Modified cas12 guides and uses thereof

Abstract

Disclosed herein are compositions, kits methods related to improved Cas12 systems, specifically including Cas12 guide RNAs. In particular, the present disclosure provides insights and technologies relating to Cas12f systems.

Description

MODIFIED CAS12 GUIDES AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 655500, filed on June 3, 2024, and U.S. Provisional Patent Application No. 63 / 688091, filed August 28, 2024, the entire contents of which are hereby incorporated by reference in their entirety.BACKGROUND

[0002] Casl2, and specifically Casl2f nuclease systems have been reported to be particularly useful, among other things in light of their relatively small size (e.g., compared with Cas9, Casl2a, etc), and because of reported low off- target cleavage and other undesirable genetic impacts (e.g., chromosomal translocations, large deletions, and unwanted integrations, for example of viral vectors from which the system is expressed). See, for example, Xin, et a / ., Comprehensive assessment of miniature CRISPR-Casl2f nucleases for gene disruption. Nat Commun 13:5623, 2022 (doi.org / 10.1038 / s41467-022-33346-l).SUMMARY

[0003] The present disclosure provides certain insights and technologies related to improved Casl2 systems, specifically including Casl2 guide RNAs. In particular, the present disclosure provides insights and technologies relating to Casl2f systems (e.g., RNAs).

[0004] In many embodiments, the present disclosure provides insights and technologies relevant to SpaCasl2fl systems (also referred to as "SpCasl2fl", "SpCasl2f", or "SpaCasl2f") and AsCasl2f systems. However, the present disclosure also surprisingly documents circumstances in which provided insights and technologies are or may be applicable to other Casl2f systems.

[0005] In certain embodiments, the present disclosure provides truncated Casl2f RNAs (and, therefore, systems containing them and methods relating to them, including methods of using such RNAs or systems containing them, methods of preparing and / or characterizing such, etc).

[0006] The present disclosure appreciates that certain Casl2f systems, specifically including SpaCasl2fl, have been reported to present challenges such as, for example, relatively low editing activity (see, for example, Wang, Y. et al., "Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Casl2fl nuclease." Cell Rep., 40(13): 111418, 2022) e.g., in mammalian cells. The present disclosure provides certain insights relating to improving Casl2f systems, specifically including SpaCasl2fl. For example, the present disclosure appreciates that many SpaCasl2fl guides are not stable and / or can be challenging to generate and or use, for example due to their size and / or to their lack of optimization for human expression.

[0007] The present disclosure provides a particular insight that improvements in Casl2f systems, and specifically in SpaCasl2fl systems, may be made through modification of the RNA. The present disclosure provides a specific insight that circular permutation of primary RNA sequence elements can achieve useful benefits, including permitting further reduction in RNA size. Indeed, the present disclosure demonstrates that such circular permutation can permit truncation of Casl2f RNA while substantially preserving, or even improving activity relative to one or more referenceCasl2f systems (e.g., a wild type Casl2f RNA). The present disclosure therefore identifies the source of a problem in various other studies endeavoring to develop truncated Casl2f RNAs; such studies typically truncate within the standard (e.g., wild type) linear arrangement of RNA sequence elements.

[0008] The present disclosure provides a further insight that identification of conserved stem-loops may inform improved RNA development, including of truncated RNAs, including circularly permuted truncated RNAs.

[0009] The present disclosure provides particular insights relating to modified Casl2f systems, including specifically modified Casl2f RNAs. The present disclosure provides insights regarding certain potential structural features of Casl2f RNAs, e.g., SpaCasl2fl RNAs, AsCasl2f RNAs, and / or of interaction points between such RNAs and other entities (e.g., Casl2f enzymes, target transcripts or elements thereof, etc). Moreover, the present disclosure describes and defines certain modified Casl2f RNAs, many of which have reduced size, for example, relative to MS13 and / or to a minimal AsCasl2fl guide (AGNT170).

[0010] The present disclosure therefore provides, among other things, Casl2f RNAs, which may be particularly useful in SpaCasl2fl systems but in some embodiments may be useful in other Casl2f systems, that are truncated, have different start and / or end points, and / or different placement of the spacer relative to one or more reference Casl2f RNAs (e.g., to one or more wild type Casl2f RNAs, such as a wild type SpaCasl2fl RNA), and / or chemical modifications to improve activity.

[0011] In many embodiments, provided Casl2f systems (e.g., incorporating a provided Casl2f RNA) show reasonably comparable or improved activity (in one or more respects) in one or more appropriate assays relative to a one or more reference Casl2f systems (e.g., to one or more wild type Casl2f systems, such as a wild type SpaCasl2fl system, a wild type AsCasl2f system, an MS13 system, and / or to an engineered AsCasl2f system). Those skilled in the art will appreciate that, in some embodiments, some decrease in activity may be acceptable, particularly for provided Casl2f RNAs that are truncated (e.g., relative to one or more wild type Casl2f RNAs, such as a wild type SpaCasl2fl RNA, and particularly if truncated relative to MS13 and / or to AGNT170).

[0012] In many embodiments, provided Casl2f systems are smaller than one or more wild type Casl2f systems. For example, in many embodiments, a provided Casl2f RNA is smaller than one or more wild type Casl2f RNA.

[0013] In many embodiments, provided Casl2f systems show activity in one or more assays (e.g., as described herein) that is comparable to or better than (in one or more respects) that of a wild type Casl2f system; in many such embodiments, the provided Casl2f system is smaller than the relevant wild type Casl2f system (e.g., utilizes a Casl2f RNA, as described herein, that is smaller than the wild type RNA - e.g., includes fewer residues than are present in a relevant set of RNA molecules that together comprise a wild type RNA of a wild type Casl2f CRISPR system).

[0014] In some embodiments, provided Casl2f systems may be smaller than MS13 systems. In some embodiments, provided Casl2f systems may be smaller than an engineered AsCasl2f systems.

[0015] In some embodiments, provided Casl2f systems (e.g., provided Casl2f systems that are smaller than MS13 systems, and / or than AGNT170) may have activity that is reasonably comparable (in one or more respects) to an appropriate reference - e.g., MS13 and / or an engineered AsCasl2f system as described herein. In some embodiments, provided Casl2f systems (e.g., provided Casl2f systems that are smaller than MS13 systems and / orthan engineered AsCasl2f systems) may have activity that is improved activity (in one or more respects) relative to MS13 or AGNT170.

[0016] The present disclosure provides a particular insight that permutation of elements, structure manipulation, sequence changes, and / or chemical modifications of Casl2f RNAs may improve activity in a system in one or more appropriate assays relative to one or more reference Casl2f systems and may therefore allow lower dosing.

[0017] The present disclosure provides, among other things, an RNA including sequence elements selected from a group comprising (a) a crispr sequence element, (b) a tracr sequence element, (c) a spacer sequence element, and combinations thereof, wherein an RNA disclosed herein includes one or more stem-loop sequence elements selected from the group consisting of: stem-loop -1, stem-loop 0, stem-loop 1, stem-loop 2, stem-loop 3, stem-loop 4, crispr tracr stem-loop and is a variant of a reference sgRNA selected from a group comprising: (i) reference sgRNA AGNT248, (ii) reference sgRNA AGNT246, or a combination thereof, wherein the arrangement of sequence elements in the variant differs from that found in reference sgRNAs, and wherein a variant disclosed herein optionally includes one or more of the following changes to the reference sgRNA sequence element relative to that found in the reference: (i) removal of ribonucleic sequences, (ii) addition of new ribonucleic sequences, or (iii) the combination thereof, wherein a variant disclosed herein maintains essential secondary structures and tertiary orientation for active ribonuclease polypeptide complexes while rearranging the primary sequence of ribonucleic acids.

[0018] Among other things, the present disclosure provides an RNA that is a variant of a reference sgRNA (e.g., AGNT246 or AGNT248) in that (a) arrangement of one or more of a crispr sequence element, a tracr sequence element, a spacer sequence element, and a stem-loop element is structurally permuted relative to its arrangement relative to other of the sequence elements in a reference sgRNA disclosed herein, and optionally, (b) one or more of sequence elements disclosed herein is altered relative to that found in a reference disclosed herein.

[0019] In some embodiments, one or more sequence elements disclosed herein is selected from the group consisting of (i) stem-loop -1, which is altered in that it is appended to a reference sequence lacking this stem-loop, its hairpin is extended, it includes one or more non-canonical residues, or is modified in length and sequence composition, or it is eliminated, (ii) stem-loop 0, which is altered in that it is appended to a reference sequence lacking this stem-loop, its hairpin is extended, a linking loop is added, it includes one or more non-canonical residues, or is modified in length and sequence composition, or it is eliminated, (iv) stem-loop 2, which is extended or truncated, or which contains one or more base pair or residue substitution that match nucleotides present within other Casl2f homologues, or contains 1, 2, or 3 unique single residue or base pair changes, and / or contains one or more residue substitution with a non-canonical residue, (v) stem-loop 3, which is extended or truncated and / or contains one or more residue substitutions with a canonical or non-canonical residue, or is eliminated, (vi) stem-loop 4, which is truncated and / or contains one or more non-canonical residues, or is eliminated, (vii) a tracncrispr stem that is truncated or extended, or which contains one or more base pair or residue substitution that match nucleotides present within other Casl2f homologues, or contains a unique base pair or residue substitution, and / or that includes one or more base pair substitutions with canonical or non-canonical residues, (viii) one or more loops is longer or shorter and / or is altered to be complementary with a single-stranded section elsewhere in the RNA, (ix) loop 1-crispr 3' end homology that is extended and / or that includes one or more residue insertions or substitutions with canonical or non-canonical residues, and (x) one or more unpaired sequences between stem-loops that is truncated orextended and / or that includes one or more nucleotide substitutions with canonical or non-canonical residues, and (xi) combinations thereof. In some embodiments, an arrangement described herein that is structurally permuted is selected from the group consisting of: (i) stem-loop -1 is positioned within the tracr sequence or at the 5' end of an RNA disclosed herein, (ii) stem-loop 0 is internalized or artificially linked, (iii) an intra-RNA covalent linkage is created by opening a loop and generating a new bond to a site elsewhere in an RNA disclosed herein, (iv) spacing between or among sequence elements is altered, and (v) combinations thereof.

[0020] In some embodiments, an RNA disclosed herein includes a stem-loop 1 sequence element whose sequence is unaltered. In some embodiments, a stem-loop 1 sequence is selected from the following: (i) 5' - AGGGCAGUUAGGUGCCCU - 3', or (ii) 5' - UCGUCGGUUCAGCGACGA - 3'.

[0021] In some embodiments, an RNA disclosed herein is characterized in that it complexes with a Casl2f polypeptide. In some embodiments, is characterized in that, when a complex of an RNA disclosed herein and a Casl2f polypeptide described herein is present in a cell, editing is detectable at a site targeted by the RNA's crispr sequence element. In some embodiments, detectable editing is greater than about 1% INDEL. In some embodiments, an RNA disclosed herein is characterized in that it complexes with a plurality of different Casl2f polypeptides. In some embodiments, a plurality of different Casl2f polypeptides described herein includes at least two Casl2f polypeptides selected from the group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

[0022] Among other things, the present disclosure provides a complex comprising an RNA disclosed herein with a Cas polypeptide. In some embodiments, a Cas polypeptide described herein is a Casl2f polypeptide. In some embodiments, a Casl2f polypeptide described herein is selected from the group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

[0023] Among other things, the present disclosure provides an in vitro system comprising a complex disclosed herein and a target nucleic acid targeted by a crispr sequence element of a RNA disclosed herein.

[0024] Among other things, the present disclosure provides a mammalian cell engineered to contain or express a complex disclosed herein.

[0025] Among other things, the present disclosure provides a plant cell engineered to contain or express a complex disclosed herein.

[0026] Among other things, the present disclosure provides a nucleic acid that encodes an RNA disclosed herein. In some embodiments, a nucleic acid disclosed herein also encodes a Casl2f polypeptide. In some embodiments, a nucleic acid disclosed herein is a template whose transcription produces the RNA. In some embodiments, a nucleic acid disclosed herein is a plasmid. In some embodiments, a nucleic acid disclosed herein is a vector.

[0027] Among other things, the present disclosure provides a viral vector that encodes an RNA disclosed herein.

[0028] Among other things, the present disclosure provides a vector system comprising a first vector that encodes an RNA disclosed herein and a second vector that encodes a Casl2f polypeptide.

[0029] Among other things, the present disclosure provides a pharmaceutical composition comprising a RNA disclosed herein and a pharmaceutically acceptable excipient.

