Serpina1-targeting engineered guide RNAS and polynucleotides

Abstract

Disclosed herein are engineered latent guide RNAs targeting SERPINA1 and compositions comprising the same for treatment of diseases or conditions (e.g. alpha-1 antitrypsin deficiency) in a subject. Also disclosed herein are methods of treating diseases or conditions (e.g. alpha-1 antitrypsin deficiency) in a subject by administering engineered latent guide RNAs or pharmaceutical compositions described herein.

Description

INTERNATIONAL PATENT APPLICATIONSERPINA1-TARGETING ENGINEERED GUIDE RNAS AND POLYNUCLEOTIDESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No. 63 / 526,074, filed July 11, 2023, and Provisional Application Serial No: 63 / 613,136 filed December 21. 2023, the disclosures of which are incorporated herein by reference in their entirety’.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 199235-762601_PCT_SL.xml which was created on July 9, 2024, and is 1,787,475 bytes in size, is hereby incorporated by reference in its entirety.SUMMARY

[0003] Disclosed herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA comprises a polynucleotide 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: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563 - SEQ ID NO: 768, SEQ ID NO: 1424 SEQ ID NO: 1536, SEQ ID NO: 1668 SEQ ID NO: 1692, SEQ ID NO: 1718 - SEQ ID NO: 1742, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1768 - SEQ ID NO: 1792, SEQ ID NO: 1793 - SEQ ID NO: 1807. or SEQ ID NO: 1850 - SEQ ID NO: 1874. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide 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: 769 - SEQ ID NO: 1112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 - SEQ ID NO: 1421, SEQ ID NO: 1537 - SEQ ID NO: 1642, SEQ ID NO: 1643 - SEQ ID NO: 1667, SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822, or SEQ ID NO: 1825 - SEQ ID NO: 1849. In some embodiments, upon hybridization of the engineered guide RNA to a target SERPINA1 RNA sequence, the engineered guide RNA facilitates RNA editing of a target adenosine in the target SERPINA1 RNA sequence by an RNA editing entity. Insome embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity’ to SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 463. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity’ to SEQ ID NO: 518. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 518. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 518. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 518. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 1116. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 1171. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequenceidentity to SEQ ID NO: 1 171. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 1171. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity’ to SEQ ID NO: 1 171. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 1171. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 1171. In some embodiments, the target SERPINA1 RNA sequence encodes an alpha- 1 antitrypsin polypeptide having an E342K mutation. In some embodiments, the engineered guide RNA upon hybridization to the target SERPINA1 RNA sequence forms a guide-target RNA scaffold that comprises one or more structural features.

[0004] Also disclosed herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA hybridizes to a target SERPINA1 RNA sequence encoding an alpha- 1 antitrypsin (AAT) polypeptide having an E342K mutation, thereby forming a guide-target RNA scaffold with the target SERPINA1 RNA sequence, wherein formation of the guide-target RNA scaffold substantially forms one or more structural features that comprises a bulge, internal loop, wobble base pair, hairpin, a mismatch, or any combination thereof. In some embodiments, the one or more structural features comprises a bulge, wherein the bulge is a symmetric bulge. In some embodiments, the one or more structural features comprises a bulge, wherein the bulge is an asymmetric bulge. In some embodiments, the one or more structural features comprises an internal loop, wherein the internal loop is a symmetric internal loop. In some embodiments, the one or more structural features comprises an internal loop, wherein the internal loop is an asymmetric internal loop. In some embodiments, the one or more structural features comprises a Wobble base pair. In some embodiments, the one or more structural features comprises a hairpin, wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin. In some embodiments, the one or more structural features comprises two or more asymmetric bulges. In some embodiments, the two or more asymmetric bulges comprise a 3- 1 asymmetric bulge at position - 10 relative to a target adenosine at position 0, and a 2-1 asymmetric bulge at position 3 relative to a target adenosine at position 0. In some embodiments, the one or more structural features comprises two or more symmetric internal loops. In some embodiments, the two or more symmetric internal loops comprises a 6-6 symmetric internal loop at position -18 and position 32 relative to a targetadenosine at position 0. In some embodiments, the one or more structural features comprises a mismatch. In some embodiments, the mismatch comprises an A / C mismatch at position 0 relative to a target adenosine at position 0, wherein the adenosine of the A / C mismatch is the target adenosine. In some embodiments, the RNA editing entity comprises an ADAR. In some embodiments, the ADAR is AD ARI, ADAR2, or a combination thereof. In some embodiments, the engineered guide RNA facilitates restoration of wildtype AAT protein.

[0005] Also disclosed herein is a vector comprising the polynucleotide encoding any one or more of the engineered guide RNAs described herein.

[0006] Also disclosed herein is a viral particle encapsidating any one or more of the vectors described herein. In some embodiments, viral particle comprises an adeno-associated virus (AAV) virion. In some embodiments, the AAV virion is an AAV1 virion, an AAV2 virion, an AAV3 virion, an AAV4 virion, an AAV5 virion, an AAV6 virion, an AAV7 virion, an AAV8 virion, an AAV9 virion, an AAV 10 virion, an AAV11 virion, or a derivative, a chimera, or a variant thereof. In some embodiments, the AAV virion is 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 is a pharmaceutical composition comprising: (a) any one or more of the engineered guide RNA or the polynucleotide encoding the engineered guide RNA described herein, any one or more of the vector described herein, or any one or more of the viral particle described herein; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent.

[0008] Also disclosed herein is a method of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one or more of the engineered guide RNA or the polynucleotide encoding the engineered guide RNA described herein, any one or more of the vector described herein, any one or more of the viral particle described herein, or any one or more of the pharmaceutical compositions described herein. In some embodiments, the disease or condition comprises alpha-1 antitrypsin deficiency. In some embodiments, the subject has a SERPINA1 gene encoding an alpha- 1 antitry psin polypeptide having an E342K mutation (in some embodiments, also referred to herein as a SERPINA1 E342K mutation). In some embodiments, the subject has a SERPINA1 gene with a G to A mutation at nucleotide position 9989 of the SERPINA1 gene. In some embodiments, the subject is human or a non-human animal.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of embodiments of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0010] FIG. 1 shows a depiction of a first engineered guide RNA (top) that forms a single mismatch with the target SERPINA1 mRNA sequence and a second exemplary engineered guide RNA (bottom) targeting SERPINA1 mRNA, where the second engineered guide RNA forms two mismatches with the target SERPINA1 mRNA sequence. Figure discloses SEQ ID NOS: 1877-1878, respectively, in order of appearance.

[0011] FIG. 2 (left) shows that the constructs containing the U7 promoter, U7 hairpin, and the SmOPT sequences exhibited the highest levels of on-target RNA editing. FIG. 2 (right) shows that the second engineered guide RNA. which formed an A / C and A / A mismatch upon hybridization to SERPINA1 mRNA, exhibited less local off-target editing. Figure discloses SEQ ID NOS: 1879-1880, respectively, in order of appearance.

[0012] FIG. 3 (left) depicts editing of SERPINA1 mRNA with various guides as a function of guide length at 24 hours and 48 hours. FIG. 3 (right) shows the editing at the target A to be edited (“0” on the x-axis) and at RNA editing at off-target positions (represented as black bars at positions that are not “0”) at 24 hours and 48 hours with 95 nucleotide and 100 nucleotide SERPINA1 guides.

[0013] FIG. 4 (left) depicts editing of SERPINA1 mRNA with three exemplary guides. FIG. 4 (right) shows the editing at the target A to be edited (“0” on the x-axis) and at RNA editing at off-target positions (represented as black bars at positions that are not “0”) with the three exemplary SERPINA1 guides. Figure discloses SEQ ID NOS 1881-1883, respectively, in order of appearance.

[0014] FIG. 5 (left) depicts editing of SERPINA1 mRNA with various guides as a function of guide length spanning 95, 99, 103, 107, 111, 115, 119, and 123 nucleotides. FIG. 5 (top right) depicts editing of SERPINA1 mRNA with various guides of guide length of 107, 111, and 119 nucleotides, with and without introduction of a bulge. FIG. 5 (bottom right) shows the editing at the target A to be edited (“0” on the x-axis) and at RNA editing at off-target positions(represented as black bars at positions that are not “0”) for the SERPINA1 guide. Figure discloses SEQ ID NO: 1884.

[0015] FIG. 6 (right) shows a schematic of the SERPINA1 target sequence and oligo tether engineered guide RNAs. FIG. 6 (left) depicts editing of SERPINA1 mRNA with the engineered guide RNAs having oligo tethers.

[0016] FIG. 7 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 1885-1886, respectively, in order of appearance.

[0017] FIGS. 8A - 8D show editing of SERPINA1 in the minigene system with different SERP1NA1 guide constructs. FIG. 8A shows percent editing of SERPINA1 with SERP-AC- AA_95-50_-10_25 guide (SEQ ID NO: 72). FIG. SB shows percent editing of SERPINA1 with SERP-AC-AA_95-50_-8_28 guide (SEQ ID NO: 73). FIG. 8C shows percent editing of SERPINA1 with SERP-AC-AA_95-50_-10_26 guide (SEQ ID NO: 71). FIG. 8D shows percent editing of SERPINA1 with a control SERP-100.50-position -20 adenosine guide (SEQ ID NO: 74).

[0018] FIG. 9 shows results of a library screen of SERPINA1 targeting guides. The AD ARI fraction edited is depicted on the Y-axis and the specificity score is depicted on the X-axis.

[0019] FIG. 10 shows the change in body weight for subjects administered an AAV vector encoding an engineered guide RNA targeting SERPINA1 and control vector over the course of the 28-day study.

[0020] FIG. 11 illustrates transduction of an engineered guide RNA pay load in the liver after administration of an AAV vector encoding an exemplary engineered guide RNA targeting SERPINA1 and control vector, as measured by expression of an mCherry reporter.

[0021] FIG. 12 depicts normalized quantitation of an engineered guide RNA after administration of an AAV vector encoding an exemplary engineered guide RNA targeting SERPINA1 and control vector.

[0022] FIG. 13 depicts quantitation of the amount of target adenosine editing of a SERPINA1 RNA via ADAR through administration of an AAV vector encoding an engineered guide RNA targeting SERP1NA1, as compared to the level of editing of the control AAV vector.

[0023] FIG. 14 depicts tiling of the position of the barbell macro-footprint along the guidetarget RNA scaffold to identify those engineered guide RNAs that facilitate the highest level of SERPINA1 editing.

[0024] FIG. 15 shows design of the right and left barbell coordinates, as well as engineering of guide length for the exemplary engineered guide 06566 RNA.

[0025] FIG. 16 depicts in cell ADAR-mediated editing of exemplary engineered guide RNAs targeting SERPINA1 using a GFP editing reporting screen. The y-axis shows all candidate engineered guide RNAs tested, and the x-axis shows the target sequence positions, with position 0 representing the target adenosine to be edited.

