their ability to facilitate editing of a specific target RNA. Engineered guide RNAs selected for their ability to facilitate editing of a specific target are capable of adopting various micro-footprint latent structures, which can vary on a target-by-target basis.Attorney Docket No.199235-774601

[0144] As disclosed herein, a “macro-footprint” sequence can be positioned such that it flanks a micro-footprint sequence. Further, while a macro-footprint sequence can flank a micro- footprint sequence, additional latent structures can be incorporated that flank either end of the macro-footprint as well. In some embodiments, such additional latent structures are included as part of the macro-footprint. In some embodiments, such additional latent structures are separate, distinct, or both separate and distinct from the macro-footprint.

[0145] In some embodiments, the presence of barbells flanking the micro-footprint can improve one or more aspects of editing. For example, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a higher amount of on target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Additionally, and or alternatively, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Further, while the effect of various micro- footprint structural features can vary on a target-by-target basis based on selection in a high throughput screen, the increase in the one or more aspects of editing provided by the barbell macro-footprint structures can be independent of the particular target RNA. For example, macro-footprints (e.g., barbell macro-footprints) and micro-footprints can provide an increased amount of on target adenosine editing relative to an otherwise comparable guide RNA lacking the barbells. In other embodiments, the presence of the barbell macro-footprint in addition to the micro-footprint described here can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA, upon hybridization of the guide RNA and target RNA to form a guide-target RNA scaffold lacking the barbells.

[0146] A dumbbell design in an engineered guide RNA comprises two symmetrical internal loops, wherein the target A to be edited is positioned between the two symmetrical loops for selective editing of the target A. The two symmetrical internal loops are each formed by 6 nucleotides on the guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a dumbbell can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0147] In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell is positioned at least about 5 bases (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases) away from the A / C mismatch with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A / CAttorney Docket No.199235-774601 mismatch. In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell is positioned at most about 50 bases away from the A / C mismatch (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A / C mismatch.

[0148] In some embodiments, a first internal loop or a second internal loop independently comprises a number of bases of at least about 5 bases or greater (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150); about 150 bases or fewer (e.g., 145, 135, 125, 115, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g., 5-150, 6- 145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-115, 13-110, 14-105, 15-100, 16-95, 17-90, 18- 85, 19-80, 20-75, 21-70, 22-65, 23-60, 24-55, 25-50) of the engineered guide RNA and a number of bases of at least about 5 bases or greater (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150); about 150 bases or fewer (e.g., 145, 135, 125, 115, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g., 5-150, 6-145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-115, 13-110, 14-105, 15-100, 16-95, 17-90, 18-85, 19-80, 20-75, 21-70, 22-65, 23-60, 24-55, 25-50) of the target RNA.

[0149] In some embodiments, provided herein are engineered guide RNAs comprising a barbell macro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a micro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a macro-footprint and a micro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a micro-footprint in the absence of a macro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a macro-footprint in the absence of a micro-footprint.

[0150] In some embodiments, a macro-footprint sequence can comprise a barbell macro- footprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop.

[0151] In some examples, a first internal loop is positioned near the 5′ end of the guide-target RNA scaffold and a second internal loop is positioned near the 3′ end of the guide-target RNA scaffold. The length of the dsRNA comprises a 5′ end and a 3′ end, where up to half of the length of the guide-target RNA scaffold at the 5′ end can be considered to be “near the 5′ end” while up to half of the length of the guide-target RNA scaffold at the 3′ end can be consideredAttorney Docket No.199235-774601 “near the 3′ end.” Non-limiting examples of the 5′ end can include about 50% or less of the total length of the dsRNA at the 5′ end, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. Non-limiting examples of the 3′ end can include about 50% or less of the total length of the dsRNA at the 3′ end about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%.

[0152] In some embodiments, the engineered guide RNAs of the disclosure comprising a barbell macro-footprint sequence (that manifests as a first internal loop and a second internal loop) can improve RNA editing efficiency, increase the amount or percentage of RNA editing generally, as well as for on-target nucleotide editing, such as on-target adenosine. In some embodiments, the engineered guide RNAs of the disclosure comprising a first internal loop and a second internal loop can also facilitate a decrease in the amount of or reduce off-target nucleotide editing, such as off-target adenosine or unintended adenosine editing. The decrease or reduction in some examples can be of the number of off-target edits or the percentage of off- target edits.

[0153] Each of the first and second internal loops of the barbell macro-footprint can independently be symmetrical or asymmetrical, where symmetry is determined by the number of bases or nucleotides of the engineered guide RNA and the number of bases or nucleotides of the target RNA, that together form each of the first and second internal loops. E. Engineered Polynucleotides Encoding Engineered Guide RNAs

[0154] An engineered polynucleotide as described herein can comprise one or more polynucleotide sequence(s) that encode one or more engineered guide RNA(s). For example, an engineered polynucleotide can comprise 1, 2, 3, 4, or more than 4 polynucleotide sequence(s) that encode 1, 2, 3, 4, or more than 4 engineered guide RNAs.

[0155] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target): (1) different target sequences of the same target RNA, or (2) different target sequences of different target RNAs. For example, a first engineered guide RNA encoded by a first polynucleotide sequence can hybridize to a target sequence of a first target RNA while a second engineered guide RNA encoded by a second polynucleotide sequence can hybridize to a target sequence of a second target RNA, in some instances resulting in ADAR- mediated editing of an adenosine in the target sequence of the first target RNA and an adenosine in the target sequence of the second target RNA.Attorney Docket No.199235-774601

[0156] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target) the same target sequence of a target RNA. For example, the one or more engineered guide RNA(s) encoded by the one or more polynucleotide sequence(s) can each independently hybridize to a target sequence of a target RNA and / or facilitate editing of the same adenosine in the target sequence of the target RNA via ADAR. In some cases, the one or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA have identical sequences (i.e., the one or more engineered guide RNAs are copies of each other).

[0157] Alternatively, two or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA can comprise different sequences. For example, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to: 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some instances, a first engineered guide RNA encoded by an engineered polynucleotide can have at least about 70% to about 99% sequence identity, at least about 60% to about 99% sequence identity, at least about 80% to about 99% sequence identity, at least about 60% to about 70% sequence identity, at least about 70% to about 80% sequence identity, at least about 75% to about 85% sequence identity, at least about 85% to about 99% sequence identity, at least about 85% to about 90% sequence identity, at least about 88% to about 93% sequence identity, at least about 90% to about 95% sequence identity, at least about 92% to about 99% sequence identity, or at least about 95% to about 99% sequence identity to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 60% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greaterAttorney Docket No.199235-774601 than, or equal to about 61% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 62% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 63% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 64% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 65% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 66% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 67% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 68% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identityAttorney Docket No.199235-774601 of less than, greater than, or equal to about 69% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 70% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 71% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 72% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 73% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 74% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 75% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 76% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotideAttorney Docket No.199235-774601 can have a sequence identity of less than, greater than, or equal to about 77% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 78% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 79% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 80% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 81% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 82% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 83% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 84% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNAAttorney Docket No.199235-774601 encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 85% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 86% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 87%, to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 88% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 89% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 90% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 91% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 92% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a firstAttorney Docket No.199235-774601 engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 93% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 94% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 95% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 96% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 97% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 98% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some embodiments, polynucleotides encoding a first engineered guide RNA, a second engineered guide RNA, or both can be delivered via an AAV. In some instances, the AAV can be formulated in a composition, such as any of the pharmaceutical compositions disclosed herein. F. Additional Engineered Guide RNA ComponentsAttorney Docket No.199235-774601

[0158] The present disclosure provides for engineered guide RNAs with additional structural features and components. For example, an engineered guide RNA described herein can be circular. In another example, an engineered guide RNA described herein can comprise a U7, an SmOPT sequence, or a combination of both sequences.