[0030] Among other things, the present disclosure provides a method of making an RNA disclosed herein comprising incubating a nucleic acid disclosed herein with (i) an RNA polymerase and (ii) ribonucleotides. In some embodiments, ribonucleotides described herein comprise one or more naturally occurring ribonucleotides. In some embodiments, ribonucleotides described herein comprise one or more modified ribonucleotides. In some embodiments, a polymerase described herein is T7 RNA polymerase.

[0031] Among other things, the present disclosure provides a method of making a complex disclosed herein, wherein the method comprises a step of contacting an RNA disclosed herein with a Cas polypeptide described herein. In some embodiments, contacting described herein is in vitro. In some embodiments, contacting described herein is in vivo. In some embodiments, contacting described herein comprises expressing an RNA disclosed herein in a cell that contains a Cas polypeptide. In some embodiments, contacting described herein comprises expressing a Cas polypeptide described herein in a cell that contains an RNA disclosed herein. In some embodiments, a Cas polypeptide is a Casl2f polypeptide. In some embodiments, a Casl2f polypeptide is selected from a group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

[0032] Among other things, the present disclosure provides a method of making a pharmaceutical composition, the method comprising a step of combining an RNA disclosed herein or a complex disclosed herein or a nucleic acid encoding an RNA disclosed herein, or a cell expressing the vector system disclosed herein, with one or more pharmaceutically acceptable carriers or excipients.

[0033] Among other things, the present disclosure provides a method of treating a disease, disorder, or condition, associated with a target sequence, the method comprising a step of modifying a target genetic sequence described herein by contacting it with a complex disclosed herein, wherein a crispr sequence element of an RNA disclosed herein is substantially complementary to said target genetic sequence.

[0034] Among other things, the present disclosure provides a method of modifying a target sequence with a complex disclosed herein. In some embodiments, a target sequence described herein is in vitro. In some embodiments, a target sequence described herein is in vivo.

[0035] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.BRIEF DESCRIPTIONS OF THE DRAWING

[0036] Figure 1 shows a proposed secondary structure diagram and stem-loop (SL) numbering scheme applied to three guide chassis: the wild type sequence, MS13 truncation sgRNA, and AGNT35. Boxed nucleotides are synthetic linker sequences.

[0037] Figure 2 shows example circularly permuted guide chassis with additional homology added to the crispr- tracr loop and the 5' end, as described in the present disclosure.

[0038] Figure 3 shows examples of circularly permuted and then truncated guide chassis, as described in the present disclosure.

[0039] Figure 4 shows example guide chassis starting from MS13 that are further truncated at one or more stemloops, as described in the present disclosure.

[0040] Figure 5 shows example guide chassis starting from circularly permuted and truncated AGNT47 with additions or modifications to the crispr-tracr stem-loop, as described in the present disclosure.

[0041] Figure 6 shows example guide chassis with modifications to the homology region between stem-loop 1 and the 3'end of the guide chassis, as described in the present disclosure.

[0042] Figure 7 shows example guide chassis permuted to internalize the spacer sequence by connecting to stemloop 0, combined with different truncations and linker sequences, as described in the present disclosure.

[0043] Figure 8 shows example guide chassis permuted to internalize the spacer sequence with truncations or adding back wild type sequence, as well as to add Twister Ribozymes, as described in the present disclosure.

[0044] Figure 9 shows a guide chassis diagram with example nucleotides identified that when synthesized with 2'- O-methyl improve, reduce or are neutral to activity, as described in the present disclosure.

[0045] Figure 10 shows example guide chassis sequences that contain MS2 hairpin insertions, as described in the present disclosure.

[0046] Figure 11 shows results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0047] Figure 12 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0048] Figure 13 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of Casl2f nucleases with guides of the present disclosure.

[0049] Figure 14 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0050] Figure 15 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0051] Figures 16A-16B shows sequencing and flow-cytometry results of T cell, RNA electroporation-based assays to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0052] Figures 17A-17C shows sequencing results of Primary Human Hepatocyte (PHH), RNA transfection-based assays to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0053] Figures 18A-18B shows sequencing results of a Primary Human Hepatocyte (PHH), LNP transfectionbased assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0054] Figure 19 shows flow-cytometry results of a HEK293T, plasmid transfection-based assay to determine gene activation with nuclease deficient Casl2f and guides of the present disclosure.

[0055] Figure 20 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0056] Figure 21 shows sequencing results of a Primary Human Hepatocyte (PHH), RNA transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0057] Figure 22 shows example guide chassis sequences engineered to contain sequences to be used as templates, denoted as Poly-X, for editing at targeted sites, as described in the present disclosure.

[0058] Figure 23 shows sequencing results of a Primary Human Hepatocyte (PHH), RNA transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0059] Figures 24A-24D show exemplary guide chassis. Figure 24A shows an exemplary AsCasl2f sgRNA from Wu et al., 2023, with a stem-loop (SL) numbering scheme applied. Figure 24B shows an exemplary truncated AsCasl2f sgRNA from Wu et al., 2023, with a stem-loop (SL) numbering scheme applied. Figure 24C shows an exemplary AsCasl2f stem-loop 2 (SL2) from Hino et aL, 2023. Figure 24D shows a proposed secondary structure diagram for SpaCasl2f sgRNA with a stem-loop (SL) numbering scheme applied.

[0060] Figures 25A-25B show sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure. Figure 25A shows cleavage activity of guide chassis generated from AGNT170 compared to MS13. Figure 25B shows cleavage activity of guide chassis generated from AGNT35 compared to MS13.

[0061] Figure 26 shows proposed secondary structure diagrams for AGNT246, AGNT164, and AGNT1196.

[0062] Figure 27 shows a proposed secondary structure diagram for AGNT246 with a PID interacting region emphasized.

[0063] Figure 28 shows sequencing results of a HEK293T, mRNA transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0064] Figure 29 shows sequencing results of a T cell, mRNA electroporation-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0065] Figure 30 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0066] Figure 31 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0067] Figure 32 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0068] Figure 33 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0069] Figure 34 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0070] Figure 35 shows sequencing results of a HEK293T and T cell, mRNA transfection and electroporation respectively based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0071] Figure 36 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.

[0072] Figure 37 shows sequencing results of a HEK293T, plasmid transfection-based assay to determine targeted cleavage activity of a Casl2f nuclease with guides of the present disclosure.CERTAIN DEFINITIONS

[0073] The scope of the present disclosure is defined by the claims appended hereto and is not limited by certain embodiments described herein. Those skilled in the art, reading the present specification, will be aware of various modifications that may be equivalent to such described embodiments, or otherwise within the scope of the claims. In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0074] Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0075] The articles "a" and "an," as used herein, should be understood to include the plural referents unless clearly indicated to the contrary. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process. It is to be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists (e.g., in Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects are referred to as "comprising" particular elements, features, etc., certain embodiments or aspects "consist," or "consist essentially of," such elements, features, etc. For purposes of simplicity, those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0076] Throughout the specification, whenever a polynucleotide, polyribonucleotide or polypeptide is represented by a sequence of letters (e.g., A, C, G, and T which denote adenosine, cytidine, guanosine, and thymidine, respectively in the case of a polynucleotide or A, C, G, and U which denote adenosine, cytidine, guanosine, and uracil respectively in the case of a polyribonucleotide), such polynucleotides, polyribonucleotide or polypeptides are presented in 5' to 3' or N-terminus to C-terminus order, from left to right.

[0077] About or approximately. As used herein, the terms "about" and "approximately," when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0078] Administration: As used herein, the term "administration" typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment. In some embodiments, an administered composition on treatment includes the agent to be delivered. In some embodiments, an administered composition or treatment may include a precursor (e.g., a prodrug of the agent, a cell that produces the agent, a nucleic acid that encodes the agent, etc) of the agent, such that the agent is released or generated upon administration of the composition or treatment.

[0079] Agent : In general, the term "agent", as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism, vesicle, micelle, viral particle, virus-like-particle, LNP, etc.] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc).

[0080] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of the polymer. In some embodiments, presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers). In some embodiments, a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share the sequence element.

[0081] Comparable: As used herein, the term "comparable" refers to two or more agents (e.g., entities or set(s) of conditions), situations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0082] Conservative: As used herein, the term "conservative" refers to instances describing a conservative amino acid substitution, including a substitution of an amino acid residue by another amino acid residue having a side chain R group with similar structural, chemical e.g., charge or hydrophobicity), and / or functional properties. Those skilled in the art will appreciate that a conservative amino acid substitution often will not substantially change functional properties of interest of a protein. Examples of groups of amino acids that have side chains that are typically considered to have similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Vai, V), leucine (Leu, L), and isoleucine (lie, I); aliphatic-hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gin, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine (Cys, C) and methionine (Met, M). In some embodiments, conservative amino acids substitution groups may include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamate / aspartate (Glu / Asp, E / D), and asparagine / glutamine (Asn / GIn, N / Q). In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et ai., Science 256: 1443, 1992, which is incorporated herein by reference in its entirety. In some embodiments, a substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix. One skilled in the art would appreciate that a change (e.g., substitution, addition, deletion, etc.) of amino acids that are not conserved between the same protein from different species is less likely to have an effect on the function of a protein and therefore, these amino acids should be selected for mutation. In some embodiments, a "conservative" substitution is considered a "homologous" residue for purposes of calculating percent homology between amino acid sequences.

[0083] Corresponding to. As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding td' a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rathercorresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify " corresponding amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term "corresponding to" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0084] Designed: As used herein, the term "designed" refers to a feature of having been conceived, created, and / or generated through an act of the hand of man. For example, the term may be used to refer to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0085] Engineered: In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man (e.g., by having been designed) to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be "engineered" if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be "engineered" if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered e.g, new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as "engineered" even though the actual manipulation was performed on a prior entity.

[0086] Homology. As used herein, the term "homology" refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules {e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules (or portions thereof) are considered to be "substantially homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous, meaning that identical or homologous (e.g., representing conservative substitutions) residues are present in corresponding positions of both molecules. Calculation of percent homology of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes {e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position. When a position in the first sequence is occupied by the same residue or by a structurally and / or functionally related residue (as will be understood by those skilled in the art, in context), then the two molecules are considered "homologous" at that position. Percent homology between two sequences is a function of the number of homologous positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. For example, percent homology between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In many embodiments, full-length sequences may be considered to be homologous if they share one or more characteristic sequence elements of particularly high homology (or identity) and / or share an overall level of homology (which may be significantly lower than that shared by the characteristic sequence elements) above a threshold; those skilled in the art are familiar with classification systems for determining when such threshold(s) have been met.

[0087] Nucleic acid'. As used herein, in its broadest sense, refers to a compound and / or substance that is, or can be incorporated into, a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is, or can be incorporated into, a polynucleotide chain with a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid' refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, " nucleic acid' refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a " nucleic acid' is or comprises ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a 0-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), or a combination thereof. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid residue analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that one or more residues, and insome embodiments, are linked together other than by a phosphodiester. For example, in some embodiments, a nucleic acid includes one or more phosphorothioate and / or phosphoroamidite (e.g., 5'-N-phosphoramidite) linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template in vivo or in vitro), enzymatic synthesis in the absence of a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0088] Nudeobase. The term "nucleobase" refers to the parts of nucleic acids that are involved in the hydrogenbonding that binds one nucleic acid strand or sequence element to another complementary strand or sequence element in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is substituted A, T, C, G or U. In some embodiments, a modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobases is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting themelting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. In some embodiments, as will be understood by those skilled in the art, the term "nucleobase" encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, a "nucleobase" refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).

[0089] Nucleoside-. The term "nucleoside" refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a "nucleoside" refers to a nucleoside unit in an oligonucleotide or a nucleic acid.

[0090] Nucleotide: The term "nucleotide" as used herein refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more internudeotidic linkages (e.g., phosphate linkages in natural DNA and RNA). The naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that, in various embodiments, as will be clear to those skilled in the art, naturally and non-naturally occurring sugar analogs are included. Nucleotides are linked via internudeotidic linkages to form nucleic acids, or polynucleotides. Various internudeotidic linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H -phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids. In some embodiments, a natural nucleotide comprises a naturally occurring base, sugar and internudeotidic linkage. As used herein, the term "nucleotide" also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, a "nucleotide" refers to a nucleotide unit in a polynucleotide.

[0091] Operably linked', as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element " operably linked' to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, " operably linked' control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.

[0092] Pharmaceutical composition-. As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in a particular form (e.g., in a solid form or a liquid form), and / or may be specifically adapted for, for example: oral administration (for example, as a drenche [aqueous or non-aqueous solutions or suspensions], tablet, capsule, bolus, powder, granule, paste, etc, which may be formulated specifically for example for buccal, sublingual, or systemic absorption); parenteral administration (for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation, etc); topical application (for example, as a cream, ointment, patch or spray applied for example to skin, lungs, or oral cavity); intravaginal or intrarectal administration (for example, as a pessary, suppository, cream, or foam); ocular administration; nasal or pulmonary administration, etc.