[0026] FIG. 17 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 127. The target A is at position “0” on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0027] FIG. 18 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 162. The target A is at position “0"’ on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0028] FIG. 19 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 194. The target A is at position “0” on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0029] FIG. 20 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 196. The target A is at position “0"’ on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0030] FIG. 21 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 221. The target A is at position “0” on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0031] FIG. 22 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 237. The target A is at position “(T on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0032] FIG. 23 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 239. The target A is at position “0” on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0033] FIG. 24 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 288. The target A is at position “0” on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0034] FIG. 25 shows editing of SERPINA1 with a guide RNA of the present disclosure having a micro-footprint sequence of SEQ ID NO: 458. The target A is at position “0"’ on the X-axis. Each dash on the X-axis indicates a base in the target RNA and its relative location to the target A position. Percent editing is indicated on the Y-axis.

[0035] FIG. 26 shows a heatmap of secondary structures formed with the target and guide RNA of the top 100 micro-footprint guide RNA designs. The designs have secondary structures which include variations of symmetric and asymmetric features appearing both on the target and guide side. The legend indicates a specific feature of the micro-footprint guide RNA. The first number indicates the feature on the target RNA. while the second number indicates the feature on the guide RNA. For example, a 3-3 feature indicates a 3-3 symmetric bulge, and a 2-1 feature indicates 2-1 asymmetric bulge (2 nucleotides in the target RNA and 1 nucleotide in the guide RNA). The target position of the SERPINA1 target is indicated on the X-axis with positionL‘0” being the target A.

[0036] FIG. 27 shows a graph of the editing efficiency and specificity of the top 100 microfootprint guide RNAs. The X-axis shows the guide RNAs. The Y-axis shows the fraction editedof on-target and off-target edits. The darker plotted points show the on-target fraction edited, while the lighter shade points indicate the off-target fraction edited.

[0037] FIG. 28 shows a heat map of the editing across the SERPINA1 target RNA. The Y-axis shows the guide RNAs. The X-axis shows the target SERPINA1 sequence relative to the target A at position 0. A darker shade indicates a high editing fraction at a position, while a lighter shade indicates a low editing fraction.

[0038] FIG. 29 shows engineered guide RNA designs of the present disclosure that exhibit high on-target RNA editing and limited RNA editing at the +1 off-target position.

[0039] FIGS. 30A-30D show in cell guide editing profiles. FIG. 30A shows in cell guide editing profiles of SEQ ID NO: 463, SEQ ID NO: 482, and SEQ ID NO: 507. FIG. 30B shows in cell guide editing profiles of SEQ ID NO: 473, SEQ ID NO: 492, and SEQ ID NO: 518. FIG. 30C shows in cell guide editing profiles of SEQ ID NO: 481, SEQ ID NO: 502, and SEQ ID NO: 561. FIG. 30D shows in cell guide editing profiles of SEQ ID NO: 555, SEQ ID NO: 556 and SEQ ID NO: 562, which were control guide sequences.

[0040] FIGS. 31A-31C show improved specificity of SERPINA1 editing (e.g.. reduced -2 and +1 position editing) with machine learning (ML) gRNAs as compared to gRNAs identified in a high throughput screen (HTS). FIG. 31A shows a percent editing graph of the SERPINA1 target. The graphs show 3 HTS gRNAs paired with their modified ML counterpart gRNAs. The top graph shows ML modified gRNA of SEQ ID NO: 1695 and HTS gRNA of SEQ ID NO 1 123; the middle graph shows ML modified gRNA of SEQ ID NO: 1697 and HTS gRNA of SEQ ID NO: 1134; the bottom graph shows ML modified gRNA of SEQ ID NO: 1700 (bottom) and the HTS gRNA of SEQ ID NO: 1137. FIG. 31B shows target position editing as compared to editing at the -2 position of HTS gRNAs and their progeny ML gRNAs. FIG. 31C shows target position editing as compared to editing at the +1 position of HTS gRNAs and their progeny ML gRNAs.

[0041] FIG. 32 shows a sequence logo (top of figure) of the per-base gRNA contributions towards -2 off-target editing in a SERPINA1 target RNA. An upright nucleotide indicates a base associated with positive contribution to the model's prediction on -2 off-target editing and a upside down nucleotide indicates a base with negative contribution to the model's prediction on -2 off-target editing. The guide target scaffold (bottom of figure) shows nucleotides that correlate positively or negatively to -2 off-target editing. Figure discloses SEQ ID NO: 1887.

[0042] FIG. 33 shows a heat map of adenosine editing across the SERPINA1 target RNA with the 22 HTS gRNAs paired with their progeny ML gRNAs. The position of the adenosines is shown on the X-axis, 0 indicates the target position, a “+” position indicates a region downstream of the target position, and aposition indicates a region upstream of the target position. The paired guides are shown on the Y -axis. A lighter color indicates increased editing at the indicated position.

[0043] FIGS. 34A-34C show in cell guide editing profdes of HTS gRNAs and their progeny ML gRNAs. FIG. 34A shows in cell guide editing profdes of HTS gRNA SEQ ID NO: 1123, and ML progeny gRNA SEQ ID NO: 1695. FIG. 34B shows in cell guide editing profdes of HTS gRNA SEQ ID NO: 1136, and ML progeny gRNA SEQ ID NO: 1699. FIG. 34C shows in cell guide editing profdes of HTS gRNA SEQ ID NO: 1200, and ML progeny gRNA SEQ ID NO: 1712.

[0044] FIGS. 35A-35B show improved specificity of SERPINA1 editing (e.g., reduced -2 and +1 position editing) with machine learning (ML) gRNAs as compared to gRNAs identified in a high throughput screen (HTS). FIG. 35A shows target position editing as compared to editing at the -2 position of HTS gRNAs (VI) and their progeny ML gRNAs (V2 and V3). FIG. 35B shows target position editing as compared to editing at the +1 position of HTS gRNAs (VI) and their progeny ML gRNAs (V2 and V3).

[0045] FIG. 36 shows a heat map of adenosine editing across the SERPINA1 target RNA with the 23 HTS gRNAs paired with their progeny ML gRNAs. The position of the adenosines is shown on the X-axis, 0 indicates the target position, a “+” position indicates a region downstream of the target position, and aposition indicates a region upstream of the target position. The paired guides are shown on the Y -axis. A lighter color indicates increased editing at the indicated position.

[0046] FIG. 37 shows a graph of the on target editing efficiency of guide RNAs (SEQ ID NO: 518, 463, 1720, and 561) to the SERPINA1 target RNA tested in HepG2 cells. The Y-axis shows the percent editing efficiency, and the X-axis shows the guide RNAs and controls.

[0047] FIG. 38 show s a sequence alignment of DNA sequences (SEQ ID NOS 1808 and 1809- 1822) encoding engineered guide RNAs (SEQ ID NO: 1793 and SEQ ID NO: 1794 - 1807) that target the SERPINA1 3’ UTR that have 12 to 30 nucleotide substitutions.

[0048] FIGS. 39A-39B shows percent RNA editing in engineered guide RNAs (SEQ ID NO: 1793 - 1807) targeting SERPINA1 3’ UTR. FIG. 39A shows total RNA editing for theSERPINA1 3’ UTR. The parental engineered guide RNA (PO), the progeny engineered guide RNAs (P12 to P30), and control (structurally diverse, GFP plasmid, and no transfection) are shown on the X-axis. The Y-axis shows percent RNA editing. FIG. 39B shows on target editing (target position 0) as compared to off-target editing in guide RNAs. FIG. 39C shows a heat map of the editing across the SERPINA1 3’ UTR target RNA. The Y-axis shows the guide RNAs. The X-axis shows the target SERPINA1 3’ UTR sequence relative to the target A at position 0. A darker shade indicates a high editing fraction at a position, while a lighter shade indicates a low editing fraction.

[0049] FIG. 40 shows percent RNA editing in engineered guide RNAs (SEQ ID NO: 1793 - 1807) targeting the SERPINA1 3’ UTR is associated with free energy of the binding between target and guide RNAs. FIG. 40 shows total RNA editing for the SERPINA1 3’ UTR target RNA. The parental engineered guide RNA (P0), the progeny engineered guide RNAs (Pl 2 to P30), and control (structurally diverse) are shown on the X-axis. The Y-axis shows percent RNA editing and free energy of ensemble in kcal / mol.

[0050] FIG. 41A-41C show RNA editing at SERPINA1 3‘ UTR target sites facilitated by engineered guide RNAs (SEQ ID NOS: 1875-1879). FIG. 41A shows percent RNA editing at 3 different SERPINA1 3’ UTR target sites (Target 1, Target 2, Target 3) facilitated by engineered guide RNAs (SEQ ID NOS: 1875-1879). FIG. 41B shows on target editing (target position 0) at a 3’ UTR target site corresponding to Target 1, as compared to off-target editing facilitated by SEQ ID NO: 1875. FIG. 41C is a schematic demonstrating 3 'UTR Target 1. FIG. 41C discloses SEQ ID NO: 1888.

[0051] FIG. 42A-42C show RNA editing facilitated by an engineered guide RNA of SEQ ID NO: 1875 inserted into different DNA vectors and administered at different vector genome concentrations. FIG. 42A show RNA editing using the engineered guide RNA of SEQ ID NO: 1875 in various vectors administered at concentrations of 500K or 100K vector genomes per cell to primary hepatocytes isolated from NSG-PiZ mice HepG2-6B5 cells. FIG. 42B show SERPINA1 3’UTR editing using the engineered guide RNA of SEQ ID NO: 1875 at concentrations of 500K, 100K, 50K, and 10K vector genomes per cell in NSG-PiZ primary hepatocytes. FIG. 42C shows on target editing (target position 0) as compared to off-target editing facilitated by SEQ ID NO: 1875 at a concentration of 500K vector genomes per cell in NSG-PiZ primary hepatocytes.

[0052] FIG. 43A-43B show RNA editing facilitated by engineered guide RNAs (SEQ ID NO: 473, 481, 482, 491, 502, 507, 555, 556, 561, and 562). FIG. 43A compares editing in HEK656 cells of SERPINA1 at a target site encoding an E342K mutation or at a +1 off-target site facilitated by engineered guide RNAs. FIG. 43B shows on target editing (target position 0, corresponding to the SERPINA1 E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 518 in HEK656 cells.

[0053] FIG. 44A-44B show RNA editing in HepG2 cells facilitated by engineered guide RNAs encoded by plasmids. FIG. 44A compares editing in HepG2 cells of SERPINA1 at a target site encoding an E342K mutation or at a +1 off-target site facilitated by engineered guide RNAs, including SEQ ID NO: 473, 481, 482, 491, 502, 507, 555, 556, 561, and 562identified from the HEK screen shown in FIG. 43A (circled triangular points from FIG. 43A). FIG. 44B shows on target editing (target position 0, corresponding to the SERPINA1 E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 518 in HepG2 6B5 cells.