[0159] In some cases, an engineered guide RNA can be circularized. In some cases, an engineered guide RNA provided herein can be circularized or in a circular configuration. In some aspects, an at least partially circular guide RNA lacks a 5’ hydroxyl or a 3’ hydroxyl. In some embodiments, a circular engineered guide RNA can comprise a guide RNA comprising a polynucleotide sequence of any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142 that targets the polyA signal sequence of DUX4.

[0160] In some examples, an engineered guide RNA can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some examples, a backbone of an engineered guide RNA can comprise a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA.

[0161] In some embodiments, a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to a solvent. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to nucleases. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes. In some instances, a backbone of an engineered guide can be represented as a polynucleotide sequence in a circular 2-dimensional format with one nucleotide after the other. In some instances, a backbone of an engineered guide can be represented as a polynucleotide sequence in a looped 2-dimensional format with one nucleotide after the other. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be joined through a phosphorus-oxygen bond. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be modified into a phosphoester with a phosphorus-containing moiety.

[0162] As described herein, an engineered guide can comprise a circular structure. An engineered polynucleotide can be circularized from a precursor engineered polynucleotide. Such a precursor engineered polynucleotide can be a precursor engineered linear polynucleotide. In some cases, a precursor engineered linear polynucleotide can be a precursor for a circular engineered guide RNA. For example, a precursor engineered linear polynucleotide can be a linear mRNA transcribed from a plasmid, which can be configured toAttorney Docket No.199235-774601 circularize within a cell using the techniques described herein. A precursor engineered linear polynucleotide can be constructed with domains such as a ribozyme domain and a ligation domain that allow for circularization when inserted into a cell. A ribozyme domain can include a domain that is capable of cleaving the linear precursor RNA at specific sites (e.g., adjacent to the ligation domain). A precursor engineered linear polynucleotide can comprise, from 5’ to 3’: a 5’ ribozyme domain, a 5’ ligation domain, a circularized region, a 3’ ligation domain, and a 3’ ribozyme domain. In some cases, a circularized region can comprise a guide RNA described herein. In some cases, the precursor polynucleotide can be specifically processed at both sites by the 5’ and the 3’ ribozymes, respectively, to free exposed ends on the 5’ and 3’ ligation domains. The free exposed ends can be ligation competent, such that the ends can be ligated to form a mature circularized structure. For instance, the free ends can include a 5’-OH and a 2’, 3’-cyclic phosphate that are ligated via RNA ligation in the cell. The linear polynucleotide with the ligation and ribozyme domains can be transfected into a cell where it can circularize via endogenous cellular enzymes. In some cases, a polynucleotide can encode an engineered guide RNA comprising the ribozyme and ligation domains described herein, which can circularize within a cell. For example, PCT / US2021 / 034301 provides a description of circular guide RNAs and their structures, sequences of circular guide RNAs, and methods of engineering circularized polynucleotide domains, and each of these descriptions in PCT / US2021 / 034301 is herein incorporated by reference.

[0163] An engineered polynucleotide as described herein (e.g., a circularized guide RNA) can include spacer domains. As described herein, a spacer domain can refer to a domain that provides space between other domains. A spacer domain can be used to between a region to be circularized and flanking ligation sequences to increase the overall size of the mature circularized guide RNA. Where the region to be circularized includes a targeting domain as described herein that is configured to associate to a target sequence, the addition of spacers can provide improvements (e.g., increased specificity, enhanced editing efficiency, etc.) for the engineered polynucleotide to the target polynucleotide, relative to a comparable engineered polynucleotide that lacks a spacer domain. In some instances, the spacer domain is configured to not hybridize with the target RNA. In some embodiments, a precursor engineered polynucleotide or a circular engineered guide, can comprise, in order of 5’ to 3’: a first ribozyme domain; a first ligation domain; a first spacer domain; a targeting domain that can be at least partially complementary to a target RNA, a second spacer domain, a second ligation domain, and a second ribozyme domain. In some cases, the first spacer domain, the secondAttorney Docket No.199235-774601 spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA.

[0164] A circular or looped RNA can be formed by employing a self-cleaving entity, such as a ribozyme, tRNA, aptamer, catalytically active fragment of any of these, or any combination thereof. For example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ end, a 5’ end, or both of a precursor engineered RNA. In another example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ terminal end, a 5’ terminal end, or both of a precursor engineered RNA. A self-cleaving ribozyme can comprise, for example, an RNase P RNA a Hammerhead ribozyme (e.g., a Schistosoma mansoni ribozyme), a glmS ribozyme, an HDV-like ribozyme, an R2 element, a peptidyl transferase 23S rRNA, a GIR1 branching ribozyme, a leadzyme, a group II intron, a hairpin ribozyme, a VS ribozyme, a CPEB3 ribozyme, a CoTC ribozyme, or a group I intron. In some cases, the self-cleaving ribozyme can be a trans-acting ribozyme that joins one RNA end on which it is present to a separate RNA end. In some embodiments, an aptamer can be added to each end of the engineered guide RNA. A ligase can be contacted with the aptamers at each end of the engineered guide RNA to form a covalent linkage between the aptamers thereby forming a circular engineered guide RNA. In some cases, a self-cleaving element or an aptamer can be configured to facilitate self-circularization of an engineered polynucleotide or a pro- polynucleotide (e.g., from a precursor engineered polypeptide) after transcription in a cell. In some instances, circularization of a guide RNA can be shown by PCR. For example, primers can be developed that bind to the end of a guide RNA and are directed outward such that a product is only formed when guides are circularized.

[0165] In some cases, circularization can occur by back-slicing and ligation of an exon. For example, an RNA can be engineered from 5’ to 3’ to comprise a forward complementary sequence intron, an exon (which can comprise the guide sequence), followed by a reverse complementary sequence intron. Once transcribed, the complementary sequence introns can hybridize and form dsRNA. The internal exon containing the guide sequence can be removed by splicing and ligated by an endogenous ligase to form a circular guide. In one example, an engineered guide RNA can initiate circularization in a cell by autocatalytic reactions of encoded ribozymes. After cleavage by one or more ribozymes, the linear polynucleotide will undergo intracellular RNA ligation of the 5’ and the 3’ end of ligation sequences by an endogenous ligase to circularize the guide RNA.Attorney Docket No.199235-774601

[0166] A suitable self-cleaving molecule can include a ribozyme. For example, a ribozyme domain can create an autocatalytic RNA. A ribozyme can comprise an RNase P, an rRNA (such as a Peptidyl transferase 23S rRNA), Leadzyme, Group I intron ribozyme, Group II intron ribozyme, a GIR1 branching ribozyme, a glmS ribozyme, a hairpin ribozyme, a Hammerhead ribozyme, an HDV ribozyme, a Twister ribozyme, a Twister sister ribozyme, a VS ribozyme, a Pistol ribozyme, a Hatchet ribozyme, a viroid, or any combination thereof. A ribozyme can include a P3 twister U2A ribozyme. A ribozyme can comprise 5’ GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGC CT 3’ (SEQ ID NO: 9). A ribozyme can comprise 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3’ (SEQ ID NO: 10). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGC CT 3’ (SEQ ID NO: 9). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3’ (SEQ ID NO: 10). A ribozyme can include a P1 Twister Ribozyme. A ribozyme can include 5’ AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 11). A ribozyme can include 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 3’ (SEQ ID NO: 12). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 11). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 3’ (SEQ ID NO: 12).