[0093] Polypeptide: As used herein refers to a polymeric chain of amino acids, typically including at least three or more amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature; in some embodiments, such a polypeptide may be referred to and / or utilized herein as a reference polypeptide. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N- terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise acharacteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0094] Polyribonucleotide-. As used herein, the term "polyribonucleotide" refers to a polymer of 3 ribonucleotides or more. In some embodiments, a polyribonucleotide is single stranded. In some embodiments, a polyribonucleotide is double stranded. In some embodiments, a polyribonucleotide comprises both single and double stranded portions. In some embodiments, a polyribonucleotide can comprise a backbone structure as described in the definition of "Nucleic acid / Oligonucleotide" above. A polyribonucleotide can be a regulatory RNA {e.g., siRNA, microRNA, etc.}, or a messenger RNA (mRNA) oligonucleotide. In some embodiments where a polyribonucleotide is a mRNA oligonucleotide, a polyribonucleotide typically comprises at its 3' end a poly(A) region. In some embodiments where a polyribonucleotide is an mRNA oligonucleotide, a polyribonucleotide typically comprises at its 5' end an art- recognized cap structure, e.g., for recognizing and attachment of an mRNA to a ribosome to initiate translation. In some embodiments, a polyribonucleotide comprises an RNA oligonucleotide. When a number of ribonucleotides is used as an indication of size, e.g., for a polyribonucleotide, a certain number of nucleotides refers to the number of ribonucleotides on a single strand.

[0095] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0096] Source: t\e term "source" as used herein, typically refers to a context in which an agent of interest {e.g., that may be or comprise a carbohydrate, a lipid, a nucleic acid, a metal, polypeptide, a small molecule, or a combination thereof) may be found in nature, or from which such agent can be or has been obtained {e.g., isolated). In some embodiments, a source may be or comprise a biological source {e.g., an organism, tissue, or cell, or sample thereof); in some embodiments a source may be an environmental source. In some embodiments, a source may be or comprise a primary sample from an organism {e.g., which may be or comprise a tissue or fluid of such organism, and / or may be or comprise cell(s) of such organism). In some embodiments, an organism may be or comprise aprokaryotic organism {e.g., a bacterium) or a eukaryotic organism e.g, a fungus or yeast, an insect, a mammal, a plant, a reptile, etc.'). In some embodiments, an infectious agent such as a virus or phage may be considered an organism for purposes of this disclosure, and in particular with respect to being a source. In some embodiments, a source may be or comprise an engineered source, such as a cell line or culture, an in vitro system, etc.

[0097] Stable: The term "stable," when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure and / or activity over a period of time under a designated set of conditions. In some embodiments, the period of time is at least about one hour; in some embodiments the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the period of time is within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. In some embodiments, the designated conditions are ambient conditions (e.g., at room temperature and ambient pressure). In some embodiments, the designated conditions are physiologic conditions (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline). In some embodiments, the designated conditions are under cold storage (e.g., at or below about 4 °C, -20 °C, or -70 °C). In some embodiments, the designated conditions are in the dark.

[0098] Treatment. As used herein, the term "treatment" (also "treat" or "treating") refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0099] Variant: As used herein, the term "variant" refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocycle core) and / or one or more characteristic pendent moieties so that a variant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and / or in types of bondspresent (single vs double, E vs Z, etc) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three- dimensional space and / or contributing to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to one another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc) covalently attached to the polypeptide backbone. In some embodiments, a variant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, optionally other than conservative amino acid substitutions. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide. In many embodiments, a polypeptide of interest is considered to be a "variant" of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in a variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue(s) as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, a parent or reference polypeptide is one found in nature.[O1OO] Vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid', which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."[O1O1] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known inthe art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0102] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated polypeptide (Cas) systems have been developed as versatile genome editing tools.

[0103] In some embodiments, CRISPR-Cas systems may be functionalized with various effector polypeptides to enable programmed editing of a genome. In some embodiments, CRISPR-Cas systems may be functionalized with various effector polypeptides to enable programmed editing of an epigenome. In some embodiments, CRISPR-Cas systems may be functionalized with various effector polypeptides to enable programmed editing of a transcriptome. In some embodiments, CRISPR-Cas systems may be functionalized with various effector polypeptides to enable programmed editing of an epitranscriptome.

[0104] The present disclosure, among other things, provides certain insights and technologies related to improved Cas systems. In particular, the present disclosure provides insights and technologies relating to Casl2f systems (e.g., RNAs).CRISPR-Cas systems

[0105] The present disclosure, among other things, provides certain Casl2 guide RNAs (e.g., engineered Casl2 guide RNAs), systems that include and / or utilize them, and related technologies (e.g., methods). In some embodiments, such provided Casl2 guide RNAs are improved in one or more respects relative to an appropriate reference guide RNA (e.g., the SpaCasl2fl guide RNA).

[0106] One of skill in the art will understand a CRISPR-Cas system comprises a guide RNA (herein "gRNA") and a Cas nuclease, which together form a ribonucleopolypeptide complex. The Cas nuclease may have nuclease activity against one strand of a target DNA sequence, against both strands of a target DNA sequence, or may not have nuclease activity against either strand of the target DNA sequence. A variety of review articles and other resources are available that described, for example, characteristics of one or more CRISPR / Cas systems, relationships between and / or among them, and uses for them (see, e.g., Hillary & Ceasar Mol. Biotechno / 65:311, March 2023, doi: 10.1007 / S12033-022-00567-0. Epub 2022 Sep 27; see also "The Complete Guide to Understanding CRISPR sgRNA" as provided by Synthego at its web site, e.g. as of March 19 2024: synthego.com / guide / how-to-use-crispr / sgrna; each of these is incorporated herein by reference in its entirety).

[0107] In some embodiments, a gRNA comprises distinct crispr RNA (herein "crRNA") and tracrRNA components; in some such embodiments, a crRNA includes a nucleotide sequence element (typically about 17 to about 20 residues in length) complementary to target DNA, and a tracrRNA (typically about 50 to about 180 residues in length) serves as a binding scaffold for a Cas polypeptide.

[0108] In some embodiments, a guide RNA comprises a single guide RNA (herein "sgRNA"), wherein a crRNA and tracrRNA components are included in a single molecule. In many embodiments, an sgRNA in accordance with the present disclosure is an engineered sgRNA.

[0109] Those skilled in the art will be aware of various technologies for producing gRNAs, specifically including sgRNAs (e.g., engineered sgRNAs). In some embodiments, an sgRNA is generated in vitro. In some embodiments, an sgRNA is generated in vivo. In some embodiments, an sgRNA is generated by chemical synthesis. In some embodiments, an sgRNA is generated by enzymatic synthesis; in some such embodiments, an sgRNA is generated by transcription. In some embodiments, an sgRNA is generated by in vitro transcription; in some embodiments, an sgRNA is generated by in vivo transcription.

[0110] One of skill in the art will recognize that in some embodiments, a Cas nuclease may recognize and bind to a specific protospacer adjacent motif (PAM) sequence in genomic DNA. The presence of a specific PAM sequence next to the sequence targeted by the guide RNA (spacer) may be required in some embodiments for the Cas nuclease to recognize and bind to a specific sequence in genomic DNA.

[0111] In some embodiments, a Cas nuclease may make a double-strand break in DNA. In some embodiments, endogenous repair mechanisms triggered by a double-strand break may result in gene knockout by deleting nucleotides from the gene, or by inserting nucleotides into the gene. In some embodiments the insertion or deletion may result in a frameshift. In some embodiments, a double strand break may result in knock-in of a desired sequence, which may be referred to herein as a "donor" sequence. See, for example, Lee, HK et al., "Development of CRISPR technology for precise single-base genome editing: a brief review," BMB Rep, Feb;54(2):98-105, 2021, which is incorporated herein by reference. In some embodiments, a donor sequence may be heterologous to the cell or system in which Cas-directed editing is taking place. In some embodiments, a donor sequence may be endogenous to the cell or system in which Cas-directed editing is taking place.

[0112] CRISPR-Cas systems are broadly distributed in bacteria and archaea. A rubric has been developed under which CRISPR-Cas systems may be divided into two classes (class 1 and 2) and six major types (I-VI) based on the numbers and configurations of the interference module. In some embodiments, class 1 systems include, but are not limited to, type I, type, II, and IV and class 2 systems comprise type II, type V, and type VI (Wang et al., 2022).

[0113] Certain Cas polypeptides, including for example Streptococcus pyogenes Cas9 (SpCas9, type II-A) and Acidaminococcussp. BV3L6 Casl2a (AsCasl2a, type V-A) have shown particularly potent gene-editing activity in a variety of environments. See, for example, Wu et al. See also, for example, Pickar-Oliver & Gersbach, The next generation of CRISPR-Cas technologies and applications. Nat Rev Mo! Cell Biol. 20 5Aug;2O(8):49O-5O7. Doi: 10.1038 / s41580-019-0131-5, which is incorporated herein by reference. One of skill in the art will recognize a challenge of Cas9 and VA-Casl2a effectors is that their large sizes limit cellular delivery that may restrict certain applications, including therapeutics. See, for example Raguram, A. et al., "Therapeutic in vivo delivery of gene editing agents," Cell. 2022 Jul 21;185(15):2806-2827. Doi: 10.1016 / j.cell.2022.03.045, which is incorporated herein by reference. See also, for example, Kabadi, A. et al., "AAV-based CRISPR-Cas9 genome editing: Challenges and engineering opportunities," Current Opinion in Biomedical Engineering, 2024, doi.org / 10.1016 / j.cobme.2023.100517, which is incorporated herein by reference.

[0114] The present disclosure appreciates that more compact CRISPR-Cas systems can simplify delivery and extend application. Among other things, the present disclosure appreciates that Casl2 systems (e.g., Casl2f system) may be particularly useful and / or valuable in certain context. The present disclosure further appreciates that improvements can be made even in Casl2 systems (e.g., Casl2f systems).

[0115] Among other things, the present disclosure provides Casl2 (e.g., Casl2f) guide RNAs, systems that include and / or utilize them, and associated technologies (e.g., methods of making and / or using them).

[0116] In many embodiments, provided guide RNAs are shorter than canonical Casl2 (e.g., Casl2f) guide RNAs (e.g., than one or more reference guide RNAs as described herein).

[0117] The present disclosure provides certain insights to advantages of shorter guide RNAs disclosed herein.

[0118] In some embodiments, shorter guide RNAs disclosed herein may have improved chemical synthesis in one or more compared to one or more reference RNAs. In some embodiments, shorter guide RNAs disclosed herein may have less synthesis-associated errors compared to one or more reference RNAs compared to one or more reference RNAs.

[0119] In some embodiments, shorter guide RNAs disclosed herein may have higher specific activity compared to one or more reference RNAs..In some embodiments, shorter guide RNAs may require lower dosing (e.g., fewer mg of RNA per unit of CRISPR / Cas activity, and / or smaller mass amount of RNA administered to achieve a comparable level of CRISPR / Cas activity, etc.) compared to one or more reference RNAs.

[0120] The present disclosure recognizes that dsRNA is recognized by innate immune systems.

[0121] In some embodiments, shorter guide RNAs may have reduced secondary or tertiary structures compared to one or more reference RNAs.

[0122] In some embodiments, shorter guide RNAs may display lower immunogenicity compared to one or more reference RNAs.

[0123] Among other things, the present disclosure demonstrates that certain provided guide RNAs are useful with more than one different Cas polypeptide (e.g., with more than one Casl2 polypeptide, e.g., with more than one Casl2f polypeptide).

[0124] In many embodiments, the present disclosure provides guide RNAs that are particularly useful with a Casl2 polypeptide (e.g., with a Casl2f polypeptide), e.g., in particular with a SpaCasl2fl.Miniature CRISPR-Cas Systems

[0125] One of skill of the art will understand that one ongoing challenge of genome editing is the ability to package CRISPR-associated Cas systems for cell delivery. One of skill in the art will appreciate that so-called "miniature" CRISPR-Cas systems have been developed for genome editing applications because their reduced size can improve cell delivery. In some embodiments, miniature CRISPR-Cas systems may be packaged for cell delivery. In some embodiments, miniature CRISPR-Cas systems are small enough to be packaged into single adeno-associated viral (AAV) particles.