[0054] FIG. 45A-45B show RNA editing of SERPINA1 at a target site encoding an E342K mutation facilitated by engineered guide RNA set forth in SEQ ID NO: 518 delivered via an AAV-DJ viral vector. FIG. 45A shows on target editing (target position 0, corresponding to the E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 518 in HEK656 cells. FIG. 45B shows on target editing (target position 0, corresponding to the E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 518 in HepG2 6B5 cells.

[0055] FIG. 46A-46B show RNA editing of SERPINA1 at a target site encoding an E342K mutation facilitated by engineered guide RNA set forth in SEQ ID NO: 463 encoded by a plasmid. FIG. 46A shows on target editing (target position 0, corresponding to the E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 463 in HEK656 cells. FIG. 46B shows on target editing (target position 0, corresponding to the E342K mutation) as compared to off-target editing facilitated by an engineered guide RNA of the sequence in SEQ ID NO: 463 in HepG2 6B5 cells.DETAILED DESCRIPTIONRNA Editing

[0056] RNA editing can refer to a process by which RNA can be 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 introduce mutations, correct missense mutations, or edit coding or non-coding regions of RNA to inhibit RNA translation and effect protein knockdown.

[0057] Described herein are engineered guide RNAs that facilitate RNA editing of a SERPINA1 RNA by an RNA editing entity (e.g., an adenosine Deaminase Acting on RNA (ADAR)) or biologically active fragments thereof. 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.Engineered Guide RNAs

[0058] Disclosed herein are engineered guide RNAs and engineered polynucleotides encoding the same for site-specific, selective editing of a SERPINA1 target RNA via an RNA editing entity or a biologically active fragment thereof. An engineered guide RNA of the present disclosure can comprise latent structures, such that when the engineered guide RNA is hybridized to the SERPINA1 target RNA to form a guide-target RNA scaffold, at least a portion of the latent structure manifests as at least a portion of a structural feature as described herein. In some cases, an engineered guide RNA herein can comprise a micro-footprint comprising latent structures, a macro-footprint comprising latent structures, or both.

[0059] An engineered guide RNA as described herein comprises a targeting domain with complementarity to a SERPINAl target RNA (e.g., a target SERPINA1 RNA sequence) 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 SERPINAl target RNA (e.g., a target SERPINAl RNA sequence) 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 SERPINAl target RNA(e.g., a target SERPINA1 RNA sequence), 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.

[0060] Hybridization of the SERP1NA1 target RNA (e.g.. a target SERP1NA1 RNA sequence) and the targeting domain of the guide RNA produces specific secondary structures in the guidetarget 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 SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence) 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 a 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 SERPINA1 target RNA (e g., a target SERPINA1 RNA sequence) with high specificity, selectivity, and robust activity.

[0061] Provided herein are engineered guide RNAs 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.

[0062] The present disclosure, in some embodiments, provides engineered guide RNAs that facilitate edits at multiple adenosines. Hydrolytic deamination of multiple adenosines in an RNA can be referred to as hyper-editing. In some cases, hyper-editing can occur in cis (e.g. in an Alu element) or in trans (e.g. in a target RNA by an engineered guide RNA). In some cases, hyper-editing can introduce edits in at least 2 or more nucleotides of a subject target RNA. In some cases, hyper-editing can introduce at least or at most about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52. 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or at least or at most about 100 edits in a region of a target RNA. In an embodiment, hyper-editing can occur in an untranslated region, translated region, 3’UTR, 5’UTR, or any combinations thereof.

[0063] 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 a SERPINA1 target RNA molecule (e.g., a target SERPINA1 RNA sequence), 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 SERPINA1 target RNA molecule (e.g., a target SERPINA1 RNA sequence) by the RNA editing entity7.

[0064] In some examples, a SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence) 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 SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence). In some embodiments, part of the engineered guide RNA (e.g., a targeting domain) hybridizes to the sequence of the SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence). 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. In some cases, administration of the engineered guide RNAs disclosed herein restore expression and / or enzymatic function of a wildtype Alpha- 1 antitry psin (AAT) protein, as compared to an inactive or defective AAT protein in a subject with an AAT deficiency.

[0065] In some embodiments, an engineered guide RNA can comprise a barbell macrofootprint. 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 endof 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. 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.

[0066] 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 / C 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.

[0067] 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, 12-1 15, 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, 1 15, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 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.

[0068] 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 editingentity (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.

[0069] 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.

[0070] 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 or focus 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 structural features 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 microfootprint 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.

[0071] 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 microfootprint 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.

[0072] 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.

[0073] In some embodiments, a macro-footprint sequence can comprise a barbell macrofootprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop.

[0074] 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 considered “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%, about25%, 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%.

[0075] 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.

[0076] 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.

[0077] As described herein, a double stranded RNA (dsRNA) substrate (e.g.. 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 symmetric internal loop of the present disclosure can be designated as a 5 / 5, 6 / 6, 7 / 7, 8 / 8, 9 / 9, 10 / 10, 11 / 11, 12 / 12, 13 / 13, 14 / 14, 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 20 / 20 symmetric internal loop, where the first number is the number of nucleotides contributed to the symmetric internal loop from the engineered guide RNA side of the guide-target RNA scaffold and the second number is the number of nucleotides contributed to the symmetric internal loop from 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 6nucleotides 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 nucleotides 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 guidetarget 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 guidetarget 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 guidetarget RNA scaffold target and 80 nucleotides on the target RNA side of the guide-target RNAscaffold. 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 guidetarget RNA scaffold. A symmetrical internal loop of the present 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 guidetarget 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 guidetarget 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 guidetarget RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by450 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 guidetarget RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered polynucleotide side of the 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 guidetarget 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.

[0078] As described herein, a double stranded RNA (dsRNA) substrate (e.g., 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.

[0079] An asymmetric internal loop of the present disclosure can be designated as a 5 / 6, 5 / 7, 5 / 8, 5 / 9, 5 / 10, 5 / 11, 5 / 12, 5 / 13, 5 / 14, 5 / 15, 5 / 16, 5 / 17, 5 / 18, 5 / 19, 5 / 20, 6 / 5, 6 / 7, 6 / 8, 6 / 9, 6 / 10, 6 / 11, 6 / 12, 6 / 13, 6 / 14, 6 / 15, 6 / 16, 6 / 17, 6 / 18, 6 / 19, 6 / 20, 7 / 5, 7 / 6, 7 / 8, 7 / 9, 7 / 10, 7 / 11, 7 / 12,7 / 13, 7 / 14, 7 / 15, 7 / 16, 7 / 17, 7 / 18, 7 / 19, 7 / 20, 8 / 5, 8 / 6, 8 / 7, 8 / 9, 8 / 10, 8 / 11, 8 / 12, 8 / 13, 8 / 14,8 / 15, 8 / 16, 8 / 17, 8 / 18, 8 / 19, 8 / 20. 9 / 5, 9 / 6, 9 / 7, 9 / 8, 9 / 10, 9 / 11, 9 / 12, 9 / 13, 9 / 14, 9 / 15, 9 / 16,9 / 17, 9 / 18, 9 / 19, 9 / 20, 10 / 5, 10 / 6, 10 / 7, 10 / 8, 10 / 9, 10 / 11, 10 / 12, 10 / 13, 10 / 14, 10 / 15, 10 / 16, 10 / 17, 10 / 18, 10 / 19, 10 / 20, 11 / 5, 11 / 6, 11 / 7, 11 / 8, 11 / 9, 11 / 10, 11 / 12, 11 / 13, 11 / 14, 11 / 15, 11 / 16, 11 / 17, 11 / 18, 11 / 19, 11 / 20, 12 / 5, 12 / 6, 12 / 7, 12 / 8, 12 / 9, 12 / 10, 12 / 11, 12 / 13, 12 / 14,12 / 15, 12 / 16, 12 / 17. 12 / 18, 12 / 19, 12 / 20, 13 / 5, 13 / 6, 13 / 7, 13 / 8, 13 / 9, 13 / 10, 13 / 11. 13 / 12,13 / 14, 13 / 15, 13 / 16, 13 / 17, 13 / 18, 13 / 19, 13 / 20, 14 / 5, 14 / 6, 14 / 7, 14 / 8, 14 / 9, 14 / 10, 14 / 11,14 / 12, 14 / 13, 14 / 15, 14 / 16, 14 / 17, 14 / 18, 14 / 19, 14 / 20, 15 / 5, 15 / 6, 15 / 7, 15 / 8, 15 / 9, 15 / 10,15 / 11, 15 / 12, 15 / 13, 15 / 14, 15 / 16, 15 / 17, 15 / 18, 15 / 19, 15 / 20, 16 / 5, 16 / 6, 16 / 7, 16 / 8, 16 / 9,16 / 10, 16 / 11, 16 / 12, 16 / 13, 16 / 14, 16 / 15, 16 / 17, 16 / 18, 16 / 19, 16 / 20, 17 / 5, 17 / 6, 17 / 7, 17 / 8,17 / 9, 17 / 10, 17 / 11, 17 / 12, 17 / 13, 17 / 14, 17 / 15, 17 / 16, 17 / 18, 17 / 19, 17 / 20, 18 / 5, 18 / 6, 18 / 7, 18 / 8, 18 / 9, 18 / 10, 18 / 11, 18 / 12, 18 / 13, 18 / 14, 18 / 15, 18 / 16, 18 / 17, 18 / 19, 18 / 20, 19 / 5, 19 / 6, 19 / 7, 19 / 8, 19 / 9, 19 / 10, 19 / 11, 19 / 12, 19 / 13, 19 / 14, 19 / 15, 19 / 16, 19 / 17, 19 / 18, 19 / 20, 20 / 5, 20 / 6, 20 / 7, 20 / 8, 20 / 9. 20 / 10, 20 / 11, 20 / 12, 20 / 13, 20 / 14, 20 / 15, 20 / 16, 20 / 17, 20 / 18, 20 / 19 asymmetric internal loop, the first number is the number of nucleotides contributed to the asymmetric internal loop from the engineered guide RNA side of the guide-target RNA scaffold and the second number is the number of nucleotides contributed to the asymmetric internal loop from 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 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-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 targetRNA 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 guidetarget 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 guidetarget 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-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-targetRNA 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 guidetarget 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 guidetarget 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 asymmetncal internal loop of the present disclosure can be 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 presentdisclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guidetarget 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 guidetarget RNA scaffold and 50 nucleotides on the engineered polynucleotide side of the guidetarget 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 5 nucleotides on the engineered polynucleotide side of the guide-target RNAscaffold. 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 polynucleotide 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 polynucleotide 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 polynucleotide side of the guide-target RNA scaffold. An asymmetncal 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 50nucleotides on the engineered polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide side of the guide-target RNA scaffold. Anasymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 guidetarget RNA scaffold and 200 nucleotides on the engineered polynucleotide side of the guidetarget 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 onthe engineered polynucleotide 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 300 nucleotides on the engineered polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide 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 guidetarget RNA scaffold and 500 nucleotides on the engineered polynucleotide side of the guidetarget RNA scaffold. Thus, an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.

[0080] In some embodiments, a first internal loop or a second internal loop can independently comprise 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, 1 10, 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 anumber 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, 12-1 15, 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.