[0167] A ligation domain can facilitate a linkage, covalent or non-covalent, of a first nucleotide to a second nucleotide. In some embodiments, a ligation domain can recruit a ligating entity to facilitate a ligation reaction. In some cases, a ligation domain can recruit a recombining entity to facilitate a homologous recombination. In some instances, a first ligation domain can facilitate a linkage, covalent or non-covalent, to a second ligation domain. In someAttorney Docket No.199235-774601 embodiments, a first ligation domain can facilitate the complementary pairing of a second ligation domain. In some cases, a ligation domain can comprise 5’ AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 13). In some embodiments, a ligation domain can comprise 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 14). In some cases, a ligation domain can comprise 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 15). In some cases, a ligation domain can comprise 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 16). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 13). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 14). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 15). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 16).

[0168] The compositions and methods of the present disclosure can provide for engineered polynucleotides encoding for guide RNAs that are operably linked to a portion of a small nuclear ribonucleic acid (snRNA) sequence. The engineered polynucleotide can include at least a portion of a small nuclear ribonucleic acid (snRNA) sequence. The U7 and U1 small nuclear RNAs, whose natural role is in spliceosomal processing of pre-mRNA, have for decades been re-engineered to alter splicing at desired disease targets. Replacing the first 18 nt of the U7 snRNA (which naturally hybridizes to the spacer element of histone pre-mRNA) with a short targeting (or antisense) sequence of a disease gene, redirects the splicing machinery to alter splicing around that target site. Furthermore, converting the wild type U7 Sm-domain binding site to an optimized consensus Sm-binding sequence (SmOPT) can increase the expression level, activity, and subcellular localization of the artificial antisense-engineered U7 snRNA. Many subsequent groups have adapted this modified U7 SmOPT snRNA chassis with antisense sequences of other genes to recruit spliceosomal elements and modify RNA splicing for additional disease targets.

[0169] An snRNA is a class of small RNA molecules found within the nucleus of eukaryotic cells. They are involved in a variety of important processes such as RNA splicing (removal of introns from pre-mRNA), regulation of transcription factors (7SK RNA) or RNA polymeraseAttorney Docket No.199235-774601 II (B2 RNA), and maintaining the telomeres. They are always associated with specific proteins, and the resulting RNA-protein complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. There are many snRNAs, which are denominated U1, U2, U3, U4, U5, U6, U7, U8, U9, and U10.

[0170] The snRNA of the U7 type is normally involved in the maturation of histone mRNA. This snRNA has been identified in a great number of eukaryotic species (56 so far) and the U7 snRNA of each of these species should be regarded as equally convenient for this disclosure.

[0171] Wild-type U7 snRNA includes a stem-loop structure, the U7-specific Sm sequence, and a sequence antisense to the 3′ end of histone pre-mRNA.

[0172] In addition to the SmOPT domain, U7 comprises a sequence antisense to the 3′ end of histone pre-mRNA. When this sequence is replaced by a targeting sequence that is antisense to another target pre-mRNA, U7 is redirected to the new target pre-mRNA. Accordingly, the stable expression of modified U7 snRNAs containing the SmOPT domain and a targeting antisense sequence has resulted in specific alteration of mRNA splicing.

[0173] The engineered polynucleotide can comprise at least in part an snRNA sequence. The snRNA sequence can be U1, U2, U3, U4, U5, U6, U7, U8, U9, or a U10 snRNA sequence.

[0174] In some instances, an engineered polynucleotide that comprises at least a portion of an snRNA sequence (e.g. an snRNA promoter, an snRNA hairpin, and the like) can have superior properties for treating or preventing a disease or condition, relative to a comparable polynucleotide lacking such features. For example, as described herein an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate exon skipping of an exon at a greater efficiency than a comparable polynucleotide lacking such features. Further, as described herein an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate an editing of a base of a nucleotide in a target RNA (e.g. a pre-mRNA or a mature RNA) at a greater efficiency than a comparable polynucleotide lacking such features. Promoters and snRNA components are described in PCT / US2021 / 028618 and PCT / US2022 / 078801, and each of these descriptions in PCT / US2021 / 028618 and PCT / US2022 / 078801 are herein incorporated by reference.

[0175] Disclosed herein are engineered RNAs comprising (a) an engineered guide RNA as described herein, and (b) a U7 snRNA hairpin sequence, a SmOPT sequence, or a combination thereof. In some embodiments, the U7 hairpin comprises a human U7 Hairpin sequence, or a mouse U7 hairpin sequence. In some cases, a human U7 hairpin sequence comprises TAGGCTTTCTGGCTTTTTACCGGAAAGCCCCT (SEQ ID NO: 17 or RNA:Attorney Docket No.199235-774601 UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU SEQ ID NO: 18). In some cases, a mouse U7 hairpin sequence comprises CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 19 or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU SEQ ID NO: 20). In some embodiments, the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 21 or RNA: AAUUUUUGGAG SEQ ID NO: 22). In some embodiments, a guide RNA comprising a polynucleotide sequence of any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 137 – SEQ ID NO: 142 that target the polyA signal sequence of DUX4 can comprise a guide RNA comprising a U7 hairpin sequence (e.g., a human or a mouse U7 hairpin sequence), an SmOPT sequence, or a combination thereof. In some cases, a combination of a U7 hairpin sequence and a SmOPT sequence can comprise a SmOPT U7 hairpin sequence, wherein the SmOPT sequence is linked to the U7 sequence. In some cases, a U7 hairpin sequence, an SmOPT sequence, or a combination thereof is downstream (e.g., 3’) of the engineered guide RNA disclosed herein.

[0176] The compositions and methods of the present disclosure can provide for engineered polynucleotides encoding for guide RNAs that are operably linked to a portion of a SmOPT and U7 hairpin sequence. The engineered polynucleotide and / or guide RNAs can include at least a portion of a SmOPT and U7 hairpin sequence. In some instances, a guide RNA comprises the SmOPT and U7 hairpin sequence that is downstream (e.g., 3’) of the guide sequence. In some cases, the sequence encoding the SmOPT and U7 hairpin sequence comprises AATTTTTGGAACAGGGTTTTCTGCCTTCGGGCGGAAAACCCCCT (SEQ ID NO: 31). In some cases, the sequence encoding the SmOPT and U7 hairpin sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31. In some cases, the sequence encoding the SmOPT and U7 hairpin sequence can be incorporated into an engineered polynucleotide in the forward direction or the reverse direction. The RNA sequence of the SmOPT and U7 hairpin sequence comprises AAUUUUUGGAACAGGGUUUUCUGCCUUCGGGCGGAAAACCCCCU (SEQ ID NO: 60). In some cases, the SmOPT and U7 hairpin sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 60.