[0126] One of skill in the art will recognize that several compact Cas polypeptides (e.g., that may be useful in a "miniature" CRISPR-Cas system) have been reported, including CasX (also known as Casl2e, V-E, 986 amino acids), Cas (also known as Casl2j, V-J, 700-800 amino acids) and Casl2f (also known as Casl4, V-F, 400-700 amino acids). Without wishing to be bound by any particular theory, Casl2f polypeptides are of particular interest given their small sizes and unique dimerization-mediated DNA-targeting mechanism.CRISPR-Casl2f systems

[0127] In many embodiments, the present disclosure provides insights and technologies relevant to CRISPR-Casl2f systems. In many embodiments, provided insights and technologies are particularly relevant to CRISPR-SpaCasl2fl systems. The present disclosure also surprisingly documents circumstances in which provided insights and technologies are or may be applicable to other Casl2f systems (e.g., as an alternative to or, particularly remarkably, in addition to, SpaCasl2fl systems).

[0128] One of skill in the art will understand CRISPR-Cas systems are broadly distributed in bacteria and archaea with functional diversity. See, for example, Wu et al. In some embodiments, CRISPR-Cas systems may be divided into two classes (class 1 and 2) and six major types (I-VI) based on the numbers and configurations of the interference module. In some embodiments, class 1 systems include type I, type, II, and IV and class 2 systems comprise type II, type V, and type VI.

[0129] One of skill in the art will appreciate small CRISPR-associated effector polypeptides belonging to the type V- F subtype have been identified, for example through mining of sequence databases, wherein locus architecture for some of these systems include genes (. casl, cas2, cas4, casl4) that encode adaption molecules. In some embodiments, a small CRISPR-associated effector family is grouped into a family. In some embodiments, a CRISPR- associated effector polypeptide is grouped into a Casl2f2 (also known as Casl4b) family. In some embodiments, a CRISPR-associated effector polypeptide is grouped into a Casl2f3 (also known as Casl4c, type V-U2 and U4) family. In some embodiments, a CRISPR-associated effector polypeptide is grouped into a Casl2fl (also known as Casl4a and type V-U3) family.

[0130] One of skill in the art will recognize that Casl2f, also known as Casl4, is a family of compact RNA-guided nucleases that belong to the class 2 type V-F CRISPR-associated effector nuclease family. In some embodiments, Casl2f polypeptides are about 400-700 amino acids in length (Wang et al.). In some embodiments, Casl2f polypeptides from V-U3 and Casl4 families) are nearly half the size of the smallest Cas9 or Casl2 nucleases. See, for example, Karvelis et al. In some embodiments, the N-terminal half of Casl2f polypeptides differs significantly in length compared to Casl2 orthologs, accounting for size differences between two groups.

[0131] A person of skill in the art will appreciate that Casl2f nucleases are one of the most compact CRISPR-Cas genome editors. In some embodiments, Casl2f nucleases cleave dsDNA with the aid of a gRNA. Alternatively or additionally, in some embodiments, Casl2f polypeptides cleave single-stranded DNA (ssDNA) with the aid of a gRNA.

[0132] In some embodiments, Casl2f polypeptides, like most other Casl2 polypeptides, are able to cleave dsDNA targets if a 5'PAM sequence is present in the vicinity of the guide RNA target. In some embodiments, Casl2f targets PAM sequences comprising 5' T-rich sequences. In some embodiments, type V-U3 PAM recognition includes C- and T-rich motifs.

[0133] Those skilled in the art will be aware of a variety of Casl2 polypeptides, and specifically Casl2f polypeptides, that have been identified in nature or otherwise described in the art; in many embodiments one or more such Cas21f polypeptides can be utilized as a Casl2 (e.g., Casl2f) polypeptide, e.g., in a CRISPR-Cas system, as described herein. Alternatively or additionally, in some embodiments, such Casl2f polypeptides can be used as a "reference" Casl2 and / or Casl2f polypeptide described utilized herein. In some embodiments, a Casl2f polypeptide(e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) (a / k / a a "Casl2f polypeptide" is MilCasl2f2 or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is UnlCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is Un2Casl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide ((e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is Mi2Casl2f2 or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is AuCasl2f2 or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is PtCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is RuCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is CnCasl2f or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is RhCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is OsCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is CnCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is AllCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is AI2Casl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is TilCasl2fl or a relevant (e.g., functional or characteristic) portion thereof.

[0134] In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is SpaCasl2fl or a relevant (e.g., functional or characteristic) portion thereof. In some embodiments, a Casl2f polypeptide (e.g., a utilized Casl2f polypeptide and / or a reference Casl2f polypeptide) is AsCasl2fl or a relevant (e.g., functional or characteristic) portion thereof.CRISPR-SpaCasl2fl and CRISPR-AsCasl2fl Systems

[0135] The present disclosure appreciates that certain Casl2f systems, specifically including SpaCasl2fl (also known as "SpCasl2fl", "SpCasl2f", or "SpaCasl2f") and AsCasl2fl, are compact genome editors. However, the present disclosure understands limitations have been reported to present challenges such as, for example, relatively low editing activity (see, for example, Wang, Y. et al., "Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Casl2fl nuclease." Cell Rep., 40(13): 111418, 2022), e.g., in mammalian cells.

[0136] For example, the present disclosure appreciates that many SpaCasl2fl guides are not stable and / or can be challenging to generate and or use, for example due to their size and / or to their lack of optimization for human expression.

[0137] A person of skill in the art will recognize that MS13 is an engineered SpaCasl2fl guide (Wang et al., 2022). In many embodiments, MS13 demonstrates significantly improved gene editing efficiency, for example as compared with an appropriate reference (e.g., a naturally-occurring SpaCasl2fl guide).

[0138] A person of skill in the art will recognize that engineered AsCasl2fl guides that also have been reported to demonstrate improved gene editing efficiency have been generated. (Wu et al., 2019).

[0139] In some embodiments, CRISPR-SpaCasl2fl tracrRNA harbors a unique head-to-toe hairpin structure. In some embodiments, MS13 harbors a stem-loop 0 truncation in the head-to-toe hairpin structure of the tracrRNA (see Wang et al., 2022).

[0140] The present disclosure recognizes that although MS13 has improved activity through reduced size, one limitation is that MS13 is physically constrained to maintain this structure.

[0141] The present disclosure, among other things, provides technologies to remove requirements of extra looping domains to enable successful truncations of additional loops.

[0142] The present disclosure, among other things, provides insights regarding improved CRISPR-Cas systems, specifically including improved "miniature" systems, such as improved CRISPR-Casl2 systems, e.g., improved CRISPR-Casl2f systems. For example, the present disclosure appreciates sequence elements found in certain CRISPR guide RNAs, specifically including in an MS13 RNA or an engineered AsCasl2fl RNA, can achieve certain benefits, including, in some embodiments, amenability to further truncation.

[0143] A person of skill in the art, will appreciate upon reading this disclosure, that in some embodiments, predicted base pairs may be conserved between AsCasl2fl guides and SpaCasl2fl guides.Provided RNAs

[0144] Among other things, the present disclosure provides engineered guide RNAs, and related technologies, including for example, CRISPR-Cas systems that include them, templates that encode them, cells that express or otherwise contain them (and / or CRISPR-Cas systems that include engineered guide RNAs), etc.

[0145] In some embodiments, the present disclosure provides engineered Casl2 guide RNAs. In some embodiments, the present disclosure provides engineered Casl2f guide RNAs, for example engineered Casl2fl RNAs, including specifically engineered SpaCasl2fl guide RNAs. In some embodiments, engineered Casl2fl RNAs include engineered AsCasl2fl guide RNAs. In some embodiments, provided RNAs are variants of a reference guide RNA, such as of a naturally occurring Casl2, e.g., Casl2f such as Casl2fl, and specifically such as SpaCasl2fl, guide RNA and / or of an engineered Casl2, e.g., Casl2f such as Casl2fl, and specifically such as SpaCasl2fl guide RNA, such as of an MS13 RNA. The present disclosure appreciates that many SpaCasl2fl guides are not stable and / or can be challenging to generate and or use, for example due to their size and / or to their lack of optimization for human expression.

[0146] In many embodiments, a provided guide RNA is a single guide RNA ("sgRNA").

[0147] In some embodiments, a sgRNA disclosed herein is engineered to comprise a chassis and a spacer element. As used herein, the term "chassis" refers to all components of a guide RNA except a spacer element. In some embodiments, a chassis comprises a tracrRNA sequence element, a crRNA sequence element, and a linker sequence element disclosed herein.

[0148] In some embodiments, a chassis is about 70 nt to about 160 nt, about 75 nt to about 160 nt about 80 nt to about 160 nt, about 85 nt to about 160 nt, about 90 nt to about 160 nt, about 95 nt to about 160 nt about 100 nt to about 160 nt, about 115 nt to about 160 nt, about 120 nt to about 160 nt, about 125 nt to about 160 nt, about 130 nt to about 160 nt, about 135 nt to about 160 nt, about 140 nt to about 160 nt, about 145 nt to about 160 nt, about 150 nt to about 160 nt, about 70 nt to about 75 nt, about 70 nt to about 80 nt, about 70 nt to about 85 nt, about 70 nt to about 90 nt, about 70 nt to about 95 nt, about 70 nt to about 100 nt, about 70 nt to about 105 nt, about 70 nt to about 110 nt, about 70 nt to about 115 nt, about 70 nt to about 120 nt, about 70 nt to about 125 nt, about 70 nt to about 130 nt, about 70 nt to about 135 nt, about 70 nt to about 140 nt, about 70 nt to about 145 nt, about 70 nt to about 150 nt, about 80 nt to about 85 nt, about 80 nt to about 90 nt, about 80 nt to about 95 nt, about 80 nt to about 100 nt, about 80 nt to about 110 nt, about 80 nt to about 115 nt, about 80 nt to about 120 nt about 80 nt to about 125 nt, about 80 nt to about 130 nt, about 80 nt to about 135 nt, about 80 nt to about 140 nt, about 80 nt to about 145 nt, about 80 nt to about 150 nt, about 80 nt to about 155 nt in length.

[0149] In some embodiments, a chassis is at most 70 nt to at most 160 nt, at most 75 nt to at most 160 nt at most80 nt to at most 160 nt, at most 85 nt to at most 160 nt, at most 90 nt to at most 160 nt, at most 95 nt to at most 160 nt at most 100 nt to at most 160 nt, at most 115 nt to at most 160 nt, at most 120 nt to at most 160 nt, at most 125 nt to at most 160 nt, at most 130 nt to at most 160 nt, at most 135 nt to at most 160 nt, at most 140 nt to at most 160 nt, at most 145 nt to at most 160 nt, at most 150 nt to at most 160 nt, at most 70 nt to at most 75 nt, at most 70 nt to at most 80 nt, at most 70 nt to at most 85 nt, at most 70 nt to at most 90 nt, at most 70 nt to at most 95 nt, at most 70 nt to at most 100 nt, at most 70 nt to at most 105 nt, at most 70 nt to at most 110 nt, at most 70 nt to at most 115 nt, at most 70 nt to at most 120 nt, at most 70 nt to at most 125 nt, at most 70 nt to at most 130 nt, at most 70 nt to at most 135 nt, at most 70 nt to at most 140 nt, at most 70 nt to at most 145 nt, at most 70 nt to at most 150 nt, at most 80 nt to at most 85 nt, at most 80 nt to at most 90 nt, at most 80 nt to at most 95 nt, at most 80 nt to at most 100 nt, at most 80 nt to at most 110 nt, at most 80 nt to at most 115 nt, at most 80 nt to at most 120 nt at most 80 nt to at most 125 nt, at most 80 nt to at most 130 nt, at most 80 nt to at most 135 nt, at most 80 nt to at most 140 nt, at most 80 nt to at most 145 nt, at most 80 nt to at most 150 nt, or is at most 80 nt to at most 155 nt in length.

[0150] In some embodiments, a chassis is at most 70 nt, at most 75 nt, at most 80 nt, at most 85 nt, at most 90 nt, at most 95 nt, at most 100 nt, at most 110 nt, at most 115 nt, at most 120 nt, at most 125 nt, at most 130 nt, at most 135 nt, at most 140 nt, at most 145 nt, at most 150 nt, at most 155 nt, or is at most 160 nt in length.

[0151] In some embodiments, a chassis is 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt,81 nt, 82 nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, or is 160 nt in length. tracrRNA Sequence Element

[0152] In some embodiments, a tracrRNA sequence element (herein "tracrRNA") serves as a binding scaffold for a Cas nuclease.

[0153] In some embodiments, a tracrRNA comprises a stem-loop 1, with a loop 1 homologous with a crispr 3' end spacer adjacent motif (herein "SAM").