[0081] In some embodiments, an engineered guide RNA comprising a barbell macro-footprint (e.g, a latent structure that manifests as a first internal loop and a second internal loop) comprises a cytosine in a micro-footprint sequence in between the macro-footprint sequence that, when the engineered guide RNA is hybridized to the target RNA, is present in the guidetarget RNA scaffold opposite an adenosine that is edited by the RNA editing entity (e.g, an on-target adenosine). In such embodiments, the cytosine of the micro-footprint is comprised in an A / C mismatch with the on-target adenosine of the target RNA in the guide-target RNA scaffold.

[0082] A first internal loop and a second internal loop of the barbell macro-footprint can be positioned a certain distance from the A / C mismatch, with respect to the base of the first internal loop and the base of the second internal loop that is the most proximal to the A / C mismatch. In some embodiments, the first internal loop and the second internal loop can be positioned the same number of bases from the A / C mismatch, with respect to the base of the first internal loop and the base of the second internal loop that is the most proximal to the A / C mismatch. In some embodiments, the first internal loop and the second internal loop can be positioned a different number of bases from the A / C mismatch, with respect to the base of the first internal loop and the base of the second internal loop that is the most proximal to the A / C mismatch.

[0083] In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell can be 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 / C mismatch. In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell can be 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.

[0084] In some embodiments, the first internal loop can be positioned from about 1 base away from the A / C mismatch to about 30 bases away from the A / C mismatch (e.g. , 1 -20, 7-30, 5-20,6-20. 5-15) with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned from about 5 bases away from the A / C mismatch to about 15 bases away from the A / C mismatch (e.g., 5-15, 6-14,7-13, 8-12, 9-11) with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some examples, the first internal loop can be positioned from about 9 bases away from the A / C mismatch to about 15 bases away from the A / C mismatch (e.g., 10-14, 11- 13) with respect to the base of the first internal loop that is the most proximal to the A / C mismatch.

[0085] In some embodiments, the second internal loop can be positioned from about 12 bases away from the A / C mismatch to about 40 bases away from the A / C mismatch (e.g.. 13-39, 14- 38. 15-40, 15-38, 15-37. 16-36, 17-35, 18-38, 18-34. 19-33, 20-32, 21-31, 22-30. 23-29, 24- 28, 25-27) with respect to the base of the second internal loop that is the most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned from about 20 bases away from the A / C mismatch to about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned 24, 26, 28, or 30 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned al least about 1 base away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 1 base away from the on-target adenosine of the target RNA to about 30 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 1 base away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned at least about 5 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 15 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned at least about 6 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 6 bases away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned at least about 7 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 7 bases away from the on-target adenosine of the target RNA to about 30 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 9 bases away from the on-target adenosine of the target RNA to about 15 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 6 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 10 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned at least about 12 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned at least about 15 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA to about 38 bases aw ay from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned at least about 18 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 18 bases away from the on-target adenosine of the target RNA to about 38 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 18 bases away from the on-target adenosine of the target RNA to about 35 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 20 bases away from the on-target adenosine of the target RNA to about 33 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 24bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 33 bases away from the on-target adenosine of the target RNA. In some embodiments, the second internal loop can be positioned about 34 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 7 bases away from the on-target adenosine of the target RNA to about 30 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 18 bases away from the on-target adenosine of the target RNA to about 34 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 1 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 6 bases away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA to about 38 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 18 bases away from the on-target adenosine of the target RNA to about 38 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 15 bases away from the on- target adenosine of the target RNA and the second internal loop can be positioned about 18 bases away from the on-target adenosine of the target RNA to about 38 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 1 base away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In someembodiments, the first internal loop can be positioned about 5 bases away from the on-target adenosine of the target RNA to about 20 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA to about 40 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 10 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 34 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 15 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 33 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 6 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 34 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 34 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 6 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 33 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 12 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 24 bases away from the on-target adenosine of the target RNA. In some embodiments, the first internal loop can be positioned about 10 bases away from the on-target adenosine of the target RNA and the second internal loop can be positioned about 33 bases away from the on-target adenosine of the target RNA. In some embodiments, the engineered guide RNA can comprise a cytosine that, when the engineered guide RNA is hybridized to the target RNA, is present in the guide-target RNA scaffold opposite the on-target adenosine that is edited by the RNA editing entity, thereby forming an A / C mismatch in the double stranded RNA substrate. In some embodiments, the first internal loop and the second internal loop can be positioned the same number of bases from the A / C mismatch with respect to the base of the first internal loop and the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned at least about 1 base away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 1 base away from the A / C mismatch to about 20 bases away from theA / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned at least about 5 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 5 bases away from the A / C mismatch to about 20 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 5 bases away from the A / C mismatch to about 15 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned at least about 6 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 6 bases away from the A / C mismatch to about 20 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned at least about 7 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 7 bases away from the A / C mismatch to about 30 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 9 bases away from the A / C mismatch to about 15 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned at least about 12 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 12 bases away from the A / C mismatch to about 40 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned at least about 15 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 15 bases away from the A / C mismatch to about 40 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 15 bases away from the A / C mismatch to about 38 bases away from the A / Cmismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned at least about 18 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 18 bases away from the A / C mismatch to about 38 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 18 bases away from the A / C mismatch to about 35 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 20 bases away from the A / C mismatch to about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 24 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the second internal loop can be positioned about 34 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 10 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 34 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 15 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 6 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 34 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 12 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and thesecond internal loop can be positioned about 34 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 6 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 12 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 24 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch. In some embodiments, the first internal loop can be positioned about 10 bases away from the A / C mismatch with respect to the base of the first internal loop that is most proximal to the A / C mismatch and the second internal loop can be positioned about 33 bases away from the A / C mismatch with respect to the base of the second internal loop that is most proximal to the A / C mismatch.A. Targeting Domain

[0086] Engineered guide RNAs useful for facilitating editing of a SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence) as 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. A targeting sequence can also be referred to as a “targeting domain” or a “targeting region”.

[0087] 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. I l l, 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.

[0088] 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.B. Engineered Guide RNAs Having a Recruiting Domain

[0089] In some examples, an engineered guide RNA useful for facilitating editing of a SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence) as described herein 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 SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence). A “recruiting domain” can be referred to herein as a “recruiting sequence” or a “recruiting region”. In some examples, an engineered guide RNA can be configured to facilitate editing of a base of a nucleotide of a polynucleotide of a region of a SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence), modulation expression of a polypeptide encoded by the SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence), or both. In some cases, an engineered guide RNA 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. In order to facilitate editing, an engineered guide RNA of the disclosure can be configured to recruit an RNA editing entity. Some embodimentsprovide for an RNA editing entity comprising an ADAR protein, where the ADAR protein can be selected from the group consisting of an AD ARI (e.g.. human or mouse), an ADAR2 (e.g., human or mouse), and any combination thereof. Various RNA editing entity recruiting domains can be utilized. In some examples, a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2) or Alu. In some embodiments of the disclosure, the RNA editing entity can have an ADAR protein. An ADAR protein can be selected from the group consisting of: an AD ARI, an ADAR2, and a combination of AD ARI and ADAR2. Other embodiments can be directed to an RNA editing entity selected from the group consisting of: a human AD ARI , a mouse ADAR1 , a human ADAR2, a mouse ADAR2, and any combination thereof.

[0090] In some examples, more than one recruiting domain can be included in an engineered guide RNA 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 target RNA after the targeting sequence, for example an antisense sequence, hybridizes to a target RNA. In some cases, a recruiting domain can allow for transient binding of the RNA editing entity' to the engineered guide RNA. In some examples, the recruiting domain allows for permanent binding of the RNA editing entity' to the engineered guide RNA. 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.

[0091] 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 orportion 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 preformed 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.

[0092] 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.

[0093] 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: 1). In some cases, a recruiting domain can comprise at least about 80% sequence homology to at least about 10, 15, 20, 25, or 30 nucleotides of SEQ ID NO: 1. In some examples, a recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence homology and / or length to SEQ ID NO: 1.

[0094] Any number of recruiting domains can be found in an engineered RNA 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 RNA. Recruiting domains can be located at any position of an engineered guide RNA. In some cases, a recruiting domain can be on an N- terminus, middle, or C-terminus of a polynucleotide. A recruiting domain can be upstream or downstream of a targeting sequence. In some cases, a recruiting domain flanks a targeting sequence of a 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

[0095] In some examples, an engineered guides disclosed herein useful for facilitating editing of a SERPINA1 target RNA (e.g., a target SERPINA1 RNA sequence) via an RNA editingentity can be an engineered latent guide RNA. A '‘micro-footprint sequence” of a guide RNA comprising latent structures can comprise a portion of sequence that, upon hybridization to a target RNA, forms at least a portion of a structural feature, other than a single A / C mismatch feature at the target adenosine to be edited. 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.

[0096] A double stranded RNA (dsRNA) substrate is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. The resulting dsRNA substrate is also referred to herein as a “guide-target RNA scaffold.”

[0097] FIG. 7 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 RNAside 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.

[0098] In some instances, an engineered latent guide RNA lacks a recruiting domain, and recruitment of the RNA editing entity can be effectuated by structural features of the guidetarget RNA scaffold formed by hybridization of the engineered guide RNA and the target RNA. 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 latent guide RNA, upon hybridization to a target RNA, form with the target RNA molecule one or more structural features present in the guide-target RNA scaffold that recruits an RNA editing entity (e.g., ADAR).

[0099] In some embodiments, provided herein are engineered guide RNAs comprising a macro-footprint and a micro-footprint, where the macro-footprint includes barbells (or internal loops) near the 5' and 3’ ends of the guide-target RNA scaffold and the micro-footprint includes other structural features including, but not limited to, mismatches, symmetric internal loops, asymmetric internal loops, symmetric bulges, or asymmetric bulges. 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).

[0100] 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 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. 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.

[0101] In some embodiments, a mismatch positioned 5’ of the edit site can facilitate baseflipping 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.

[0102] 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.

[0103] 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 canbe 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 of the engineered guide RNAs of the present disclosure, or any combination thereof.

[0104] In some aspects, a structural feature comprises a non-recruitment hairpin. A nonrecruitment 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 °C 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.

[0105] 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, 1 10, 1 1 1 , 112, 1 13, 1 14, 115, 1 16, 1 17, 1 18, 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, 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.

[0106] 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 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 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 guideRNA 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 guidetarget RNA scaffold of the present disclosure has 2 bulges. In some embodiments, the guidetarget RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guidetarget 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.

[0107] 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.

[0108] 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 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 guidetarget 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-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.

[0109] 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. 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 guidetarget 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 guidetarget 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 disclosure 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 RNAscaffold. 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 guidetarget 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.

[0110] 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 guidetarget 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 isno 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.

[0111] 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 therebetween. 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 by 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 by800 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.