[0177] Also disclosed herein are promoters for driving the expression of a guide RNA disclosed herein. In some cases, the promoters for driving expression can be upstream (e.g., 5’) to the guide RNA sequence disclosed herein. In some cases, a promoter can comprise a U1Attorney Docket No.199235-774601 promoter, a U7 promoter, a U6 promoter or any combination thereof. In some cases, a promoter can comprise a CMV promoter. In some cases, a U7 promoter, or a U6 promoter can be a mouse U7 promoter, or a mouse U6 promoter. In some cases, a U1 promoter, a U7 promoter, or a U6 promoter can be a human U1 promoter, a human U7 promoter, or a human U6 promoter. In some cases, a U7 promoter can be an engineered mouse U7 promoter. In some cases, a U1 promoter can be an engineered human U1 promoter. In some cases, a promoter herein can be oriented in the forward or the reverse direction on a polynucleotide encoding a guide RNA.

[0178] In some embodiments, a polynucleotide herein (e.g., a plasmid) can comprise an engineered mU7 promoter. In some cases, an engineered mU7 promoter comprises the sequence: TAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACTGACTCATGC AAATCAAGAGAAATGCAAATAGCCTTTACAAGCGGTCACAAACTCAAGAAACGA GCGGTTTTAATAGTCTTTTAGAATATTGTTTATCGAACCGAATAAGGAACTGTGC TTTGTGATTCACATATCAGTGGAGGGGTGTGGAAATGGCACCTTGATAAGTCACC ATGAGTGTAAAGGGAGTTGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCCGC (SEQ ID NO: 30). In some cases, an engineered mU7 promoter has at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30.

[0179] In some embodiments, a polynucleotide herein (e.g., a plasmid) can comprise an engineered promoter sequence. In some cases, an engineered promoter sequence comprises the sequence: ATTTAATAGCAGTCTTTATTTAAAAGAAATCAAACTCAGACGTACAAATACACAA AACAGATAAAACCCGAGTCTCTGACCAGGAAAGCGTTATTTTCCAGCCAGCCAG TCTTCGGCTTCGCCCCCTAACGGTGACATAAGGCACTCTGTGAAATGCTCTGTTC CGGAATCAAAAGATTGATCCGATTATTTGCATACCCATAATGCACTGCTCACAGT ACAAATTTAAAAAGGCAAAATCAAACATTTTTATTCTAAGCATATTCTGTGAAAG TTAGACTTTTGTTTAAACAATACTCTTAAAATTTTTTTCTAGGTATAGAACCTTGG CATTCACTAGTCACCATCACTATACTAGGAGTTTCTGTTACCCGAGAAACGAGTT ATGAAATTAACAAGC (SEQ ID NO: 33). In some cases, an engineered promoter sequence has at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 33.Attorney Docket No.199235-774601

[0180] In some cases, a human U6 promoter comprises a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTA GAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA CGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTT AAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTT ATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 23). In some cases, a mouse U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: GTACTGAGTCGCCCAGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGC CGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTA ATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACA GATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTA TAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTC ACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAA AGCCTTGTTTG (SEQ ID NO: 24). In some cases, a human U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TTAACAACAACGAAGGGGCTGTGACTGGCTGCTTTCTCAACCAATCAGCACCGA ACTCATTTGCATGGGCTGAGAACAAATGTTCGCGAACTCTAGAAATGAATGACTT AAGTAAGTTCCTTAGAATATTATTTTTCCTACTGAAAGTTACCACATGCGTCGTTG TTTATACAGTAATAGGAACAAGAAAAAAGTCACCTAAGCTCACCCTCATCAATT GTGGAGTTCCTTTATATCCCATCTTCTCTCCAAACACATACGCA (SEQ ID NO: 25). In some cases, a mouse U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TTAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACTGACTCATT TGCATAGCCTTTACAAGCGGTCACAAACTCAAGAAACGAGCGGTTTTAATAGTCT TTTAGAATATTGTTTATCGAACCGAATAAGGAACTGTGCTTTGTGATTCACATAT CAGTGGAGGGGTGTGGAAATGGCACCTTGATCTCACCCTCATCGAAAGTGGAGT TGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCCGC (SEQ ID NO: 26).Attorney Docket No.199235-774601

[0181] In some cases, a human U1 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGG GAGAGGCAGACGTCACTTCCTCTTGGCGACTCTGGCAGCAGATTGGTCGGTTGAG TGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGAC AGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCT GCTTCGCCACGAAGGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGAT CGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGC GGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCA GAGCCCGAAGATCTC (SEQ ID NO: 27). In some cases, a CMV promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: ATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAA CCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAC CGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACC (SEQ ID NO: 28).

[0182] Also described herein are terminator sequences (also called termination sequences) for enhanced expression of a guide RNA disclosed herein. In some cases, a polynucleotide encoding a guide RNA can comprise a terminator sequence. In some cases, the terminator sequence can be located downstream (e.g., 3’) of the sequence encoding the SmOPT and U7 hairpin sequence or a sequence encoding a guide RNA sequence. In some cases, the terminator sequence comprises a terminator sequence 1, a terminator sequence 2, a terminator sequence 3, a terminator sequence 4, or any combination thereof. In some cases, a terminator sequenceAttorney Docket No.199235-774601 1 can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: AATTTTTGTAATGAAAAAATAGACGGCAAGGGTTATTCTTAAAACTGCAGTTTTG TAGCTTGGGTGGCATGTTAAGTGTTCTCCTTACAGTCGCAACGATGGGAAACAGA AAGTAACGTGTTATCCTCTCCGCCGCCGTGAGCTCTTTTAACACTAGCTAAGTGG CCGCAGGGCTCTTCTCTTTCCTTTCCACTTGGGGC (SEQ ID NO: 32). In some cases, a terminator sequence 2 can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: ATCATGTTTTATAAAAAAAGACTTAAAGAGGAAAACATTATGGTGCAACTTTAG GCTTAAGTGATTCATTGTCACTGTTTGTTTAAACATTGTGTAACAGAACTTGCAA AGACAGTTAACTCTTGTTTTCCATGTCAAAGGTCTGAATACTTGCATGATAAAAG TCTGTGTAACTTTCCCTGGTGACATCTGACTTGCTA (SEQ ID NO: 34). In some cases, a terminator sequence 3 can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: CTTAGTAAGTTTAAAAACAGAAAAAAAACCGTGTTGCTACAGCTATAAACTTCA AACATGCAGTTTATAGCAGTGGGCAACACGTCTCATCTCAAAAATTCAGCACTCA AACATTACACAGGAAGAGGATGTAATTTTTTAAAATGAAAGCTCTGAAGTAATTT GAGTATTCTCTGTCCTTTTTGTAAAAAAAATTACGA (SEQ ID NO: 65). In some cases, a terminator sequence 4 can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TATAAAGCTGTTAAAAAATCAGATTGACTTCATTTAGGGTGTTTCTTACAGATAT CGTTTAAGTTTTCGGTTCTGCTTGTAAACGCTTCAATCGCTCATGCGTTTAAACTT TGTGTAAGTGCAAGCGTTCTTTTTCTAGTGAAGCTTCTTCCAACAAGTTAGAAGT ATTTACTTCAGTGTCATTAATGTGAAAGATTCTT (SEQ ID NO: 66). In some cases, a terminator sequence can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TATAAAGCTGTTAAAAAATCAGATTGACTTCATTTAGGGTGTTTCTTACAGATAT CGTTTAAGTTTTCGGTTCTGCTTGTAAACGCTTCAATCGCTCATGCGTTTAAACTT TGTGTAAGTGCAAGCGTTCTTTTTCTAGTGAAGCTTCTTCCAACAAGTTAGAAGT ATTTACTTCAGTGTCATTAATGTGAAAGATTCTT (SEQ ID NO: 67). In some cases, a terminator sequence can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: TATAAAGCTGTTAAAAAATCAGATTGACTTCATTTAGGGTGTTTCTTACAGATATAttorney Docket No.199235-774601 CGTTTAAGTTTTCGGTTCTGCTTGTAAACGCTTCAATCGCTCATGCGTTTAAACTT TGTGTAAGTGCAAGCGTTCTTTTTCTAGTGAAGCTTCTTCCAACAAGTTAGAAGT ATTTACTTCAGTGTCATTAATGTGAAAGATTCTT (SEQ ID NO: 68).