[0154] In some embodiments, a tracrRNA comprises a stem-loop 2 where, starting from linker to stem-loop 1, there is a 5-6bp stem, followed by a bulge, where at least one unpaired nucleotide of the bulge is an A. Downstream of this bulge is a stem-loop that can be a varying length and can be modified with additional break points or hairpins.

[0155] In some embodiments, a tracrRNA comprises a strand to hybridize with crisprRNA in a crisprRNA:tracrRNA (herein "cr:tr") hairpin.

[0156] In some embodiments, a tracrRNA comprises at least stem-loop 1, stem-loop 2, and crispr-complementary sequence elements (e.g., sequence elements with sufficient sequence identity and / or other common structural characteristics, for example as noted herein, that those skilled in the art, reading the present disclosure, would recognize them as corresponding to a stem-loop 1, stem-loop 2, or crispr-complementary sequence element, as is relevant, in accordance with the present disclosure). In some embodiments, a stem-loop 1, stem-loop 2, or crispr- complementary sequence element may harbor a base modification.

[0157] In some embodiments, a tracrRNA may have additional stem-loops added. In some embodiments, a tracrRNA may have a permuted order of stem-loops within a primary sequence. In some embodiments, the elements of a stem-loop may be separated in the primary sequence.

[0158] In some embodiments, a tracrRNA may have a stem-loop 3 (i.e., a sequence element with sufficient sequence identity and / or other common structural characteristics, for example as noted herein, that those skilled in the art, reading the present disclosure, would recognize it as corresponding to a stem-loop 3 sequence element in accordance with the present disclosure). In some embodiments, a tracrRNA stem-loop 3 is greater than 1 nt with a 3 nt loop. In some embodiments, a tracrRNA stem-loop 3 may harbor a base modification.

[0159] In some embodiments, a tracrRNA may have a stem-loop 4 (e.g., a sequence element with sufficient sequence identity and / or other common structural characteristics, for example as noted herein, that those skilled in the art, reading the present disclosure, would recognize it as corresponding to a stem-loop 4 sequence element in accordance with the present disclosure). In some embodiments, a tracrRNA stem-loop 4 is greater than 4 bp with a 3 nt loop. In some embodiments, a tracrRNA stem-loop 4 may harbor a base modification.

[0160] In some embodiments, a tracrRNA may have additional hairpin(s) upstream of a stem-loop 1 sequence element; the present disclosure teaches that, in some embodiments, such additional hairpin(s) may contribute improved stability and / or polypeptide recruitment. In some embodiments, an additional hairpin may harbor a base modification. crRNA Sequence Element

[0161] In some embodiments, a crRNA sequence element (also known as "crispr RNA" or "crRNA") includes a nucleotide sequence element (typically about 17 to about 20 residues in length) complementary to target DNA and a sequence that interacts with a Cas polypeptide. In some embodiments, a crRNA has a sequence that interacts with a tracrRNA.

[0162] In some embodiments, a crRNA immediately upstream of the spacer (3' end) has homology to loop 1.

[0163] In some embodiments, a crRNA has a 3' end spacer adjacent motif (herein "SAM").

[0164] In some embodiments, a crRNA SAM utilized in accordance with the present disclosure can be from 3 to 6 nucleotides with homology to stem-loop 1. In some embodiments, a crRNA SAM utilized in accordance with the present invention has some homology with stem-loop 1 and is extended with additional sequence(s) that may, in some embodiments have some homology with a separate element in the tracr.

[0165] In some embodiments, a crRNA may have an insertion (e.g., relative to an appropriate reference crRNA). In some embodiments, a crRNA may include one or more elements (e.g., hairpin element(s)) bound (e.g., specifically bound) by an RNA binding polypeptide (herein "RBP"); those skilled in the art will be familiar with various such elements, including, for example, Cas6, Csy4, MS2, PCP, TAR, etc., and / or combinations thereof. In some embodiments, an insertion includes a hairpin without an RBP domain.

[0166] In many embodiments, a crRNA and tracrRNA form a cr:tr hairpin.

[0167] In some embodiments, a cr:tr hairpin comprises at least 5 nucleotides from each component hybridized together. In some embodiments, a cr:tr hairpin may contain unpaired bulge(s).

[0168] In some embodiments, a cr:tr hairpin can be a target for base modification,

[0169] In some embodiments, a cr:tr hairpin can be linked onto a single strand. In some embodiments, a cr:tr hairpin can be in adjacent sequence. In some embodiments, a cr:tr hairpin can be non-adjacent sequences.

[0170] In some embodiments, a cr:tr hairpin can be separate RNA strands that hybridize.Spacer Element

[0171] As used herein, the term "spacer" refers to a sequence that may be engineered and / or selected to direct a guide to a target sequence. In many cases, a spacer will have a length within a range between a lower end of about 7 nt, about 8 nt, about 9 nt, about 10 nt, about 11 nt, about 12 nt , about 13 nt , about 14 nt, about 15 nt, 16 nt, about 17 nt, about 18nt, about 19nt, or about 20 nt and an upper end of about 21 nt, about 22 nt, about 23 nt, about 24 nt, or about 25 nt. In some embodiments, a spacer will have a length within a range of about 7 nt to about 24 nt, about 8 nt to about 25 nt, about 9 nt to about 25 nt , about 10 nt to about 25 nt, about 11 nt to about 25 nt, about 12 nt to about 25 nt, about 13 nt to about 25 nt, about 14 nt to about 25 nt, about 15 nt to about 25 nt, about 16 nt to about 25 nt, about 17 nt to about 25 nt, about 18 nt to about 25 nt, about 19 nt to about 25 nt, about 20 nt to about 25 nt, 21 nt to about 25 nt, about 22 nt to 25 nt, about 23 nt to about 25 nt, about 24 nt to about 25 nt, about 7 to about 24 nt, about 7 to about 23 nt, about 7 to about 22 nt, about 7 to about 21 nt, about 7 to about 20 nt, about 7 to about 19 nt, about 7 to about 18 nt, about 7 to about 17 nt, about 7 to about 16 nt, about 7 to about 15 nt, about 7 to about 14 nt, about 7 to about 13 nt, about 7 to about 12 nt, about 7 to about 11 nt, about 7 to about 10 nt, about 7 to about 9 nt, or about 7 to about 8 nt. In some embodiments, a spacer sequence can be anexact match to the genomic coordinates; in some embodiments, a spacer may or contain one or a small number of mismatches, insertions, or deletions. In some embodiments, additions to a spacer end are not homologous to a target site.

[0172] In some embodiments, a spacer can be a 3' terminal sequence.

[0173] In some embodiments, a spacer can be incorporated internally in the primary sequence, for example, with a linker to stem-loop 0.

[0174] In some embodiments, a spacer can be modified. In some embodiments, base identify of a spacer may be changed or one or more chemical modifications to a spacer may be utilized.Linker Sequence Element

[0175] As used herein, the term "linker" refers to a sequence element between a crRNA element and a tracrRNA.

[0176] In some embodiments, a linker can be composed of adenine and guanine nucleotides.

[0177] In some embodiments, a linker can be 1 nucleotide in length. In some embodiments, a linker can be 2 nucleotides in length. In some embodiments, a linker can be 3 nucleotides in length. In some embodiments, a linker can be 4 nucleotides in length. In some embodiments, a linker can be 5 nucleotides in length. In some embodiments, a linker can be 6 nucleotides in length. In some embodiments, a linker can be 7 nucleotides in length. In some embodiments, a linker can be 8 nucleotides in length. In some embodiments, a linker can be 9 nucleotides in length. In some embodiments, a linker can be 10 nucleotides in length. In some embodiments, a linker can be 11 nucleotides in length. In some embodiments, a linker can be 12 nucleotides in length. In some embodiments, a linker can be 13 nucleotides in length. In some embodiments, a linker can be 14 nucleotides in length. In some embodiments, a linker can be 15 nucleotides in length. In some embodiments, a linker can be 16 nucleotides in length. In some embodiments, a linker can be 17 nucleotides in length. In some embodiments, a linker can be 18 nucleotides in length. In some embodiments, a linker can be 19 nucleotides in length. In some embodiments, a linker can be 20 nucleotides in length.

[0178] In some embodiments, a linker can be a spacer.

[0179] In some embodiments, a linker can be a template for a polymerase.

[0180] In some embodiments, a linker can contain hairpins.

[0181] In some embodiments, a linker can be chemically modified.

[0182] In some embodiments, a linker can be 5' to 3'. In some embodiments, a linker can be 3' to 5'.

[0183] In some embodiments, a linker can be formed in a cell through base-pair hybridization; alternatively or additionally, in some embodiments, a linker can be formed by hybridization outside of a cell.

[0184] In some embodiments, a linker can be a target for cellular polypeptides.

[0185] Among other things, the present disclosure also provides insights relating to desirable features of certain guide RNAs (e.g., engineered guide RNAs which, in many embodiments, may be Casl2f guide RNAs), including, for example relating to relative arrangement of structural element(s) in three-dimensional space.

[0186] In some embodiments, the present disclosure teaches that nucleotide sequence of one or more elements of an RNA may be relatively less important than one or more other structural features such as, for example, such relative arrangement in three-dimensional space.

[0187] In some embodiments, the present disclosure teaches that the beginning of both stem-loop 2 and the first 5nt of the spacer run parallel to each other.

[0188] In some embodiments, the present disclosure teaches that the distance between the (backbones of) parallel stem-loop 2 and the spacer is approximately the width of a b-form DNA helix (about 15 to about 27 Angstroms).

[0189] In some embodiments, the present disclosure teaches that the stem-loop 1 is of appropriate sequence and length to allow the loop to hybridize with the SAM, and / or additionally the SAM and other tracr sequences, and orient the spacer in parallel to stem-loop 2, forming a bridge between stem-loop 2 and the spacer.

[0190] In some embodiments, the loop of stem-loop 1 accomplishes proper positioning of the SAM by bending about 90deg relative to stem-loop 2. In some embodiments, the un-paired nucleotides (about 3nt to about 6nt) between the end of stem-loop 1 and start of stem-loop 2 allow stem-loop 1 to accomplish this fold.

[0191] In some embodiments, a bulge in stem-loop 2, about 6nt to about 7nt downstream of the start of stem-loop 2 contains a nucleotide that is flipped out of the double-helix and oriented towards the spacer. In some embodiments, the opposite strand has a nucleotide that is flipped out. In some embodiments, the opposite strand has a nucleotide that is flipped out that is about 1 to about 3nt upstream of stem-loop 2, between stem-loops 1 and 2.

[0192] In some embodiments, starting at approximately the 6thnucleotide in the spacer, the spacer bends up and around stem-loop 2, decreasing the distance between the strands.

[0193] In some embodiments, with the position of the beginning of step-loop 2 and the spacer at z=0, each forming a vector in the z-dimension with only positive z values, stem-loops 0, -1, 3, 4 (if present), as well as the cr:tr stem-loop, are situated behind a plane at z=0 in the x and y dimensions. In some embodiments, these stem-loops (other than stem-loop 2) have the flexibility to adopt vectors in any x,y orientation, but can only have negative z values.RNA Modifications

[0194] In some embodiments, a provided guide RNA may include one or more modified residues and / or linkages.

[0195] Those skilled in the art are aware of a variety of modifications that can be introduced into sugar, base, and / or intra-residue linkages in polynucleotides.

[0196] Those skilled in the art are further aware that incorporation of one or more such modifications may require or exclude one or more particular synthesis methodologies, and therefore may or may not be applicable to certain embodiments of the present disclosure. For example, embodiments in which a chemically synthetized RNA is utilized may include modifications that may not be available when an RNA is synthesized inside a cell or organism, for example, by expression from a template (e.g., a DNA template) that encodes it.

[0197] In some embodiments, an RNA modification is or comprises a modification to a sugar. Without wishing to be bound by any particular theory, the present disclosure appreciates that, in some embodiments, modified sugarsmay provide improved RNA stability. In some embodiments, modified sugars can be utilized to alter and / or optimize one or more hybridization characteristics. In some embodiments, modified sugars can be utilized to alter and / or optimize target recognition. In some embodiments, modified sugars can be utilized to optimize Tm. In some embodiments, modified sugars can be utilized to improve one or more RNA properties or activities.

[0198] A natural RNA sugar has a structure of, wherein a nucleobase is attached to the 1' position, and the 3' and 5' positions are connected to internucleotidic linkages (as appreciated by those skilled in the art, if at the 5'-end of a nucleic acid molecule, the 5' position may be connected to a 5'-end group (e.g., -OH), and if at the 3'-end of an oligonucleotide, the 3' position may be connected to a 3'-end group (e.g., -OH).