[0112] 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 guidetarget 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 nucleotides 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA side of the guide-target RNA scaffold target and 40 nucleotides on the target RNA sideof the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the engineered guide RNA 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 guide RNA 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 guide RNA side of the guidetarget 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA 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 present disclosure can be formed by 120 nucleotides on the engineered guide RNA 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA side of the guidetarget 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 guide RNA side of the guide-target RNA scaffold target and 250nucleotides 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA 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 guide RNA side of the guidetarget 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 guide RNA side of the 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 guide RNA 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 guide RNA 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 guide RNA 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.

[0113] 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 asy mmetrical 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-targetRNA 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.

[0114] An asymmetrical internal loop of the present disclosure can be formed by from 5 to 150 nucleotides on the engineered guide RNA 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 guide RNA 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-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 presentdisclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guidetarget 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 guidetarget 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-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 guidetarget 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 asymmetncal 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 be 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 guidetarget 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 guidetarget 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 guidetarget 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 onthe 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 guidetarget 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 guidetarget 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 guidetarget 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 present 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 guidetarget 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 guidetarget 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 guidetarget 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 guidetarget 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 guidetarget 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 be 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 guidetarget 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 guidetarget RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-targetRNA 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 guidetarget 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 guidetarget 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 be 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 guidetarget 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 guideRNA 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 guidetarget 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 guidetarget 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 present 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 guidetarget 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 guidetarget 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 guidetarget 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 guidetarget 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 guidetarget 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 be 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 guideRNA 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.

[0115] 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 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 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, 1 16, 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.D. Engineered Polynucleotides Encoding Engineered Guide RNAs

[0116] 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.

[0117] 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.

[0118] 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).

[0119] 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 identity7, at least about 90% to about 95% sequence identity7, at least about 92% to about 99% sequence identity, or at least about 95% to about 99% sequence identity7to a second engineered guide RNA encoded by7the 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 identity7of 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 identity7of less than, greater 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 identity7of 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 identity7of less than, greater than, or equal to about 64% to a second engineered guideRNA 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 identity 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 toabout 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 polynucleotide 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 identity7of 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 RNA 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 asequence 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 first 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 anengineered 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.E. Additional Engineered Guide RNA Components

[0120] Also disclosed herein are 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.

[0121] 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 from any one of SEQ ID NOS: 2 - 62, SEQ ID NOS: 65 -75, SEQ ID NO: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563 - SEQ ID NO: 768. SEQ ID NO: 1424 - SEQ ID NO: 1536, SEQ ID NO: 1668 - SEQ ID NO: 1692. SEQ ID NO: 1718 - SEQ ID NO: 1742, SEQ ID NO: 1768 - SEQ ID NO: 1792, SEQ ID NO: 1793 - SEQ ID NO: 1807, SEQ ID NO:1850 - SEQ ID NO: 1875, or SEQ ID NOS: 1889 - 1892. In some embodiments, a circular engineered guide RNA can comprise a DNA encoding a guide RNA from any one of SEQ ID NO: 769 - SEQ ID NO: 1112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 - SEQ ID NO: 1421. SEQ ID NO: 1537 - SEQ ID NO: 1642. SEQ ID NO: 1643 - SEQ ID NO: 1667, SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822, SEQ ID NO: 1825 - SEQ ID NO: 1849, SEQ ID NO: 1876, or SEQ ID NO: 1893 - 1896.

[0122] 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. 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.

[0123] 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 to 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 includea 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’ ribozy me 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.

[0124] 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 second spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA

[0125] In some instances, an engineered guide RNA can be circularized. A circularized engineered guide RNA can be produced from a precursor engineered polynucleotide. In some cases, a precursor engineered polynucleotide can be a precursor engineered linear polynucleotide. In some cases, a precursor engineered polynucleotide can be linear. For example, a precursor engineered polynucleotide can be a linear mRNA transcribed from a plasmid. In another example, a precursor engineered polynucleotide can be constructed to be a linear polynucleotide with domains such as a ribozy me domain and a ligation domain that allow for circularization in a 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 precursor engineered polynucleotide can be circular. In some cases, a precursor engineered polynucleotide can comprise DNA, RNA or both. In some cases, a precursor engineered polynucleotide can comprise a precursor engineered guide RNA. In some cases, a precursor engineered guide RNA can be used to produce an engineered guide RNA.

[0126] A circular or looped engineered guide polynucleotide, such as an engineered guide RNA can be formed directly or indirectly by forming a linkage (such as a covalent linkage) between more than one end of a RNA sequence, such as a 5’ end and a 3’ end. An RNA sequence can comprise an engineered guide RNA (such as a recruiting domain, targeting domain, or both). A linkage can be formed by employing an enzyme, such as a ligase. A suitable ligase (or synthetase) can include a ligase that forms a covalent bond. A covalent bond can include a carbon-oxygen bond, a carbon-sulfur bond, a carbon-nitrogen bond, a carboncarbon bond, a phosphoric ester bond, or any combination thereof. A linkage can also be formed by employing a recombinase. An enzyme can be recruited to an RNA sequence to form a linkage. A circular or looped RNA can be formed by ligating more than one end of an RNA sequence using a linkage element. In some embodiments, a linkage can be formed by a ligation reaction. In some instances, a linkage can be formed by a homologous recombination reaction. A linkage element can employ click chemistry to form a circular or looped RNA. A linkage element can be an azide-based linkage. A circular or looped RNA can be formed by genetically encoding or chemically synthesizing the circular or looped RNA.

[0127] 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 ribozy me, 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 activefragment 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), agltnS ribozyme, an HDV -like ribozyme, an R2 element, apeptidyl transferase 23S rRNA, a GIRI branching ribozyme, a, leadzyme, a group II intron, a hairpin ribozyme, a VS ribozyme, a CPEB3 ribozy me, a CoTC ribozyme, or a group I intron. In some cases, the selfcleaving ribozyme can be a / ram-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 propolynucleotide (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 by 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.

[0128] 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.

[0129] 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 GIRI branching ribozyme, a glmS ribozyme, a hairpin ribozyme, a Hammerhead ribozyme, an HDV ribozyme, a Twister ribozyme, a Twister sister ribozyme, a V S riboz me, 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‘GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT 3’ (SEQ ID NO: 88). A ribozyme can comprise 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3?(SEQ ID NO: 89). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGC CT 3’ (SEQ ID NO: 88). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5' GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCU 3’ (SEQ ID NO: 89). A ribozyme can include a Pl Twister Ribozyme. A ribozyme can include 5’AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 92). A ribozyme can include 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 3’ (SEQ ID NO: 93). A ribozy me can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACG C 3’ (SEQ ID NO: 92). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 3’ (SEQ ID NO: 93)

[0130] 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 some 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: 96). In some embodiments, a ligation domain can comprise 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 97). In some cases, a ligation domain can comprise 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 98). In some cases, a ligation domain can comprise 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 99). In some cases, a ligationdomain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 96). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 97). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 98). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 99).

[0131] 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 second spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA.

[0132] The compositions and methods of the present disclosure provide 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 altersplicing 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.

[0133] 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 polymerase 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 Ul, U2, U3. U4, U5, U6, U7, U8, U9, and U10.

[0134] 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.

[0135] 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.

[0136] 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. While AAV-2 / 1 based vectors expressing an appropriately modified murine U7 gene along with its natural promoter and 3' elements have enabled high efficiency gene transfer into the skeletal muscle and complete dystrophin rescue by covering and skipping mouse Dmd exon 23, the engineered polynucleotides as described herein (whether directly administered or administered via, for example. AAV vectors) can facilitate editing of target RNA by a deaminase.

[0137] The engineered polynucleotide can comprise at least in part an snRNA sequence. The snRNA sequence can be Ul. U2, U3, U4, U5, U6. U7, U8, U9. or a U10 snRNA sequence.

[0138] 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 havesuperior 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 descnptions in PCT / US2021 / 028618 and PCT / US2022 / 078801 are herein incorporated by reference.

[0139] 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: 78 or RNA: UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU (SEQ ID NO: 79). In some cases, a mouse U7 hairpin sequence comprises CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 63 or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU SEQ ID NO: 80). In some embodiments, the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 64 or RNA: AAUUUUUGGAG SEQ ID NO: 81 ). In some embodiments, a guide RNA from any one of SEQ ID NOS: 2 - 62 or SEQ ID NOS: 65 -75 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.

[0140] 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 5’ to the guide RNA sequence disclosed herein. In some cases, a promoter can comprise a U1 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 canbe a human U1 promoter, a human U7 promoter, or a human U6 promoter. In some cases, a human U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTA GAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA CGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTT AAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTT ATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 82). In some cases, a mouse U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:GTACTGAGTCGCCCAGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGC CGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTA ATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACA GATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTA TAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTC ACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAA AGCCTTGTTTG (SEQ ID NO: 83). In some cases, a human U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TTAACAACAACGAAGGGGCTGTGACTGGCTGCTTTCTCAACCAATCAGCACCGA ACTCATTTGCATGGGCTGAGAACAAATGTTCGCGAACTCTAGAAATGAATGACTT AAGTAAGTTCCTTAGAATATTATTTTTCCTACTGAAAGTTACCACATGCGTCGTTG TTTATACAGTAATAGGAACAAGAAAAAAGTCACCTAAGCTCACCCTCATCAATT GTGGAGTTCCTTTATATCCCATCTTCTCTCCAAACACATACGCA (SEQ ID NO: 84). In some cases, a mouse U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%. or 99% sequence identity' to:TTAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACTGACTCATT TGCATAGCCTTTACAAGCGGTCACAAACTCAAGAAACGAGCGGTTTTAATAGTCT TTTAGAATATTGTTTATCGAACCGAATAAGGAACTGTGCTTTGTGATTCACATAT CAGTGGAGGGGTGTGGAAATGGCACCTTGATCTCACCCTCATCGAAAGTGGAGT TGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCCGC (SEQ ID NO: 85). In some cases, a human U1 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:TAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGGGAGAGGCAGACGTCACTTCCTCTTGGCGACTCTGGCAGCAGATTGGTCGGTTGAG TGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGAC AGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCT GCTTCGCCACGAAGGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGAT CGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGC GGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCA GAGCCCGAAGATCTC (SEQ ID NO: 86). In some cases, a CMV promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: ATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAA CCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAC CGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACC (SEQ ID NO: 87).Chemical Modification(s)

[0141] 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 macrofootprints can be manufactured, chemically modified, and delivered directly to a subject in need thereof as RNA (w ithout a vector, such as an AAV).

[0142] Exemplary chemical modifications comprise any one of: 5' adenylate, 5' guanosinetriphosphate 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, 2'0-methyl RNA, methylphosphonate, phosphodi ester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5-methylcytidine-5'- triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof.

[0143] 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, 60,61, 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, 1 10, 11 1 , 1 12, 1 13, 114, 1 15, 1 16, 1 17, 118, 1 19, 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.

[0144] In some embodiments, a chemical modification can also be a phosphorothioate substitute. In some cases, a natural phosphodi ester bond can be susceptible to rapid degradation by cellular nucleases and; a modification of intemucleotide 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 caninhibit RNase A, RNase Tl, 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.