[0183] In some cases, a terminator sequence is in the forward direction or the reverse direction on a polynucleotide encoding a guide RNA. G. Chemically modified guide RNAs

[0184] An engineered guide RNA as described herein for use in treating a disease or condition in a subject can comprise at least one chemical modification. In some embodiments, the engineered guide RNA can comprise at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 50, 100, or more chemical modifications. In some embodiments, the engineered guide RNA described herein may not comprise a chemical modification. In some cases, the engineered guide RNAs disclosed herein with barbell macro- footprints can be manufactured, chemically modified, and delivered directly to a subject in need thereof as RNA (without a vector, such as an AAV).

[0185] Exemplary chemical modifications comprise any one of: 5′ adenylate, 5′ guanosine- triphosphate cap, 5′ N7-Methylguanosine-triphosphate cap, 5′ triphosphate cap, 3′ phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′- 5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′deoxyribonucleoside analog purine, 2′deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble / universal bases, fluorescent dye label, 2′fluoro RNA, 2′O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′-triphosphate, 5-methylcytidine-5′- triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof.

[0186] A chemical modification can be made at any location of the engineered guide RNA. In some cases, a modification may be located in a 5’ or 3’ end, or both. In some cases, a polynucleotide can comprise a modification at a base selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,Attorney Docket No.199235-774601 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150. In some cases, more than one modification can be made to the engineered guide RNA. In some cases, a modification can be permanent. In other cases, a modification can be transient. In some cases, multiple modifications may be made to the engineered guide RNA. The engineered guide RNA modification can alter physio-chemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.

[0187] In some embodiments, a chemical modification can also be a phosphorothioate substitute. In some cases, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a polynucleic acid. A modification can also enhance biological activity. In some cases, a phosphorothioate enhanced RNA polynucleic acid can inhibit RNase A, RNase T1, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA polynucleic acids to be used in applications where exposure to nucleases may be of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5′-or 3′-end of a polynucleic acid which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases.

[0188] In some embodiments, a chemical modification can occur at 3’OΗ group, 5’OΗ group, at the backbone, at the sugar component, or at the nucleotide base. Chemical modification can include non-naturally occurring linker molecules of interstrand or intrastrand cross links. In one aspect, the chemically modified nucleic acid comprises modification of one or more of the 3’OΗ or 5’OΗ group, the backbone, the sugar component, or the nucleotide base, or addition of non-naturally occurring linker molecules. In some embodiments, a chemically modified backbone comprises a backbone other than a phosphodiester backbone. In some embodiments, a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In some embodiments, a modified base comprises a base other than adenine, guanine, cytosine, thymine or uracil. In some embodiments, the engineered guide RNA comprises at least one chemically modified base. In some instances, an engineered guideAttorney Docket No.199235-774601 RNA can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more modified bases. In some cases, chemical modifications to the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, and purine or pyrimidine bases.

[0189] In some embodiments, a chemical modification of the engineered guide RNA can comprise a modification of any one of or any combination of: modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage; modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; modification of a constituent of the ribose sugar; replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring nucleobase; modification of the ribose-phosphate backbone; modification of 5’ end of polynucleotide; modification of 3’ end of polynucleotide; modification of the deoxyribose phosphate backbone; substitution of the phosphate group; modification of the ribophosphate backbone; modifications to the sugar of a nucleotide; modifications to the base of a nucleotide; or stereopure of nucleotide. Chemical modifications to the engineered guide RNA include any modification contained herein, while some exemplary modifications are recited in Table 2. Table 2 - Exemplary Chemical ModificationAttorney Docket No.199235-774601Modification of phosphate backbone

[0190] In some embodiments, the chemical modification can comprise modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage orAttorney Docket No.199235-774601 modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage. As used herein, “alkyl” may be meant to refer to a saturated hydrocarbon group which may be straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n- pentyl, isopentyl, or neopentyl). An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. As used herein, “aryl” may refer to monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, aryl groups have from 6 to about 20 carbon atoms. As used herein, “alkenyl” may refer to an aliphatic group containing at least one double bond. As used herein, “alkynyl” may refer to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups can include ethynyl, propargyl, or 3-hexynyl. “Arylalkyl” or “aralkyl” may refer to an alkyl moiety in which an alkyl hydrogen atom may be replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2- phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups. “Cycloalkyl” may refer to a cyclic, bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. “Heterocyclyl” may refer to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. “Heteroaryl” may refer to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties can include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

[0191] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. In some embodiments, the chemically modified nucleotide can include replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Examples ofAttorney Docket No.199235-774601 modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group can be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. A phosphorous atom in a phosphate group modified in this way may be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some cases, the engineered guide RNA can comprise stereopure nucleotides comprising S conformation of phosphorothioate or R conformation of phosphorothioate. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 95%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 96%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 97%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 98%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 99%. In some embodiments, both non-bridging oxygens of phosphorodithioates can be replaced by sulfur. The phosphorus center in the phosphorodithioates can be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non- bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). In some embodiments, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). In some cases, the replacement can occur at either or both of the linking oxygens.

[0192] In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any inter nucleic acid linkage. The two main classes of interAttorney Docket No.199235-774601 nucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0193] In some cases, backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non- bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and can be used in any combination. Other non-phosphate linkages may also be used.

[0194] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and / or enhance their stability in vivo.

[0195] In some instances, a phosphorous derivative (or modified phosphate group) may be attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like.

[0196] In some cases, backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Examples of such modifications include: anionic internucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos. A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g., a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.Attorney Docket No.199235-774601

[0197] In some cases, substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts. It may be also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). It may be also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. In some cases, conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[0198] In some embodiments, a chemical modification described herein can comprise modification of a phosphate backbone. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified phosphate backbone. Exemplary chemical modification of the phosphate group or backbone can include replacing one or more of the oxygens with a different substituent. Furthermore, the modified nucleotide present in the engineered guide RNA can include the replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations resulting in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Exemplary modified phosphate groups can include, phosphorothioate, phosphonothioacetate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), CAttorney Docket No.199235-774601 (e.g., an alkyl group, an aryl group, and the like), H, NR2(wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group may be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that may be to say that a phosphorous atom in a phosphate group modified in this way may be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In such case, the chemically modified engineered guide RNA can be stereopure (e.g., S or R confirmation). In some cases, a chemically modified engineered guide RNA comprises stereopure phosphate modification. For example, the chemically modified engineered guide RNA can comprise S conformation of phosphorothioate or R conformation of phosphorothioate.