[0199] Sugars can be bonded to internucleotidic linkages at various positions. As non-limiting examples, internucleotidic linkages can be bonded to the 2', 3', 4' or 5' positions of sugars. In some embodiments, as most commonly in natural nucleic acids, an internucleotidic linkage connects with one sugar at the 5' position and another sugar at the 3' position unless otherwise indicated.

[0200] In some embodiments, a sugar is an optionally substituted natural RNA sugar. In some embodiments, a sugar is optionally substituted. In some embodiments, the 2' position is optionally substituted. In some embodiments, a sugarsome embodiments, a sugar has the structure ofwherein each of Rls, R2s, R3s, R4s, and R5sis independently -H, a suitable substituent or suitable sugar modification (e.g., those described in US 9394333, US 9744183, US 9605019, US9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 032612, and / or WO 2020 / 191252, the substituents, sugar modifications, descriptions of Rls, R2s, R3s, R4s, and R5s, and modified sugars of each of which are independently incorporated herein by reference). In some embodiments, a sugar has the structure on some embodiments, R4sis -H. In someembodiments, a sugar has the structure, wherein R2sis -H, halogen, or -OR, wherein R is optionally substituted Ci-6 aliphatic. In some embodiments, R2sis -H. In some embodiments, R2sis -F. In some embodiments, R2sis -OMe. In some embodiments, R2sis -OCH2CH2OMe..0.4k^ 271R4S. I n

[0201] In some embodiments, a sugar has the structure of , wherein R2sand R4sare taken together to form -Ls-, wherein Lsis a covalent bond or optionally substituted bivalent Ci-6 aliphatic or heteroaliphatic having 1-4 heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen or sulfur). In some embodiments, Lsis optionally substituted C2-O-CH2-C4. In some embodiments, Lsis C2-O-CH2-C4. In some embodiments, Lsis C2-O-( / ?)-CH(CH2CH3)-C4. In some embodiments, Lsis C2-0-(S)- CH(CH2CH3)-C4.

[0202] In some embodiments, a sugar is a bicyclic sugar, e.g., sugars wherein R2sand R4sare taken together to form a link as described in the present disclosure. In some embodiments, a sugar is selected from LNA sugars, BNA sugars, cEt sugars, etc. In some embodiments, a bridge is between the 2' and 4'-carbon atoms (corresponding to R2sand R4staken together with their intervening atoms to form an optionally substituted ring as described herein). In some embodiments, examples of bicyclic sugars include alpha-L-methyleneoxy (4'-CH2-O-2') LNA, beta-D- methyleneoxy (4'-CH2-O-2') LNA, ethyleneoxy (4' -(CH2)2-O-2') LNA, aminooxy (4' -CH2-O-N(R)-2') LNA, and oxyamino (4'-CH2-N(R)-O-2') LNA. In some embodiments, a bicyclic sugar, e.g., a LNA or BNA sugar, is sugar having at least one bridge between two sugar carbons. In some embodiments, a bicyclic sugar in a nucleoside may have the stereochemical configurations of alpha-L-ribofuranose or beta-D-ribofuranose. In some embodiments, a sugar is a sugar described in WO 1999014226. In some embodiments, a 4'-2' bicyclic sugar or 4' to 2' bicyclic sugar is a bicyclic sugar comprising a furanose ring which comprises a bridge connecting the 2' carbon atom and the 4' carbon atom of the sugar ring. In some embodiments, a bicyclic sugar, e.g., a LNA or BNA sugar, comprises at least one bridge between two pentofuranosyl sugar carbons. In some embodiments, a LNA or BNA sugar, comprises at least one bridge between the 4' and the 2' pentofuranosyl sugar carbons.

[0203] In some embodiments, a bicyclic sugar is a sugar of alpha-L-methyleneoxy (4'-CH2-O-2') BNA, beta-D- methyleneoxy (4'-CH2-O-2 / ) BNA, ethyleneoxy (4'-(CH2)2-O-2') BNA, aminooxy (4'-CH2-O-N(R)-2') BNA, oxyamino (4'- CH2-N(R)-O-2') BNA, methyl(methyleneoxy) (4'-CH(CH3)-O-2') BNA (also referred to as constrained ethyl or cEt), methylene-thio (4'-CH2-S-2') BNA, methylene-amino (4'-CH2-N(R)-2') BNA, methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, propylene carbocyclic (4'-(CH2)3-2') BNA, or vinyl BNA.

[0204] In some embodiments, a sugar modification is 2'-0Me, 2'-M0E, 2'-LNA, 2'-F, 5'-vinyl, or S-cEt. In some embodiments, a modified sugar is a sugar of FRNA, FANA, or morpholino. In some embodiments, an oligonucleotide comprises a nucleic acid analog, e.g., GNA, LNA, PNA, TNA, F-HNA (F-THP or 3'-fluoro tetrahydropyran), MNA (mannitol nucleic acid, e.g., Leumann 2002 Bioorg. Med. Chem. 10: 841-854), ANA (anitol nucleic acid), ormorpholino, or a portion thereof. In some embodiments, a sugar modification replaces a natural sugar with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, e.g., those used in morpholino, glycol nucleic acids, etc. and may be utilized in accordance with the present disclosure. As appreciated by those skilled in the art, when utilized with modified sugars, in some embodiments internucleotidic linkages may be modified, e.g., as in morpholino, PNA, etc.

[0205] In some embodiments, a sugar is a 6'-modified bicyclic sugar that have either (R) or (S)-chirality at the 6- position, e.g., those described in US 7399845. In some embodiments, a sugar is a 5'-modified bicyclic sugar that has either (R) or (S)-chirality at the 5-position, e.g., those described in US 20070287831.

[0206] In some embodiments, a modified sugar contains one or more substituents at the 2' position (typically one substituent, and often at the axial position) independently selected from -F; -CF3, -CN, -N3, -NO, -NO?, -OR', -SR', or -N R^, wherein each R' is independently optionally substituted CMO aliphatic; -0-(Ci-Cio alkyl), -S-(Ci-Cio alkyl), -NH-(Ci-Cio alkyl), or -N(Ci-Cio alkyl)2; -0-(C2-Cio alkenyl), -S-(C2-Cio alkenyl), -NH-(C2-CIO alkenyl), or - N(C2-CIO alkenyl)2; -0-(C2-Cio alkynyl), -S-(C2-Cio alkynyl), -NH-(C2-CIO alkynyl), or -N(C2-CIO alkynyl)2; or -0 — (C1-C10 alkylene)-0 — (C1-C10 alkyl), -0-(Ci-Cio alkylene)-NH-(Ci-Cio alkyl) or -0-(Ci-Cio alkylene)-NH(Ci-Cio alkyl)2, -NH-(Ci-Cio alkylene)-0-(Ci-Cio alkyl), or -N(Ci-Cio alkyl)- (C1-C10 alkylene)-0-(Ci-Cio alkyl), wherein each of the alkyl, alkylene, alkenyl and alkynyl is independently and optionally substituted. In some embodiments, a substituent is -O(CH2)nOCH3, -O(CH2)nNH2, MOE, DMAOE, or DMAEOE, wherein wherein n is from 1 to about 10. In some embodiments, a modified sugar is one described in WO 2001 / 088198; and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, a modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, a group for improving the pharmacodynamic properties of a nucleic acid, or other substituents having similar properties. In some embodiments, modifications are made at one or more of the 2', 3', 4', or 5' positions, including the 3' position of the sugar on the 3'-terminal nucleoside or in the 5' position of the 5'-terminal nucleoside.

[0207] In some embodiments, the 2'-OH of a ribose is replaced with a group selected from -H, -F; -CF3, -CN, -N3, -NO, -NO2, -OR', -SR', or -NCR^, wherein each R' is independently described in the present disclosure; -0-(Ci-Cio alkyl), -S-(Ci-Cio alkyl), -NH-(Ci-Cio alkyl), or -N(Ci-Cw alkyl)2; -0-(C2-Cw alkenyl), -S-(C2-Cio alkenyl), -NH- (C2-C10 alkenyl), or -N(C2-Cio alkenyl)2; -0-(C2-Cio alkynyl), -S-(C2-Cio alkynyl), -NH-(C2-C alkynyl), or -N(C2- C10 alkynyl)2; or -0 — (C1-C10 alkylene)-0 — (C1-C10 alkyl), -0-(Ci-Cio alkylene)-NH-(Ci-Cio alkyl) or -0-(Ci-Cio alkylene)-NH(Ci-Cio alkyl)2, -NH-(Ci-Cio alkylene)-0-(Ci-Cio alkyl), or -N(Ci-Cio alkyl)— (C1—C10 alkylene)-O-(Ci- C10 alkyl), wherein each of the alkyl, alkylene, alkenyl and alkynyl is independently and optionally substituted. In some embodiments, the 2'-OH is replaced with -H (deoxyribose). In some embodiments, the 2 -OH is replaced with -F. In some embodiments, the 2 -OH is replaced with -OR'. In some embodiments, the 2 -OH is replaced with - OMe. In some embodiments, the 2'-OH is replaced with -OCH2CH2OMe.

[0208] In some embodiments, a sugar modification is a 2'-modification. Commonly used 2'-modifications include but are not limited to 2'-OR, wherein R is optionally substituted Ci-5aliphatic. In some embodiments, a modification is 2'-OR, wherein R is optionally substituted C1-6 alkyl. In some embodiments, a modification is 2'-OMe. In some embodiments, a modification is 2'-MOE. In some embodiments, a 2'-modification is S-cEt. In some embodiments, amodified sugar is an LNA sugar. In some embodiments, a 2'-modification is -F. In some embodiments, a 2'- modification is FANA. In some embodiments, a 2'-modification is FRNA. In some embodiments, a sugar modification is a 5'-modification, e.g., 5'-Me. In some embodiments, a sugar modification changes the size of the sugar ring. In some embodiments, a sugar modification is the sugar moiety in FHNA.

[0209] In some embodiments, a sugar modification replaces a sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, including but not limited to those used in morpholino (optionally with its phosphorodiamidate linkage), glycol nucleic acids, etc.

[0210] In some embodiments, an RNA modification is or comprises a nucleobase modification.

[0211] In some embodiments, a nucleobase is optionally substituted A, C, G or U, or a substituted tautomer of A, C, G or U. In some embodiments, a nucleobase is optionally substituted A, C, G or U, e.g., 5mC, 5-hydroxymethyl C, etc. In some embodiments, a nucleobase is alkyl-substituted A, C, G or U. In some embodiments, a modified nucleobase is a modified A. In some embodiments, a modified nucleobase is a modified C. In some embodiments, a modified nucleobase is a modified G. In some embodiments, a modified nucleobase is a modified U. In some embodiments, a modified nucleobase is 5mC.

[0212] In some embodiments, a modified nucleobase is substituted A, C, G or U. In some embodiments, a nucleobase is a substituted tautomer of A, C, G or U. In some embodiments, substitution protects certain functional groups in nucleobases, for example to minimize undesired reactions during chemical synthesis of an RNA. Suitable technologies for nucleobase protection in chemical synthesis are known in the art and may be utilized in accordance with the present disclosure. In some embodiments, modified nucleobases improves properties and / or activities of RNAs as provided and / or utilized in accordance with the present disclosure. For example, in many cases, 5mC may be utilized in place of C and may modulate certain undesired biological effects, e.g., immune responses. In some embodiments, when determining sequence identity, a substituted nucleobase having the same hydrogen-bonding pattern is treated as the same as the unsubstituted nucleobase, e.g., 5mC may be treated the same as C [e.g., an RNA having 5mC in place of C (e.g., AU5mCG) is considered to have the same base sequence as an oligonucleotide having C at the corresponding location(s) (e.g., AUCG)].

[0213] In some embodiments, a modified nucleobase is derived from a natural nucleobase. Examples include uracil, adenine, cytosine, and guanine optionally having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). Certain examples of modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313.

[0214] In some embodiments, a modified nucleobase is substituted uracil, adenine, cytosine, or guanine. In some embodiments, a modified nucleobase is a functional replacement, e.g., in terms of hydrogen bonding and / or base pairing, of uracil, adenine, cytosine, or guanine. In some embodiments, a nucleobase is optionally substituted uracil, adenine, cytosine, 5-methylcytosine, or guanine.

[0215] In some embodiments, a modified base is optionally substituted adenine, cytosine, guanine, or uracil, or a tautomer thereof. In some embodiments, a modified nucleobase is a modified adenine, cytosine, guanine, or uracil, modified by one or more modifications by which:(1) a nucleobase is modified by one or more optionally substituted groups independently selected from acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof;(2) one or more atoms of a nucleobase are independently replaced with a different atom selected from carbon, nitrogen and sulfur;(3) one or more double bonds in a nucleobase are independently hydrogenated; or(4) one or more aryl or heteroaryl rings are independently inserted into a nucleobase.