[0145] In some embodiments, a chemical modification can occur at 3 ’OH, group, 5 ’OH 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 ’OH or 5 ’OH 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, an engineered guide RNA comprises at least one chemically modified base. In some instances, the engineered guide 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.

[0146] 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 1.TABLE 1 - Exemplary Chemical Modification(s) of engineered guide RNAA. Modification of phosphate backbone

[0147] In some embodiments, the chemical modification can comprise modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage or 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 alky nyl 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 "arylalkyd" 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. “Heterocyclyd” 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. “Heteroary l” 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

[0148] 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 of modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyd or aryl phosphonates and phosphotri esters. 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, alkyd, 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 ofphosphorothioate 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. 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 of modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroami dates, 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 nonbridging 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 diastereomericexcess 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). The replacement can occur at either or both of the linking oxygensenter 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 nonbridging 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), wi th 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.

[0149] 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 inter 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.

[0150] In some cases, backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or nonbridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and can be used in any combination. Other non-phosphate linkages may also be used.

[0151] In some embodiments, backbone modifications (e.g.. methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate intemucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and / or enhance their stability in vivo.

[0152] 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.

[0153] 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 intemucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral intemucleoside 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.

[0154] In some cases, substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside 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 CEE 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 anamide 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 1-di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecyl amine or hexylamino-carbonyl-oxycholesterol moiety.

[0155] 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 chemically 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), 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 OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group may be achiral. How ever, 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

[0156] 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 nonbridging 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., alkyd or aryl).

[0157] In some cases, the phosphate linker can also be modified by replacement of a bridging oxygen, (z.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.B. Replacement of phosphate moiety

[0158] 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 nonphosphorus 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. phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3 ’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g. 3 '-amino phosphoramidate and aminoalkylphosphorami dates), 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‘.C. Substitution of phosphate group

[0159] 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 chemically 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.D. Modification of the Ribophosphate Backbone

[0160] 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 yvherein 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.E. Modification of sugar

[0161] 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 can include 2’ hydroxyl group (OH) modified or replaced yvith 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(CEl2CH2O)nCH2CH2OR, wherein R can be, e.g., El or optionally substituted alky l, 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; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, 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(CEl2CH2NH)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 T position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack anucleobase at C-. The abasic sugars 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 (<?.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).F. Modification of a constituent of the ribose sugar

[0162] 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’-M0E), 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 S’-CEU (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 can 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(CHs)3) (and analogs thereof); amethyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof).

[0163] 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(RI)(R.2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof.

[0164] In some instances, the engineered guide RNA described herein can compnse 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’-0-alkyl-substituted deoxyribonucleosides and 2 -0- ethy 1 enegly col -li ke ribonucl eosi des .

[0165] 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 Ci to C10, alkyl or C2 to C10 alkenyl and alkynyl. 2’ sugar modifications also include but are not limited to-O[(CH2)nO]mCH3,-O(CH2)nOCHs,- 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: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyL SH, SCH3, OCN, Cl, Br, CN, CFs, OCFs, SOCH3, SO2 CHs, ONO2, NO2, Ns, 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-(Ci-Cio alkyl), OCF3. O(CH2)2SCH3. O(CH2)2-O- N(Rm)(Rn), and 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.

[0166] 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)- 0-2 ’and analogs thereof.G. Modifications on the base of nucleotide

[0167] 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 to in the engineered guide RNA described herein. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can be naturally-occurring or synthetic derivatives of a base.

[0168] 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 -aminoally 1-uridine, 5-halo-uridine (<?.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-sel eno-uridine, 5 -carbamoy Imethyl-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-l -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. l-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-uri dine, 2’-O-methyl-pseudouridine, 2-thio-2’-O- methyl-uridine, 5 -methoxy carbony lmethyl-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, 1-thio-uridine, deoxythymidine, 2’-F-ara- uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-( 1-E- propenylamino)uridine, pyrazolo [3 ,4-d] pyrimidines, xanthine, and hypoxanthine.

[0169] In some embodiments, the chemical modification described herein can comprise modifying a cytosine. In some embodiments, the engineered guide RNA described herein can 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-l-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-l-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O- methyl-cytidine, 5,2’ -O-dimethyl-cyti dine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O- dimethyl-cytidine, 5-formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1-thio- cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0170] In some embodiments, the chemical modification described herein can comprise modifying a adenine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified adenine. Exemplary' chemically modified adenine can include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g, 2-amino-6- chloro-purine), 6-halo-purine (e.g, 6-chloi-purine), 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza- 2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine. 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl) adenosine , 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyl- adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6, N6-dimethyl-adenosine, N6- hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl- adenosine, N6-acetyl-ad enosine, 7-methyl-adenine, 2-methylthio-adenine, 2 -methoxy - adenine, a-thio-adenosine, 2’-O-methyl-adenosine, N6, 2’-O-dimethyl-adenosine, N6-Methyl- 2’-deoxyadenosine, N6, N6, 2’-O-trimethyl-adenosine, 1 ,2’-O-dimethyl-adenosine, 2’-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine. 8-azido- adenosine, 2’-F-ara-adenosine, 2’ -F -adenosine, 2’-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0171] In some embodiments, the chemical modification described herein can comprise modifying a guanine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified guanine. Exemplary chemically modified guanine can include inosine, 1 -methyl-inosine. wyosine, methylwyosine, 4-demethyl-wyosine,isowyosine, wybutosine. peroxywybutosine. hydroxywybutosine, undemriodified hydroxy wybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1 -methyl-guanosine, N2-methyl-guanosine, N2, N2-dimethyl-guanosine, N2, 7 -dimethyl -guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1 -meththio-guanosine, N2-methyl-6-thio-guanosine. N2,N2-dimethyl-6-thio- guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2, 7-dimethyl- 2’-O-methyl-guanosine, 2’-O-methyl-inosine, 1 , 2'-O-dimethyl-inosine, 6-O-phenyl-2’- deoxyinosine, 2'-O-ribosylguanosine, 1 -thio-guanosine, 6-O-methyguanosine, O6-Methyl-2’- deoxy guanosine. 2’-F-ara-guanosine, and 2’-F-guanosine.

[0172] In some cases, the chemical modification of the engineered guide RNA can include introducing or substituting a nucleic acid analog or an unnatural nucleic acid into the engineered guide RNA. In some embodiments, nucleic acid analog can be any one of the chemically modified nucleic acid described herein. Exemplary7nucleic acid analog can be found in PCT / US2021 / 034272, PCT / US2015 / 025175, PCT / US2014 / 050423,PCT / US2016 / 067353, PCT / US2018 / 041503. PCT / US18 / 041509, PCT / US2004 / 011786. or PCT / US2004 / 01 1833, all of which are expressly incorporated by reference in their entireties. In some cases, the chemically modified nucleotide described herein can include a variant of guanosine, uridine, adenosine, thymidine, and cytosine, including any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytidine that has been altered chemically, for example by acetylation, methylation, hydroxylation. Exemplary chemically modified nucleotide can include 1-methyl-adenosine, 1 -methyl-guanosine, 1- methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2’-amino-2’-deoxyadenosine, 2’- amino-2'-deoxy cytidine. 2’ -amino-2’ -deoxy guanosine, 2'-amino-2'-deoxyuridine, 2-amino-6- chloropurineriboside, 2-aminopurine-riboside, 2’-araadenosine, 2’-aracytidine, 2’-arauridine. 2’-azido-2’-deoxyadenosine, 2’-azido-2’-deoxycytidine, 2’-azido-2’-deoxyguanosine, 2’- azido-2’-deoxyuridine, 2-chloroadenosine, 2’-fluoro-2’-deoxyadenosine, 2’-fluoro-2’- deoxycytidine, 2’ -fl uoro-2’ -deoxyguanosine, 2’-fluoro-2’-deoxyuridine, 2 ’-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2’-O- methyl-2-aminoadenosine, 2’-O-methyl-2’-deoxyadenosine, 2’-O-methyl-2’-deoxy cytidine, 2‘-O-methyl-2’ -deoxy guanosine, 2,-O-methyl-2’-deoxyuridine, 2’-O-methyl-5-methyluridine, 2’-O-methylinosine, 2’-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl- cytidine, 4-acetyl-cytidine. 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6- dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5- carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-chloro- ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy- uridine, 5-methyl-2-thio-uridine. 6-Azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6- chloropurineriboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2’- deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo- adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazoleriboside, beta-D-mannosyl-queosine, dihydro-uridine, inosine, N1 -methyladenosine, N6-([6- ami nohexyl] carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine. uracil-5- oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xyloadenosine. In some embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from 2-amino-6- chloropurineriboside-5 ’ -triphosphate, 2-aminopurine-riboside-5 ’ -triphosphate. 2- aminoadenosine-5’ -triphosphate, 2’-amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5’- triphosphate, 2-thiouridine-5’-triphosphate, 2’-fluorothymidine-5’-triphosphate, 2’-O-methyl- inosine-5’ -triphosphate, 4-thiouridine- ’-triphosphate, 5-aminoallylcytidine-5’ -triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’- triphosphate. 5-bromo-2’-deoxycytidine-5 ’-triphosphate, 5-bromo-2’-deoxyuridine-5’- triphosphate, 5 -iodocytidine-5’ -triphosphate, 5-iodo-2’-deoxycytidine-5’-triphosphate, 5- iodouridine-5 ’ -triphosphate, 5-iodo-2’-deoxyuridine-5 ’-triphosphate, 5-methylcytidine-5’- triphosphate, 5-methyluridine-5’-triphosphate, 5-propynyl-2’-deoxycytidine-5’-triphosphate, 5-propynyl-2’-deoxyuridine-5’-triphosphate, 6-azacytidine-5’ -triphosphate, 6-azauridine-5’- triphosphate, 6-chloropurineriboside-5’-triphosphate, 7-deazaadenosine-5 ’-triphosphate, 7- deazaguanosine-5 ’ -triphosphate, 8-azaadenosine- ’-triphosphate, 8-azidoadenosine-5’- triphosphate, benzimidazole-riboside-5’ -triphosphate, Nl-methyladenosine-5’ -triphosphate, Nl-methylguanosine-5’ -triphosphate, N6-methyladenosine-5 ’-triphosphate, 6- methylguanosine-5 ’ -triphosphate, pseudouridine-5 ’ -triphosphate, puromy cin-5 ’ -triphosphate, or xanthosine-5 ’ -triphosphate. In some embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected frompyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5- taurinomethyluridine, 1-tauri nomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudoundine. In some embodiments, the artificial nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine. 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine. pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine. 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-th io-l-methyl-l-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, and 4-methoxy- 1-methyl- pseudoisocytidine. In some embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7- deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine,1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and2-methoxy -adenine. In other embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from inosine, 1- methyl-inosine, wyosine, wybutosine. 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine. 1 -methylguanosine. N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In certain embodiments, the chemically modified nucleic acid as described hereincan comprise at least one chemically modified nucleotide selected from 6-aza-cytidine, 2-thio- cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5 -aminoally l-uri dine, 5-iodo-uridine, Nl- methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine. deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, Nl- methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza- adenosine.