[0199] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates may be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non- bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

[0200] In some cases, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. Replacement of phosphate moiety

[0201] In some embodiments, at least one phosphate group of the engineered guide RNA can be chemically modified. In some embodiments, the phosphate group can be replaced by non- phosphorus containing connectors. In some embodiments, the phosphate moiety can be replaced by dephospho linker. In some embodiments, the charge phosphate group can be replaced by a neutral group. In some cases, the phosphate group can be replaced by methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In some embodiments, nucleotide analogs described herein can also be modified at the phosphate group. Modified phosphate group can include modification at the linkage between two nucleotides with phosphorothioate, chiral phosphorothioate,Attorney Docket No.199235-774601 phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g., 3’-amino phosphoramidate and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In some cases, the phosphate or modified phosphate linkage between two nucleotides can be through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Substitution of phosphate group

[0202] In some embodiments, a chemical modification described herein can comprise modification by replacement of a phosphate group. In some embodiments, the engineered guide RNA described herein can comprise at least one chemical modification comprising a phosphate group substitution or replacement. Exemplary phosphate group replacement can include non- phosphorus containing connectors. In some embodiments, the phosphate group substitution or replacement can include replacing charged phosphate group can by a neutral moiety. Exemplary moieties which can replace the phosphate group can include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Modification of the Ribophosphate Backbone

[0203] In some embodiments, the chemical modification described herein can comprise modifying ribophosphate backbone of the engineered guide RNA. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified ribophosphate backbone. Exemplary chemically modified ribophosphate backbone can include scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar may be replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. Modification of sugar

[0204] In some embodiments, the chemical modification described herein can comprise modifying of sugar. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified sugar. Exemplary chemically modified sugar canAttorney Docket No.199235-774601 include 2’ hydroxyl group (OH) modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2’ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2’-alkoxide ion. The 2’-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxy"-2’ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"- 2’ hydroxyl group modification can include (LNA, in which the 2’ hydroxyl can be connected, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2’ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative). In some cases, the deoxy modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-amino (wherein amino can be, e.g., as described herein),NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which can be optionally substituted with e.g., an amino as described herein. In some instances, the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the Γ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C-. The abasic sugarsAttorney Docket No.199235-774601 can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that may be in the L form, e.g., L-nucleosides. In some aspects, the engineered guide RNA described herein includes the sugar group ribose, which may be a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose may be replaced by glycol units attached to phosphodiester bonds), threose nucleic acid. In some embodiments, the modifications to the sugar of the engineered guide RNA comprises modifying the engineered guide RNA to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), or bridged nucleic acid (BNA). Modification of a constituent of the ribose sugar

[0205] In some embodiments, the engineered guide RNA described herein can comprise at least one chemical modification of a constituent of the ribose sugar. In some embodiments, the chemical modification of the constituent of the ribose sugar can include 2’-O-methyl, 2’-O- methoxy-ethyl (2’-MOE), 2’-fluoro, 2’-aminoethyl, 2’-deoxy-2’-fuloarabinou-cleic acid, 2′- deoxy, 2′-O-methyl, 3′-phosphorothioate, 3′-phosphonoacetate (PACE), or 3′- phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the constituent of the ribose sugar comprises unnatural nucleic acid. In some instances, the unnatural nucleic acids include modifications at the 5’-position and the 2’-position of the sugar ring, such as 5’-CH2-substituted 2’-O-protected nucleosides. In some cases, unnatural nucleic acids include amide linked nucleoside dimers that can be prepared for incorporation into oligonucleotides. In some cases, the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’- OCH3 and a 5’-(S)-CH3. Unnatural nucleic acids can include 2’-substituted 5’-CH2 (or O) modified nucleosides. Unnatural nucleic acids can include 5’-methylenephosphonate DNA and RNA monomers, and dimers. Unnatural nucleic acids can include 5’-phosphonate monomers having a 2’-substitution and other modified 5’-phosphonate monomers. Unnatural nucleic acids can include 5’-modified methylenephosphonate monomers. Unnatural nucleic acids canAttorney Docket No.199235-774601 include analogs of 5’ or 6’-phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and / or 6’-position. Unnatural nucleic acids can include 5’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group. Unnatural nucleic acids can include nucleosides having a 6’-phosphonate group wherein the 5’ or / and 6’-position may be unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof).

[0206] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids can contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids can comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5’ and / or 2’ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids; replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof.

[0207] In some instances, the engineered guide RNA described herein can comprise modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. The sugar can be in a pyranosyl or furanosyl form. The sugar moiety can be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’- amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy-or amino-RNA / DNA chimeras. For example, a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O- ethyleneglycol-like ribonucleosides.

[0208] In some cases, modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O- , S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl and alkynyl.2’ sugar modifications also include but are not limited to-O[(CH2)nO]m CH3,-O(CH2)nOCH3,-Attorney Docket No.199235-774601 O(CH2)nNH2,-O(CH2)nCH3,-O(CH2)nONH2, and-O(CH2)nON[(CH2)n CH3)]2, where n and m may be from 1 to about 10. Other chemical modifications at the 2’ position include but are not limited to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of the 5’ terminal nucleotide. Chemically modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Examples of nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’-O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-(C1-C1Oalkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O- N(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rmand Rnis, independently, H or substituted or unsubstituted C1-C10 alkyl.

[0209] In certain embodiments, nucleic acids described herein can include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms. In certain embodiments, nucleic acids provided herein can include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid. Examples of such 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4’-(CH2)-O-2’ (LNA); 4’-(CH2)-S-2’; 4’-(CH2)2-O-2’ (ENA); 4’-CH(CH3)-O-2’ and 4’-CH(CH2OCH3)-O-2’, and analogs thereof; 4’-C(CH3)(CH3)- O-2’ and analogs thereof. Modifications on the base of nucleotide

[0210] In some embodiments, the chemical modification described herein can comprise modification of the base of nucleotide (e.g., the nucleobase). Exemplary nucleobases can include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or replaced in the engineered guide RNA described herein. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine orAttorney Docket No.199235-774601 pyrimidine analog. In some embodiments, the nucleobase can be naturally-occurring or synthetic derivatives of a base.

[0211] In some embodiments, the chemical modification described herein can comprise modifying an uracil. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified uracil. Exemplary chemically modified uracil can include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5- aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo- uridine), 3-methyl-uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5- methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2- thio-uridine, 5-methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5- methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5- carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-pseudouridine, 5- methyl-uridine, 1 methyl-pseudouridine, 5-methyl-2-thio-uridine, l-methyl-4-thio- pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydroundine, dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl) uridine, 1-methyl-3-(3-amino-3-carboxypropy pseudouridine, 5- (isopentenylaminomethyl) uridine, 5-(isopentenylaminomethy])-2-thio-uridine, a-thio-uridine, 2’-O-methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl-pseudouridine, 2-thio-2’-O- methyl-uridine, 5-methoxycarbonylmethyl-2’-O-methyl-uridine, 5-carbamoylmethyl-2’-O- methyl-uridine, 5-carboxymethylaminomethyl-2’-O-methyl-uridine, 3,2’-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2’-O-methyl-uridine, l-thio-uridine, deoxythymidine, 2’-F-ara- uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-( l-E- propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

[0212] In some embodiments, the chemical modification described herein can comprise modifying a cytosine. In some embodiments, the engineered guide RNA described herein canAttorney Docket No.199235-774601 comprise at least one chemically modified cytosine. Exemplary chemically modified cytosine can include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl- cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5- hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l-methyl-1-deaza-pseudoisocytidine, 1-methyl-l-deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O- methyl-cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O- dimethyl-cytidine, 5-formyl-2’-O-methyl-cytidin...

Claims

1. Attorney Docket No.199235-774601 WHAT IS CLAIMED IS:

1. A recombinant AAV encapsidating a vector, wherein the vector comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat.

2. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

81.

3. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

72.

4. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

121.

5. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

122.

6. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

123.

7. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

124.

8. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

125.

9. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

126.

10. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

127.

11. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

128.

12. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO: 129.Attorney Docket No.199235-774601 13. The recombinant AAV encapsidating the vector of claim 1, wherein the sequence of the vector comprises SEQ ID NO:

130.

14. The recombinant AAV encapsidating the vector of claim 1, wherein the vector encodes an engineered guide RNA, and wherein the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

15. A recombinant AAV encapsidating a vector, wherein the vector comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO:

111.

16. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

74.

17. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

63.

18. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

102.

19. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

103.

20. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

104.

21. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

105.

22. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

106.

23. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

107.

24. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO: 109.Attorney Docket No.199235-774601 25. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

110.

26. The recombinant AAV encapsidating the vector of claim 15, wherein the sequence of the vector comprises SEQ ID NO:

111.

27. The recombinant AAV encapsidating the vector of claim 1 or claim 15, wherein the vector encodes an engineered guide RNA, wherein the engineered guide RNA, upon hybridization to a region of a target DUX4-FL RNA, forms a guide-target RNA scaffold that comprises one or more structural features.

28. The recombinant AAV encapsidating the vector of claim 27, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of DUX4 protein encoded by the target DUX4-FL RNA.

29. The recombinant AAV encapsidating the vector of claim 27, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNA or protein downstream of DUX4 is encoded by a gene comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, or MBD3L2.

30. The recombinant AAV encapsidating the vector of claim 27, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA, and wherein the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene.

31. The recombinant AAV encapsidating the vector of claim 27, wherein the target DUX4-FL RNA comprises a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA.

32. The recombinant AAV encapsidating the vector of claim 31, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence.

33. The recombinant AAV encapsidating the vector of claim 32, wherein the RNA editing entity comprises a human ADAR1, or a human ADAR2.Attorney Docket No.199235-774601 34. The recombinant AAV encapsidating the vector of claim 27, wherein the one or more structural features comprises a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof.

35. The recombinant AAV encapsidating the vector of claim 34, comprising the one or more structural features which comprise a bulge.

36. The recombinant AAV encapsidating the vector of claim 35, wherein the bulge is an asymmetric bulge.

37. The recombinant AAV encapsidating the vector of claim 35, wherein the bulge is a symmetric bulge.

38. The recombinant AAV encapsidating the vector of claim 34, comprising the one or more structural features which comprise an internal loop.

39. The recombinant AAV encapsidating the vector of claim 38, wherein the internal loop is a symmetric internal loop.

40. The recombinant AAV encapsidating the vector of claim 38, wherein the internal loop is an asymmetric internal loop.

41. The recombinant AAV encapsidating the vector of claim 34, comprising the one or more structural features which comprise a hairpin.

42. The recombinant AAV encapsidating the vector of claim 41, wherein the hairpin is a recruitment hairpin.

43. The recombinant AAV encapsidating the vector of claim 41, wherein the hairpin is a non- recruitment hairpin.

44. The recombinant AAV encapsidating the vector of claim 15, wherein the vector encodes an engineered guide RNA, and wherein the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

45. A recombinant AAV encapsidating a vector, wherein the vector comprises a sequence encoding an engineered guide RNA sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 36 – SEQ ID NO: 43, or SEQ ID NO: 137 – SEQ ID NO: 142.Attorney Docket No.199235-774601 46. The recombinant AAV encapsidating the vector of claim 45, wherein the vector further comprises a sequence encoding a SmOPT and U7 hairpin sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

31.

47. The recombinant AAV encapsidating the vector of claim 45, wherein the vector further comprises a sequence encoding a SmOPT and U5 hairpin sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

73.

48. The recombinant AAV encapsidating the vector of any one of claims 45-46, wherein the vector further comprises a terminator sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 65 – SEQ ID NO:

68.

49. The recombinant AAV encapsidating the vector of any one of claims 45-47, wherein the vector further comprises an engineered mU7 promoter sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

30.

50. The recombinant AAV encapsidating the vector of any one of claims 45-47, wherein the vector further comprises an engineered promoter sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

33.

51. The recombinant AAV encapsidating the vector of any one of claims 45-50, wherein the vector further comprises an ITR-to-ITR sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO: 111..

52. The recombinant AAV encapsidating the vector of any one of claims 45-51, wherein the recombinant AAV comprises an AAV1 virion, AAV2 virion, AAV3 virion, AAV4 virion, AAV5 virion, AAV6 virion, AAV7 virion, AAV8 virion, AAV9 virion, AAV10 virion, AAV11 virion, or a derivative, a chimera, or a variant thereof.Attorney Docket No.199235-774601 53. The recombinant AAV encapsidating the vector of any one of claims 45-52, wherein the recombinant AAV comprises a recombinant AAV (rAAV) virion, a hybrid AAV virion, a chimeric AAV virion, a self-complementary AAV (scAAV) virion, or any combination thereof.

54. A plasmid encoding an engineered guide RNA, wherein the plasmid comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat.

55. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

81.

56. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

72.

57. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

121.

58. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

122.

59. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

123.

60. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

124.

61. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

125.

62. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

126.

63. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

127.

64. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO: 128.Attorney Docket No.199235-774601 65. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

129.

66. The plasmid encoding the engineered guide RNA of claim 54, wherein the sequence of the plasmid comprises SEQ ID NO:

130.

67. The plasmid encoding the engineered guide RNA of claim 54, wherein the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

68. A plasmid encoding an engineered guide RNA, wherein the plasmid comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO:

111.

69. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

74.

70. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

63.

71. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

102.

72. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

103.

73. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

104.

74. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

105.

75. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

106.

76. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

107.

77. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO: 108.Attorney Docket No.199235-774601 78. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

109.

79. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

110.

80. The plasmid encoding the engineered guide RNA of claim 68, wherein the sequence of the plasmid comprises SEQ ID NO:

111.

81. The plasmid encoding the engineered guide RNA of claim 54 or claim 68, wherein the engineered guide RNA, upon hybridization to a region of a target DUX4-FL RNA, forms a guide-target RNA scaffold that comprises one or more structural features.

82. The plasmid encoding the engineered guide RNA of claim 81, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of DUX4 protein encoded by the target DUX4-FL RNA.

83. The plasmid encoding the engineered guide RNA of claim 81, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNA or protein downstream of DUX4 is encoded by a gene comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, MYH1 or MBD3L2.

84. The plasmid encoding the engineered guide RNA of claim 81, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA, and wherein the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene.

85. The plasmid encoding the engineered guide RNA of claim 81, wherein the target DUX4-FL RNA comprises a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA.

86. The plasmid encoding the engineered guide RNA of claim 85, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence.

87. The plasmid encoding the engineered guide RNA of claim 86, wherein the RNA editing entity comprises a human ADAR1, or a human ADAR2.Attorney Docket No.199235-774601 88. The plasmid encoding the engineered guide RNA of claim 81, wherein the one or more structural features comprises a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof.

89. The plasmid encoding the engineered guide RNA of claim 88, comprising the one or more structural features which comprise a bulge.

90. The plasmid encoding the engineered guide RNA of claim 89, wherein the bulge is an asymmetric bulge.

91. The plasmid encoding the engineered guide RNA of claim 89, wherein the bulge is a symmetric bulge.

92. The plasmid encoding the engineered guide RNA of claim 88, comprising the one or more structural features which comprise an internal loop.