[0216] In some embodiments, a modified nucleobase is a modified nucleobase known in the art, e.g., WO2017 / 210647. In some embodiments, modified nucleobases are expanded-size nucleobases in which one or more aryl and / or heteroaryl rings, such as phenyl rings, have been added.

[0217] In some embodiments, a modified nucleobase is selected from 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-propynyl (-CsC-CHa) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6- azothymine, 5-ribosyluracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7- methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. In some embodiments, modified nucleobases are tricyclic pyrimidines, such as 1,3- diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one or 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2- one (G-damp). In some embodiments, modified nucleobases are those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine or 2- pyridone.

[0218] In some embodiments, a modified nucleobase is substituted. In some embodiments, a modified nucleobase is substituted such that it contains, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other polypeptide or peptides. In some embodiments, a modified nucleobase is a "universal base" that is not a nucleobase in the most classical sense, but that functions similarly to a nucleobase. One example of a universal base is 3-nitropyrrole.

[0219] In some embodiments, nucleosides that can be utilized in provided technologies comprise modified nucleobases and / or modified sugars, e.g., 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2'-0-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2'-0- methylpseudouridine; beta,D-galactosylqueosine; 2'-O-methylguanosine; N6-isopentenyladenosine; 1- methyladenosine; 1-methylpseudouridine; 1-methylguanosine; l-methylinosine; 2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N7-methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5- hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine; N6-methyladenosine; 7-methylguanosine; 5- methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine; 5- methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N6-isopentenyladenosine; N-((9-beta,D- ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine; N-((9-beta,D-ribofuranosylpurine-6-yl)-N- methylcarbamoyl)threonine; uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl-5- methyluridine; and 2'-O-methyluridine.

[0220] In some embodiments, a nucleobase, e.g., a modified nucleobase comprises one or more biomolecule binding moieties such as e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In other embodiments, a nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, a nucleobase comprises substitution with a fluorescent or biomolecule binding moiety. In some embodiments, a substituent is a fluorescent moiety. In some embodiments, a substituent is biotin or avidin.

[0221] In some embodiments, a nucleobase is one described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US 10450568, US 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, and / or WO 2020 / 191252, the nucleobases of each of which is incorporated herein by reference.

[0222] In some embodiments, oligonucleotides comprise base modifications, sugar modifications, and / or internucleotidic linkage modifications.

[0223] In some embodiments an RNA can comprise both one or more modified internucleotidic linkages and one or more natural phosphate linkages. As widely known by those skilled in the art, natural phosphate linkages are widely found in natural RNA molecules; they have the structure of -OP(O)(OH)O-, connect sugars in the nucleosides in the RNA, and may be in various salt forms, for example, at physiological pH (about 7.4), natural phosphate linkages are predominantly exist in salt forms with the anion being -OP(O)(O“)O-. A modified internucleotidic linkage, or a nonnatural phosphate linkage, is an internucleotidic linkage that is not natural phosphate linkage or a salt form thereof. Modified internucleotidic linkages, depending on their structures, may also be in their salt forms. For example, as appreciated by those skilled in the art, phosphorothioate internucleotidic linkages which have the structure of -OP(O)(SH)O- may be in various salt forms, e.g., at physiological pH (about 7.4) with the anion being -OP(O)(S-)O-

[0224] In some embodiments, an RNA comprises an internucleotidic linkage which is a modified internucleotidic linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroamidate, thiophosphate, 3'- thiophosphate, or 5'-thiophosphate.

[0225] In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage which comprises a chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage.In some embodiments, a chiral internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is stereochemically pure with respect to its chiral linkage phosphorus. In some embodiments, a pattern of backbone chiral centers comprises or consists of positions and linkage phosphorus configurations of chirally controlled internucleotidic linkages ( / ?p or 5p) and positions of achiral internucleotidic linkages (e.g., natural phosphate linkages).

[0226] In some embodiments, a modified internucleotidic linkage comprises optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage comprises optionally substituted alkynyl. In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In some embodiments, a triazole moiety, (e.g., a triazolyl group) is substituted. In some embodiments, a triazole moiety is unsubstituted.

[0227] In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety and has the structure of:wherein W is 0 or S. In some embodiments, W is 0. In some embodiments, W is S. In some embodiments, a non- negatively charged internucleotidic linkage is stereochemically controlled.

[0228] In some embodiments, a modified internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, an internucleotidic linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) has the structure on some embodiments, an internucleotidic linkage comprising a triazole moiety has the structuresome embodiments, an internucleotidic linkage, comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety has the structuresome embodiments, a modified internucleotidiclinkage, is or comprising a structure selected fromwherein W is O or S.

[0229] In some embodiments, a modified internucleotidic linkage comprises a Tmg groupsome embodiments, a modified internucleotidic linkage comprises a Tmg group and has the structure of(the "Tmg internucleotidic linkage"). In some embodiments, modified internucleotidic linkages include internucleotidic linkages of PNA and PMO, and an Tmg internucleotidic linkage.

[0230] In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In some embodiments, such a heterocyclyl or heteroaryl group is of a 6- membered ring.

[0231] In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a heteroaryl group is directly bonded to a linkage phosphorus. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an unsubstituted triazolyl group, e.g.,In some embodiments, a non- negatively charged internucleotidic linkage comprises a substituted triazolyl group, e.g.,

[0232] In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In some embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., = N- when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its =N-. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substitutedgroup. In some embodiments, a non-negatively charged internucleotidic linkage comprises an substitutedgroup. In some embodiments, a non-negatively charged internucleotidic linkage comprises agroup. In some embodiments, each R1is independently optionally substituted with Ci-e alkyl. In some embodiments, each R1is independently methyl.

[0233] In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, a modified internucleotidic linkage comprises a triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an unsubstituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a substituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an alkyl moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises an unsubstituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises a substituted alkynyl group. In some embodiments, an alkynyl group is directly bonded to a linkage phosphorus.

[0234] In some embodiments, an RNA comprises different types of internucleotidic phosphorus linkages. In some embodiments, an RNA comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotidic linkage. In some embodiments, an RNA comprises at least one natural phosphate linkage and at least one phosphorothioate.

[0235] Without wishing to be bound by any particular theory, the present disclosure appreciates that certain modified internucleotidic linkages may improve one or more pharmaceutical properties and / or activities of an RNA or a CRISPR-Cas complex including it.

[0236] In some embodiments, one or more types of modified internucleotidic linkages may be utilized in combination with one or more other modified structural elements, e.g., sugars, to achieve desired properties and / or activities.RNA Production

[0237] Those skilled in the art will appreciate that the art provides a variety of technologies that may be useful to product RNA, e.g., a provided RNA as described herein.

[0238] For example, in some embodiments, a provided RNA may be produced by transcription of a template that encodes it. In some embodiments, such transcription occurs inside a cell. In some embodiments, such transcription is performed in vitro.

[0239] In some embodiments, a guide RNA may be generated by IVT from a plasmid template. In some embodiments, a guide template may be generated by PCR.

[0240] In some embodiments, transcription is performed with an RNA polymerase that can incorporate one or more nucleotide analogs; those skilled in the art will be aware of such RNA polymerases. In some embodiments, a polymerase is an RNA dependent RNA-polymerase. In some embodiments, an RNA polymerase is RNA polymerase I, RNA polymerase II, RNA polymerase III, T7 RNA polymerase, POLRMT, Primase, PrimPol, or a combination thereof. In some embodiments, an RNA polymerase is T7 RNA polymerase.

[0241] In some embodiments, a guide RNA may be synthesized via non-templated chemical synthesis. In some embodiments, a guide RNA may be synthesized via enzymatic synthesis technologies see, for example, Damiano Verardo et al. Multiplex enzymatic synthesis of DNA with single-base resolution. Sci.Adv.9,eadi0263(2023).DOI: 10.1126 / sciadv.adi0263, which is herein incorporated by reference in its entirety).Compositions

[0242] Among other things, the present disclosure provides compositions. Compositions disclosed herein comprise one or more RNAs (e.g., sgRNAs) described herein.

[0243] In some embodiments, a composition comprising one or more polyribonucleotides (e.g., sgRNAs) is a pharmaceutical composition.

[0244] In some embodiments, a composition comprising one or more polyribonucleotides (e.g., sgRNAs) is a gene therapy.

[0245] In some embodiments, a composition includes RNA and a template that encodes it. In some embodiments, a template is an amplicon plasmid. In some embodiments, a template is a plasmid. In some embodiments, a template is a viral vector.

[0246] In some embodiments, a composition comprises a CRISPR-Cas complex and cells that contain said complexes.Pharmaceutical Compositions

[0247] In some embodiments, a composition comprising one or more polyribonucleotides (e.g., sgRNAs) is a pharmaceutical composition. In some embodiments, a pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Pharmaceutical compositions of the present disclosure may comprise a polypeptide disclosed herein, a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide disclosed herein.Kits

[0248] Another aspect of the present disclosure further provides a pack or kit, for example where a provided RNA and / or one or more other components of a CRISPR-Cas system, or a nucleic acid that encodes one or more of the forgoing. In some embodiments, individual components of a kit may be separately packaged; in some embodiments, two or more separately packaged components may be positioned and / or enclosed within a common container (e.g., a box).

[0249] Those skilled in the art will be aware of a variety of contexts in which a provided kit may be useful, e.g., in the practice of one or more applications (e.g., methods) as described herein. In some embodiments, a kit may comprise (i) a reaction buffer, (ii) ribose nucleic acids ATP, GTP, CTP and UTP, (iii) a dsDNA template encoding a provided nucleic acid, and / or T7 polymerase (particularly if an included dsDNA template has a T7 promoter). In some embodiments, a guide RNA can be synthesized from dsDNA template using commercial T7 polymerase kits. In some embodiments, a commercial kit is the Precision gRNA Synthesis Kit from Thermo Fisher.Cells

[0250] Among other things, the present disclosure provides cells, or preparations thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Casl2f system), e.g., that include and / or otherwise utilize a provided RNA.

[0251] In some embodiments, a provided cell preparation is used to produce a provided RNA and / or a CRISPR-Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Casl2f system), e.g., that include and / or otherwise utilize a provided RNA.

[0252] Alternatively or additionally, in some embodiments, a provided cell preparation is formulated for administration to a subject, e.g., as a therapeutic.

[0253] Still further alternatively or additionally, in some embodiments, a provided cell preparation is or comprises a plurality of cells that have been edited by a CRISPR-Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Casl2f system) that includes a provided RNA.

[0254] In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is an archaeal cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a bacterial cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a eukaryotic cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a eukaryotic single-cell organism. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or aCRISPR-Cas system, is a somatic cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a germ cell. In some embodiments, a cell, or preparations thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a stem cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a plant cell. Plant cells can be from monocots, e.g., corn, rice, rye, sorghum, millet, proso millet, foxtail millet, finger millet, wheat, sugarcane, oat, barley, switchgrass, pineapple, banana, palm, ornamentals, turfgrasses, and other grasses. Plant cells can be from dicots, e.g., soybean, Brassica species (e.g., oilseed rape or Canola and mustard), alfalfa, tobacco, Arabidopsis, sunflower, cotton, peanut, tomato, and potato. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is an algal cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is an animal cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is an invertebrate cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a vertebrate cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a fish cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a frog cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a bird cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a mammalian cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a porcine cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a bovine cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a rodent cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a non-human primate cell. In some embodiments, a cell, or preparation thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system, is a human cell.

[0255] In some embodiments, a cell engineered to contain or express an RNA provided herein and / or a CRISPR- Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Casl2f system), is in vitro (e.g., a cultured cell or population thereof). In some embodiments, an engineer cell disclosed herein is in vivo (e.g., in a subject). In some embodiments, an engineered cell is ex vivo e.g., isolated from a subject; in some such embodiments, an ex vivo engineered cell (or population thereof) may subsequently be administered to the subject from which the original cell was obtained (e.g., as an autologous therapy), or to a different subject, or population of subjects, e.g., as an allogeneic therapy).

[0256] Those skilled in the art, reading the present disclosure, will appreciate the wide variety of cell types and cell lines and that are able to be used in accordance with the present disclosure (See, for example, Valenti et al. CRISPR / Cas system: An emerging technology in stem cell research. World J Stem Cells. 11(11):937, Nov 26, 2019; doi: 10.4252 / wjsc.vll.ill.937, which is incorporated herein in its entirety; See also, for example, Chehelgerdi, etal. Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy. Mol Cancer 23:9 , 2024). doi.org / 10.1186 / sl2943-023-01925-5, which is incorporated herein in its entirety).