[0173] In some embodiments, a modified base of a unnatural nucleic acid includes, but may be not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5 -methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine. 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3 -deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines. 6-azapyrimi dines andN-2 substituted purines, N-6 substituted purines, 0-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5- propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine. 5-halouracil, 5-halocytosine, 5-propynyl (-C=C-CHs) uracil. 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6- azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl. 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo. 5- trifluoromethyl, other 5-substituted uracils and cytosines. 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine( [5,4- b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin- 2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4- b][l,4]benzoxazin-2(3H)-one). carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base may be replaced with other heterocycles, 7-deaza- adenine, 7- deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5- chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5 -fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino- adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4- ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine. 5 -hydroxy cytosine, 2’-deoxyuridine, or 2-amino-2’-deoxy adenosine.

[0174] In some cases, the at least one chemical modification can comprise chemically modifying the 5’ or 3’ end such as 5‘ cap or 3’ tail of the engineered guide RNA. In some embodiments, the engineered guide RNA can comprise a chemical modification comprising 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridines can be replaced with modified uridines, e.g., 5-(2-amino) propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g, 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O-and N-alkylated nucleotides, e.g, N6-methyladenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2’ OH-group may be replaced by a group selected from H,-OR,-R (wherein R can be, e.g, alkyd, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be. e.g, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be. e.g, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g, with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’ -sugar modified, such as,2-F 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’-O- methoxyethyladenosine (Aeo), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo), or any combinations thereof.Targets and Methods of Treatment

[0175] Also disclosed herein are methods of treating a subject by administering a guide RNA targeting SERPINA1 or a polynucleotide encoding the same. Administration of a guide RNA targeting SERPINA1 as described herein can be used to treat a disease or condition associated with a mutation of SERPINA1 as described herein (e.g. alpha-1 antitrypsin deficiency). In some cases, a disease or condition associated with a mutation of SERPINA1 is an alpha- 1- antitrypsin deficiency, a pulmonary emphysema, an early-onset pulmonary emphysema, an obstructive pulmonary disease, a liver disease, a cirrhosis, a liver fibrosis, a hepatocellular carcinoma, a lung disease, a chronic obstructive pulmonary disease (COPD), a neonatal jaundice, a jaundice, a hepatomegaly, a splenomegaly, a panniculitis, a necrotizing panniculitis, a granulomatosis with polyangiitis, a bronchitis, a chronic bronchitis, a bronchiectasis, or any combination thereof.

[0176] In some embodiments, a target tissue for a guide RNA targeting SERPINA1 can be the liver, the respiratory system, the gastrointestinal system, or any combination thereof. In some embodiments, a target cell for a guide RNA targeting SERPINA1 can comprise a hepatocyte. In some embodiments, a target cell for a guide RNA targeting SERPINA1 can comprise a mononuclear phagocyte, a neutrophil, an epithelial cell (e.g., in the airway or intestine), or any combination thereof. In some cases, a target cell can comprise a HEK 293 cell, a Hep G2 cell, a human primary hepatocyte, or a human primary epithelial cell. In some embodiments, a cell herein can be used in an assay. For example, a cell herein can be used to determine the editing of a target RNA by a guide RNA. In some cases, editing of a target RNA in a cell or in vitro by a guide RNA can be determined by next generation sequencing (NGS) and / or by Sanger sequencing. In some cases, the 3’ UTR of SERPINA1 can be targeted. In some cases, targeting the 3’ UTR can knockdown SERPINA1 protein and / or mRNA levels. In some cases, editing of SERPINA1 can be determined by droplet digital PCR and / or next generation sequencing. In some cases, the restoration of SERPINA1 can be determined by measuring protein levels of SERPINA1 before and after treatment with a guide RNA. In some cases, the restoration of protein levels can be measured by Western blot and / or flow cytometry.

[0177] In some examples, the disease or condition can be an AAT deficiency or an associated lung or liver pathology' (e.g., chronic obstructive pulmonary' disease, cirrhosis, hepatocellular carcinoma) caused, at least in part, by a mutation in a SERPINA1 gene. In some examples, the mutation can be a substitution of a G with an A at nucleotide position 9989 within a wildtype SERPINA1 gene. In some examples, the mutation can be a substitution of a G with an A at nucleotide position 1096 within a SERPINA1 transcript. In some examples, the SERPINA1 mutation results in an E342K mutation in the alpha- 1 antitry psin (AAT) polypeptide encoded by the SERPINA1 gene (also referred to herein as a SERPINA1 E342K mutation). In some examples, the mutation can be a missense mutation in the SERPINA1 gene or transcript. In some examples, administration of the engineered guides disclosed herein restore expression of a normal AAT protein as compared to an inactive or defective AAT in a subject with an AAT deficiency. In some examples, administration of the engineered guides disclosed herein restore enzymatic function of AAT protein as compared to an inactive or defective AAT protein in a subject with an AAT deficiency. In some examples, a guide-target RNA scaffold is formed upon hybridization of an engineered guide of the present disclosure to a target RNA. In some examples, the target RNA forming the guide-target RNA scaffold comprises a portion of an mRNA or pre-mRNA molecule encoded by the SERPINA1 gene. In some examples, the targeting region of the engineered guide forming the guide-target RNA scaffold is, at least in part, complementary to a portion of an mRNA or pre-mRNA molecule encoded by the SERPINA1 gene. An example of the wildtype SERPINA1 transcript can be found on Genbank from the National Library of Medicine (US), National Center for Biotechnology Information, Accession number: NM_001127701. 1.

[0178] The present disclosure provides engineered guide RNAs that facilitate ADAR- mediated RNA editing of at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the SERPINA1 E342K mutation and which exhibit limited off-target editing at the +1 position relative to the target adenosine. In some cases, the present disclosure provides engineered guide RNAs that facilitate ADAR-mediated RNA editing of more than about, or equal to about: 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%, or 75% of the SERPINA1 E342K mutation. In some embodiments, the engineered guide RNAs provided herein facilitate off-target editing (e.g., +1 position relative to the target adenosine) of less than or equal to about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the engineered guide RNAs provided herein facilitate on-target editing of greater than 30% and off-target editing at the +1 position relative to the target adenosine of less than 20%. In some embodiments, the engineered guide RNAs provided herein facilitate on-target editing of greater than 40% and off-target editing at the +1 position relative to the target adenosine of less than 20%. In some embodiments, the engineered guide RNAs provided herein facilitate on-target editing of greater than 50% and off-target editing at the +1 position relative to the target adenosine of less than 20%. In some embodiments, the engineered guide RNAs provided herein facilitate on-target editing of greater than 60% and off-target editing at the +1 position relative to the target adenosine of less than 20%. In some embodiments, the engineered guide RNAs provided herein facilitate on-target editing of greater than 70% and off-target editing at the +1 position relative to the target adenosine of less than 20%. In some embodiments, the present disclosure provides a method of editing the SERPINA1 E342K mutation, wherein the method includes providing an engineered guide RNA of the present disclosure to a subject in need thereof, wherein upon administration of the engineered guide RNA, said engineered guide RNA facilitates ADAR- mediated on-target editing of the SERPINA1 E342K mutation of from about 30% to about 70% and off-target editing at the +1 position of less than about 20%. The subject may have a liver or lung disease. The disease may be alpha- 1 antitrypsin deficiency.

[0179] Guide RNAs can facilitate correction of a G to A mutation at nucleotide position 9989 of a SERPINA1 gene. In some embodiments, a guide RNA of the present disclosure can target, for example, the SERPINA1 E342K mutation. Said guide RNAs targeting a site in SERPINA1 can be encoded by an engineered polynucleotide construct of the present disclosure. An engineered guide RNA targeting SERPINA1 can comprise a polynucleotide of any of the sequences recited in TABLES 2-24 of the examples provided in the present disclosure. Polynucleotide sequences encoding the guides disclosed in TABLES 2-24 are also encompassed herein. Any sequence disclosed herein also comprises the RNA sequence and the DNA sequence. For example, a DNA sequence of a disclosed RNA sequence comprises all Us substituted for Ts. In another example, a RNA sequence of a disclosed DNA sequence comprises all Ts substituted to Us.

[0180] Further, a guide RNA targeting SERPINA1 can comprise any one of SEQ ID NO: 2 - SEQ ID NO: 62. In some cases, a guide RNA targeting SERPINA1 can comprise any one of SEQ ID NO: 65 - SEQ ID NO: 75. In some cases, a guide RNA targeting SERPINA1 can comprise any one of SEQ ID NO: 104 - SEQ ID NO: 115. In some examples, the engineeredguide comprises a polynucleotide having at least 99% identity, at least 98% identity, at least 97% identity, at least 96% identity, at least 95% identity', at least 90% identity, at least 85% identity, at least 80% identity', or at least 70% identity to any one of SEQ ID NO: 2 - SEQ ID NO: 62. In some examples, the engineered guide comprises a polynucleotide having at least 99% identity, at least 98% identity, at least 97% identity, at least 96% identity, at least 95% identity', at least 90% identity', at least 85% identity, at least 80% identity, or at least 70% identity to any one of SEQ ID NO: 65 - SEQ ID NO: 75. In some examples, the engineered guide comprises a polynucleotide having at least 99% identity, at least 98% identity, at least 97% identity, at least 96% identity, at least 95% identity, at least 90% identity, at least 85% identity', at least 80% identity, or at least 70% identity to any one of SEQ ID NO: 104 - SEQ ID NO: 115.

[0181] In some examples, the engineered guide comprises a polynucleotide having at least 99% length, at least 98% length, at least 97% length, at least 96% length, at least 95% length, at least 90% length, at least 85% length, at least 80% length, or at least 70% length to the above SEQ ID NOS: 1-62. In some examples, the engineered guide comprises a polynucleotide having at least 99% length, at least 98% length, at least 97% length, at least 96% length, at least 95% length, at least 90% length, at least 85% length, at least 80% length, or at least 70% length to any one of SEQ ID NOS: 65-75. In some examples, the engineered guide comprises a polynucleotide having at least 99% length, at least 98% length, at least 97% length, at least 96% length, at least 95% length, at least 90% length, at least 85% length, at least 80% length, or at least 70% length to the above SEQ ID NOS: 104-115.