93. The plasmid encoding the engineered guide RNA of claim 92, wherein the internal loop is a symmetric internal loop.

94. The plasmid encoding the engineered guide RNA of claim 92, wherein the internal loop is an asymmetric internal loop.

95. The plasmid encoding the engineered guide RNA of claim 88, comprising the one or more structural features which comprise a hairpin.

96. The plasmid encoding the engineered guide RNA of claim 95, wherein the hairpin is a recruitment hairpin.

97. The plasmid encoding the engineered guide RNA of claim 95, wherein the hairpin is a non- recruitment hairpin.

98. The plasmid encoding the engineered guide RNA of claim 68, wherein the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

99. A DNA sequence comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 81, SEQ ID NO: 72, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 69 – SEQ ID NO: 71, SEQ ID NO: 82 – SEQ ID NO: 87, SEQ ID NO: 92 – SEQ ID NO: 97, or SEQ ID NO: 123 – SEQ ID NO: 130 and an AAV inverted terminal repeat.

100. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO: 81.Attorney Docket No.199235-774601 101. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

72.

102. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

121.

103. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

122.

104. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

123.

105. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

124.

106. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

125.

107. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

126.

108. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

127.

109. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

128.

110. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

129.

111. The DNA sequence of claim 99, wherein the DNA sequence comprises SEQ ID NO:

130.

112. The DNA sequence of claim 99, wherein the DNA sequence encodes an engineered guide RNA and the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

113. A DNA sequence, wherein the DNA sequence comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 74, SEQ ID NO: 63, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 44 – SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75 – SEQ ID NO: 80, or SEQ ID NO: 104 – SEQ ID NO:

111.

114. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

74.

115. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

63.

116. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

102.

117. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

103.

118. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

104.

119. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

105.

120. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

106.

121. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

107.

122. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

108.

123. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO: 109.Attorney Docket No.199235-774601 124. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

110.

125. The DNA sequence of claim 113, wherein the DNA sequence comprises SEQ ID NO:

111.

126. The DNA sequence of claim 99 or claim 113, wherein the DNA sequence encodes an engineered guide RNA, and wherein the engineered guide RNA upon hybridization to a region of a target DUX4-FL RNA, forms a guide-target RNA scaffold that comprises one or more structural features.

127. The DNA sequence of claim 126, wherein the engineered guide RNA comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 52 – SEQ ID NO: 59, or SEQ ID NO: 131 – SEQ ID NO:

136.

128. The DNA sequence of claim 126, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of DUX4 protein encoded by the target DUX4- FL RNA.

129. The DNA sequence of claim 126, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates a knockdown of an mRNA or a protein downstream of DUX4, and wherein the mRNA or protein downstream of DUX4 is encoded by a gene comprising SLC34A2, LEUTX, ZSCAN4, PRAMEF12, TRIM43, KHDC1L, DEFB103, WFDC3, AGTR2, or MBD3L2.

130. The DNA sequence of claim 126, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates an increase in a level of expression of an mRNA or a protein downstream of DUX4, as compared to a level of expression of the mRNA or the protein downstream of DUX4 with a comparable guide RNA, and wherein the mRNA or protein downstream of DUX4 is encoded by a MYH1 gene.

131. The DNA sequence of claim 126, wherein the target DUX4-FL RNA comprises a polyA signal sequence which comprises the polynucleotide sequence of ATTAAA.

132. The DNA sequence of claim 131, wherein the engineered guide RNA when hybridized to the target DUX4-FL RNA facilitates RNA editing by an RNA editing entity of one or more adenosines in the target DUX4-FL polyA signal sequence.

133. The DNA sequence of claim 132, wherein the RNA editing entity comprises a human ADAR1, or a human ADAR2.Attorney Docket No.199235-774601 134. The DNA sequence of claim 126, wherein the one or more structural features comprises a bulge, an internal loop, a wobble base pair, a hairpin, or any combination thereof.

135. The DNA sequence of claim 134, comprising the one or more structural features which comprise a bulge.

136. The DNA sequence of claim 135, wherein the bulge is an asymmetric bulge.

137. The DNA sequence of claim 135, wherein the bulge is a symmetric bulge.

138. The DNA sequence of claim 134, comprising the one or more structural features which comprise an internal loop.

139. The DNA sequence of claim 138, wherein the internal loop is a symmetric internal loop.

140. The DNA sequence of claim 138, wherein the internal loop is an asymmetric internal loop.

141. The DNA sequence of claim 134, comprising the one or more structural features which comprise a hairpin.

142. The DNA sequence of claim 141, wherein the hairpin is a recruitment hairpin.

143. The DNA sequence of claim 141, wherein the hairpin is a non-recruitment hairpin.

144. A pharmaceutical composition comprising: a) the recombinant AAV encapsidating the vector of any one of claims 1-53, the plasmid encoding the engineered guide RNA of any one of claims 54-98, or the DNA sequence of any one of claims 99-143; and b) a pharmaceutically acceptable: excipient, carrier, or diluent.

145. A method of administering to a subject an effective amount of the recombinant AAV encapsidating the vector of any one of claims 1-53, the plasmid encoding the engineered guide RNA of any one of claims 54-98, the DNA sequence of any one of claims 99-143, or the pharmaceutical composition of claim 144.

146. The method of claim 145, wherein the subject is a mouse, a non-human primate, or a human.

147. The method of any one of claims 145-146, comprising administering the pharmaceutical composition of claim 112, and wherein the pharmaceutical composition is in unit dose form.

148. A method of treating a facioscapulohumeral muscular dystrophy (FSHD) in a subject comprising administering to a subject an effective amount of the recombinant AAV encapsidating the vector of any one of claims 1-53, the plasmid encoding the engineered guide RNA of any one of claims 54-98, the DNA sequence of any one of claims 99-143, or theAttorney Docket No.199235-774601 pharmaceutical composition of claim 144, wherein the administering treats the FSHD in the subject.

149. The method of claim 148, wherein the subject is a mouse, a non-human primate, or a human.

150. The method of any one of claims 148-149, comprising administering the pharmaceutical composition of claim 112, wherein the pharmaceutical composition is in unit dose form.

151. A method of editing an DUX4-FL RNA transcript in a subject comprising administering to a subject an effective amount of the recombinant AAV encapsidating the vector of any one of claims 1-53, the plasmid encoding the engineered guide RNA of any one of claims 54-98, the DNA sequence of any one of claims 99-143, or the pharmaceutical composition of claim 144, wherein after the administering the DUX4-FL RNA transcript is edited in the subject.

152. The method of claim 151, wherein the subject is a mouse, a non-human primate, or a human.

153. The method of any one of claims 151-152, comprising administering the pharmaceutical composition of claim 112, wherein the pharmaceutical composition is in unit dose form.

154. The method of any one of claims 151-153, wherein the editing of the DUX4-FL RNA transcript comprises editing of a polyA signal sequence.

155. The method of claim 154, wherein the editing of polyA signal sequence comprises editing of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of a total DUX4-FL transcripts in a muscle of the subject.

156. A kit comprising the recombinant AAV encapsidating the vector of any one of claims 1-53, the plasmid encoding the engineered guide RNA of any one of claims 54-98, the DNA sequence of any one of claims 99-143, or the pharmaceutical composition of claim 144 and a container.

157. The pharmaceutical composition of claim 144, wherein the pharmaceutical composition is in unit dose form.

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