[0182] In some embodiments, a guide RNA targeting SERPINA1 can comprise any one of SEQ ID NO: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563- SEQ ID NO: 768, SEQ ID NO: 1424 - SEQ ID NO: 1536, SEQ ID NO: 1668 - SEQ ID NO: 1692, SEQ ID NO: 1718 - SEQ ID NO: 1742, SEQ ID NO: 1768 - SEQ ID NO: 1792. SEQ ID NO: 1793 - SEQ ID NO: 1807, SEQ ID NO: 1850 - SEQ ID NO: 1875, or SEQ ID NOS: 1889 - 1892. In some embodiments, a DNA encoding a guide RNA targeting SERPINA1 can comprise any one of SEQ ID NO: 769 - SEQ ID NO: 1112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 - SEQ ID NO: 1421, SEQ ID NO: 1537 - SEQ ID NO: 1642. SEQ ID NO: 1643 - SEQ ID NO: 1667, SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743- SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822, SEQ ID NO: 1825 - SEQ ID NO: 1849, SEQ ID NO: 1876, or SEQ ID NO: 1893 - 1896. In some examples, the engineered guide comprises a polynucleotide having at least 99% identity, at least 98% identity, at least97% identity, at least 96% identity, at least 95% identity, at least 90% identity, at least 85% identity, at least 80% identity, or at least 70% identity to any one of SEQ ID NO: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563 - SEQ ID NO: 768, SEQ ID NO: 1424 - SEQ ID NO: 1536. SEQ ID NO: 1668 - SEQ ID NO: 1692. SEQ ID NO: 1718- SEQ ID NO: 1742, SEQ ID NO: 1768 - SEQ ID NO: 1792, SEQ ID NO: 1793 - SEQ ID NO: 1807, SEQ ID NO: 1850 - SEQ ID NO: 1875, or SEQ ID NOS: 1889 - 1892. In some examples, a DNA encoding an engineered guide comprises a polynucleotide having at least 99% identity, at least 98% identity, at least 97% identity, at least 96% identity, at least 95% identity, at least 90% identity, at least 85% identity, at least 80% identity, or at least 70% identity to any one of SEQ ID NO: 769 - SEQ ID NO: 1 112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 SEQ ID NO: 1421, SEQ ID NO: 1537 SEQ ID NO: 1642, SEQ ID NO: 1643 - SEQ ID NO: 1667. SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743- SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822. SEQ ID NO: 1825 - SEQ ID NO: 1849, SEQ ID NO: 1876, or SEQ ID NO: 1893 - 1896. In some examples, the engineered guide comprises a polynucleotide having at least 99% length, at least 98% length, at least 97% length, at least 96% length, at least 95% length, at least 90% length, at least 85% length, at least 80% length, or at least 70% length to any one of SEQ ID NO: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563 - SEQ ID NO: 768, SEQ ID NO: 1424- SEQ ID NO: 1536, SEQ ID NO: 1668 - SEQ ID NO: 1692, SEQ ID NO: 1718 - SEQ ID NO: 1742, SEQ ID NO: 1768 - SEQ ID NO: 1792, SEQ ID NO: 1793 - SEQ ID NO: 1807, SEQ ID NO: 1850 - SEQ ID NO: 1875, or SEQ ID NOS: 1889 - 1892. In some examples, a DNA encoding an engineered guide comprises a polynucleotide having at least 99% length, at least 98% length, at least 97% length, at least 96% length, at least 95% length, at least 90% length, at least 85% length, at least 80% length, or at least 70% length to any one of SEQ ID NO: 769 - SEQ ID NO: 1112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 - SEQ ID NO: 1421. SEQ ID NO: 1537 - SEQ ID NO: 1642. SEQ ID NO: 1643 - SEQ ID NO: 1667, SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822, SEQ ID NO: 1825 - SEQ ID NO: 1849, SEQ ID NO: 1876, or SEQ ID NO: 1893 - 1896.

[0183] Said engineered guide RNA can be delivered via a vector encapidated in a viral particle (e.g, encoded for and delivered via AAV) as disclosed herein and can be administered via any route of administration disclosed herein to a subject in need thereof. The subject may be human and may be at risk of developing or has developed alpha- 1 antitrypsin deficiency.Such alpha-1 antitrypsin deficiency can be at least partially caused by a mutation of SERPINA1, for which an engineered guide RNA described herein can facilitate editing in, thus correcting the mutation in SERPINA1 and reducing the incidence of alpha- 1 antitrypsin deficiency in the subject. Thus, the guide RNAs of the present disclosure can be used in a method of treatment of alpha- 1 antitrypsin deficiency.Pharmaceutical Compositions

[0184] An engineered guide RNA targeting SERPINA1 described herein or a polynucleotide encoding an engineered guide RNA described herein can be formulated with a pharmaceutically acceptable carrier for administration to a subject (e.g., a human or a nonhuman animal). In some embodiments, provided herein is a pharmaceutical composition comprising an engineered guide RNA targeting SERPINA1 described herein or a polynucleotide encoding an engineered guide RNA described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition provided herein can be in unit dose form. A pharmaceutically acceptable carrier can include, but is not limited to, phosphate buffered saline solution, water, emulsions (e.g, an oil / water emulsion or a water / oil emulsions), glycerol, liquid polyethylene glycols, aprotic ...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA comprises a polynucleotide 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 identity7to any one of SEQ ID NO: 116 - SEQ ID NO: 459, SEQ ID NO: 460 - SEQ ID NO: 562, SEQ ID NO: 563 - SEQ ID NO: 768, SEQ ID NO: 1424 - SEQ ID NO: 1536, SEQ ID NO: 1668 - SEQ ID NO: 1692, SEQ ID NO: 1718 - SEQ ID NO: 1742, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1768 SEQ ID NO: 1792, SEQ ID NO: 1793 SEQ ID NO: 1807, or SEQ ID NO: 1850 - SEQ ID NO: 1874.

2. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide 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 identity7to any one of SEQ ID NO: 769 - SEQ ID NO: 1112, SEQ ID NO: 1113 - SEQ ID NO: 1215, SEQ ID NO: 1216 - SEQ ID NO: 1421, SEQ ID NO: 1537 - SEQ ID NO: 1642, SEQ ID NO: 1643 - SEQ ID NO: 1667, SEQ ID NO: 1693 - SEQ ID NO: 1717, SEQ ID NO: 1743 - SEQ ID NO: 1767, SEQ ID NO: 1808 - SEQ ID NO: 1822, or SEQ ID NO: 1825 - SEQ ID NO: 1849.

3. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1 or 2, wherein upon hybridization of the engineered guide RNA to a target SERPINA1 RNA sequence, the engineered guide RNA facilitates RNA editing of a target adenosine in the target SERPINA1 RNA sequence by an RNA editing entity.

4. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 463.

5. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity7to SEQ ID NO: 463.

6. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity' to SEQ ID NO: 463.

7. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 463.

8. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 463.

9. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 463.

10. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 518.

11. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 518.

12. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 518.

13. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 518.

14. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 518.

15. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 1, wherein the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 518.

16. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 1116.

17. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1116.

18. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 1116.

19. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 1116.

20. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 1116.

21. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 1116.

22. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 85% sequence identity to SEQ ID NO: 1171.

23. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1171.

24. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO: 1171.

25. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 97% sequence identity to SEQ ID NO: 1171.

26. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence with at least 99% sequence identity to SEQ ID NO: 1171.

27. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 2, wherein the polynucleotide encoding the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 1 171.

28. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 3, wherein the target SERPINA1 RNA sequence encodes an alpha- 1 antitrypsin polypeptide having an E342K mutation.

29. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 3, wherein the engineered guide RNA upon hybridization to the target SERPINA1 RNA sequence forms a guide-target RNA scaffold that comprises one or more structural features.

30. An engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA hybridizes to a target SERPINA1 RNA sequence encoding an alpha- 1 antitrypsin (AAT) polypeptide having an E342K mutation, thereby forming a guide-target RNA scaffold with the target SERPINA1 RNA sequence, wherein formation of the guide-target RNA scaffold substantially forms one or more structural features that comprises a bulge, internal loop, wobble base pair, hairpin, a mismatch, or any combination thereof.

31. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises a bulge, wherein the bulge is a symmetric bulge.

32. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises a bulge, wherein the bulge is an asymmetric bulge.

33. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises an internal loop, wherein the internal loop is a symmetric internal loop.

34. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises an internal loop, wherein the internal loop is an asymmetric internal loop.

35. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises a Wobble base pair.

36. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises a hairpin, wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin.

37. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises two or more asymmetric bulges.

38. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 37, wherein the two or more asymmetric bulges comprise a 3-1 asymmetric bulge at position -10 relative to a target adenosine at position 0, and a 2-1 asymmetric bulge at position 3 relative to a target adenosine at position 0.

39. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 33, wherein the one or more structural features comprises two or more symmetric internal loops.

40. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 39, wherein the two or more symmetric internal loops comprises a 6-6 symmetric internal loop at position -18 and position 32 relative to a target adenosine at position 0.

41. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 29 or 30, wherein the one or more structural features comprises a mismatch.

42. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 41, wherein the mismatch comprises an A / C mismatch at position 0 relative to a target adenosine at position 0, wherein the adenosine of the A / C mismatch is the target adenosine.

43. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 30, wherein upon hybridization of the engineered guide RNA to a targetSERPINA1 RNA sequence, the engineered guide RNA facilitates RNA editing of a target adenosine in the target SERPINA1 RNA sequence by an RNA editing entity.

44. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of claims 3-29, or 43 wherein the RNA editing entity comprises an ADAR.

45. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of claim 44, wherein the ADAR is ADAR1, ADAR2, or a combination thereof.

46. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of claims 1-45, wherein the engineered guide RNA facilitates restoration of wildtype AAT protein.

47. A vector comprising the polynucleotide encoding the engineered guide RNA of any one of claims 1 -46.

48. A viral particle encapsidating the vector of claim 47.

49. The viral particle of claim 48, wherein viral particle comprises an adeno-associated virus (AAV) virion.

50. The viral particle of claim 49, wherein the AAV virion is an AAV1 virion, an AAV2 virion, an AAV3 virion, an AAV4 virion, an AAV5 virion, an AAV6 virion, an AAV7 virion, an AAV8 virion, an AAV9 virion, an AAV 10 virion, an AAV 11 virion, or a derivative, a chimera, or a variant thereof.

51. The viral particle of claim 49 or 50, wherein the AAV virion is a recombinant AAV (rAAV) virion, a hybrid AAV virion, a chimeric AAV virion, a self-complementary AAV (scAAV) virion, or any combination thereof.

52. A pharmaceutical composition comprising:(a) the engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of claims 1-46, the vector of claim 47, or the viral particle of any one of claims 48-51; and(b) a pharmaceutically acceptable: excipient, carrier, or diluent.

53. A method of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the engineered guide RNA or the polynucleotide encoding the engineered guide RNA ofany one of claims 1-46, the vector of claim 47, the viral particle of any one of claims 48-51, or the pharmaceutical composition of claim 52.

54. The method of claim 53, wherein the disease or condition comprises alpha-1 antitrypsin deficiency.

55. The method of claim 53, wherein the subject has a SERPINA1 gene encoding an alpha- 1 antitry psin polypeptide having an E342K mutation.

56. The method of claim 53, wherein the subject has a SERPINA1 gene with a G to A mutation at nucleotide position 9989 of the SERPINA1 gene.

57. The method of any one of claims 53-56, wherein the subject is human or a non-human animal.

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