[0257] Among other things, the present disclosure provides non-limiting examples of cells that may be engineered or preparations thereof, engineered to contain or express an RNA provided herein and / or a CRISPR-Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Cas 12f system), e.g., that include and / or otherwise utilize a provided RNA, such as HEK293T cells, human T cells, and / or primary human hepatocytes.Vectors

[0258] Among other things, the present disclosure provides vectors engineered to encode a provided RNA and / or another element of a CRISPR-Cas system (e.g., a CRISPR-Casl2 system, and particularly a CRISPR-Casl2f system) that includes it.

[0259] In some embodiments, a provided vector system is or comprises a DNA or an RNA.

[0260] In some embodiments, a vector is or comprises a plasmid vector, viral vector, a transposon, a retrotransposon (e.g., piggyback, sleeping beauty), etc. Those skilled in the art, reading the present disclosure, will appreciate that a variety of vector systems are available in the art that may be useful in accordance with the present disclosure.

[0261] For example, among other things, those of ordinary skill, reading the present disclosure, will recognize that various viral vectors, especially retroviral vectors, are commonly used for inserting genes into mammalian cells (e.g., human cells). In some embodiments, viral vectors are often derived from (e.g., contain one or more elements and / or components of) lentivirus, poxviruses, herpes simplex virus I, adenoviruses (e.g., Adf535), or adeno- associated viruses (See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety). Typically, for example, retroviral vectors, such as lentivirus, are suitable tools to achieve long-term gene transfer that allow for long-term, stable integration of a transgene and its propagation in daughter cells.

[0262] In some embodiments, plant viral-based vectors may be used for transient polypeptide expression and virus induced gene silencing (or "VIGS"). Examples of plant viral based vectors may include, but are not limited to, Tobacco Rattle Virus (or "TRV"), Barley Stripe Mosaic Virus ("BSMV"), and Bean Yellow Dwarf Virus (or "BeYDV").Methods of Delivery

[0263] The present disclosure, among other things, provides technologies comprising delivering one or more polynucleotides.

[0264] A person of skill in the art will recognize that a CRISPR-Cas system can be delivered into cells in various formats. See, for example, Huang J, Zhou Y, Li .1, Lu A, Liang C. CRISPR / Cas systems: Delivery and application in gene therapy. Front Bioeng Biotechnol. 2022 Nov 22;10:942325. doi: 10.3389 / fbioe.2022.942325, which is herein incorporated by reference in its entirety.

[0265] In some embodiments, CRISPR-Cas systems may be delivered through in vitro, in vivo, and ex vivo formats.

[0266] In some embodiments, a DNA expression plasmid encoding a Cas polypeptide and sgRNA may be delivered into cells. In some embodiments, a DNA expression plasmid encoding a Cas polypeptide and sgRNA is a singleplasmid. In some embodiments, a DNA expression plasmid encoding a Cas polypeptide and sgRNA are separate plasmids.

[0267] In some embodiments, a ribonucleoprotein ("RNP") complex may be assembled ex vivo and delivered to cells.

[0268] In some embodiments, a sgRNA and mRNA of a Cas polypeptide may be co-delivered into cells.

[0269] The present disclosure, among other things, provides biological methods for delivering CRISPR-Cas systems.

[0270] In some embodiments, a recombinant virus vector (e.g., adeno-associated virus vector, adenovirus vectors, lentivirus vectors, retrovirus vectors, or herpes simplex virus vectors) may be used for delivering CRISPR-Cas systems. In some embodiments bacterial-mediated delivery of CRISPR / Cas components, may be used for delivering CRISPR-Cas systems into plant cells. See, for example, Prestwich, B.D. et al., "Novel Delivery Methods for CRISPR- Based Plant Genome Editing", In: Ricroch, A., (eds) "A Roadmap for Plant Genome Editing," Springer, Cham, 2024, (doi.org / 10.1007 / 978-3-031-46150-7_3), which is incorporated by reference in its entirety. In some embodiments, Agrobacterium-mediated transformation (AMT) may be used for gene editing. Methods of delivering CRISPR-Cas systems to plants are described, e.g., in WO 2023 / 244992.

[0271] The present disclosure, among other things, provides physical methods for introducing a nucleic acid construct into a cell (e.g., a HEK293T cell, a human T cell, or a primary human hepatocyte). In some embodiments, a physical method of delivery may comprise microinjection, hydrodynamic injection, calcium phosphate precipitation, electroporation, membrane deformation, sonoporation, lance array nanoinjection, gene gun / biolistic-based delivery, silicon carbide whiskers, or a combination thereof.

[0272] Those skilled in the art will also be aware that various chemical means are available for introducing a nucleic acid construct as described herein into a cell. For example, available technologies include polyethylene glycol (PEG) delivery, colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[0273] An exemplary system for delivery of a nucleic acid construct as described herein is a lipid-based system. A nucleic acid construct as described herein may be encapsulated in an aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension comprising a lipid, mixed with a lipid, complexed with a micelle, or otherwise associated with a lipid. Lipids for use in methods described herein may be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources as disclosed herein. In some embodiments, a lipid-based system may comprise one or more lipids that facilitate targeting of the composition to a desired cell type or cell types. In some embodiments, a delivery vehicle allows a composition to be preferentially taken up (e.g. endocytosed, phagocytosed) by a cell relative to a composition that does not comprise the delivery vehicle.Characterization

[0274] In some embodiments, a provided RNA, or a CRISPR-Cas system that includes it, may be characterized by one or more assays.

[0275] For example, in some embodiments, ability of a provided RNA to complex with one or more Cas polypeptides is assessed, e.g., by direct binding studies and / or via assessing editing efficiency of the CRISPR-Cas complex, see for example, Examples 10, 12, 14, 16, and 17.

[0276] A person of skill in the art will recognize that there are several methods to characterize editing efficiency of a CRISPR-Cas system, for example by assessing frameshift insertions and deletions (herein "INDELs") caused by CRISPR-Cas activity See, for example, Carrington B, Bishop K, Sood R. A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases. Genes (Basel). 2022 May 11;13(5):857. Doi: 10.3390 / genesl3050857. PMID: 35627242; PMCID: PMC9141975., which is herein incorporated by reference in its entirety).

[0277] In some embodiments, INDELs are detected based on direct analysis of PCR products. In some embodiments, INDELS are detected by methods including, but not limited to, polyacrylamide gel electrophoresis (PAGE) of PCR products, heteroduplex mobility assays ("HMAs"), fluorescent PCR and capillary electrophoresis, high resolution melting analysis C'HRMA"), or quantitative PCR.

[0278] In some embodiments, INDELs are detected based on analysis of post-processing PCR products. In some embodiments, INDELS are detected by methods including, but not limited to, Sanger sequencing, T7 Endonuclease I Digestion assay, next generation sequencing, or targeted amplicon sequencing. In some embodiments, activity of a CRISPR-Cas system may be characterized through gene expression changes in cells. A variety of assays may be performed to determine gene expression changes in cells, such as flow cytometry, quantitative PCR, microarrays, or RNAseq.

[0279] In some embodiments, ability of a provided RNA to complex with one or more Cas polypeptides may be assessed, for example, via in vitro reconstitution assays.

[0280] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through in vitro RNA, polypeptide, and / or complex stability assays.

[0281] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through target binding / engagement assays. In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through gene activation. In some embodiments, a provided RNA or a CRISPR- Cas system disclosed herein may be characterized through gene inhibition.

[0282] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through in vitro cleavage assays. In some embodiments, a provided RNA or a CRISPR-Cas system may be characterized through cell-based genomic cleavage assays.

[0283] In some embodiments, a provided RNA or a CRISPR-Cas system may be characterized through cell-based gene disruption assays.

[0284] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through RNA protection assays.

[0285] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through secondary recruitment via a guide RNA.

[0286] In some embodiments, a provided RNA or a CRISPR-Cas system disclosed herein may be characterized through 3D structure determination of a RNP complex.

[0287] A person of skill in the art will recognize various methods that are useful in determining 3D structure of RNP complexes, i.e., cryo-EM, x-ray crystallography, NMR, and Al-based algorithms (e.g., AlphaFold). See, for example, Dimitrova-Paternoga, L, "Integrative Structural Biology of Protein-RNA Complexes,", Structure, Pages 6-28, 2020 (doi.org / 10.1016 / j.str.2019.11.017), which is incorporated herein by reference in its entirety.

[0288] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.Applications

[0289] The present disclosure appreciates the value in improving core technologies underpinning all editing produces, enabling improved therapeutic applications of CRISPR-Cas, systems, in particular, CRISPR-Casl2f systems.

[0290] Those skilled in the art will be familiar with a variety of applications of provided RNA and other CRISPR-Cas technologies.

[0291] Without wishing to be bound by theory, CRISPR-associated system genome editing may be used in basic research, medicines, therapeutic drugs, infectious studies, and agricultural biotechnology. See, for example, Hillary & Ceasar Mol. Biotechno! 65:311, March 2023, doi: 10.1007 / S12033-022-00567-0. Epub 2022 Sep 27 , which is incorporated herein in its entirety. See also, Pickar-Oliver, A., Gersbach, C.A. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Bio! 20, 490-507 (2019). Which is incorporated herein in its entirety.

[0292] In some embodiments, CRISPR-Casl2f systems may also be used for disease diagnosis. See for example, Kaminski, M.M., Abudayyeh, O.O., Gootenberg, IS. et al. CRISPR-based diagnostics. Nat Biomed Eng 5, 643-656 (2021). Which is incorporated herein in its entirety. See also, for example, Xu Y, Li Z. CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. 2020 Sep 8;18:2401-2415. Doi: 10.1016 / j.csbj.2020.08.031., which is incorporated herein in its entirety.

[0293] In some embodiments, CRISPR-Cas systems may be utilized for animals. In some embodiments, CRISPR- Cas system may be used for improved animal welfare, i.e., disease resistance. In some embodiments, CRISPR-Cas systems may be utilized for improved livestock animals, i.e., improved productive traits and / or increased mass. In some embodiments, CRISPR-Cas systems may be utilized for improved working animals, i.e., increased muscle mass.

[0294] In some embodiments, CRISPR-Cas systems may be utilized for studying and developing model organisms. In some embodiments, CRISPR-Cas systems may be utilized for developing and testing efficacy of humanized model organisms.

[0295] In some embodiments, CRISPR-Cas systems may be utilized for somatic gene editing.

[0296] In some embodiments, CRISPR-Cas systems may be utilized for manufacturing biofuels.

[0297] In some embodiments, CRISPR-Cas systems may be utilized for improved food sources. In some embodiments, CRISPR-Cas systems may be utilized for engineering improved crops.

[0298] In some embodiments, CRISPR-Cas systems may be utilized for viral screening.

[0299] In some embodiments, CRISPR-Cas systems may be utilized for viral diagnosis.

[0300] A person of skill in the art will recognize one advantage of CRISPR-Casl2f systems is that their small size allows them to be efficiently packaged into vectors for therapeutic applications (e.g., gene therapy). In some embodiments, CRISPR-Casl2f systems may be effectively packaged into adeno-associated vectors. In some embodiments, CRISPR-Casl2f systems may be effectively packaged into LNPs.

[0301] In some embodiments, CRISPR-Casl2f systems can be administered for gene therapy.

[0302] In some embodiments, CRISPR-Casl2f systems can be used in gene therapy for monogenic diseases.

[0303] In some embodiments, CRISPR-Casl2f systems can be used in gene therapy for infectious diseases.

[0304] In some embodiments, CRISPR-Casl2f systems can be used in gene therapy for cancer treatment.

[0305] In some embodiments, disclosed herein is a gene therapy method comprising administering an engineered polypeptide, a polynucleotide, and / or cell described herein to a cell, tissue or subject. In some embodiments, a gene therapy method comprises delivery of one or more components of a gene therapy, e.g., a guide RNA and / or a Cas polypeptide.

[0306] In some embodiments, catalytically active and / or inactive CRISPR-Casl2f systems can be fused to a heterologous sequence (US20140068797 published 06 March 2014). Suitable fusion partners include, but are not limited to polypeptides that provides for increased or decreased transcriptionof a target nucleic acid (e.g., a transcription activator or a fragment thereof, recruiter of a transcription activator, a small molecule / drug-responsive transcription regulator, etc.), or for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, polymerase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. A partially active or catalytically inactive Cas endonuclease can also be fused to another polypeptide or domain, for example Clo51 or FokI nuclease, to generate double-strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).

[0307] In some embodiments, catalytically active and / or inactive CRISPR-Casl2f systems can be fused with a molecule that directs editing of single or multiple bases in a polynucleotide sequence. Such molecules include, for example, site-specific deaminases that can change the identity of a nucleotide from C»G to T«A or an A»T to G«C (Gaudelli et al. 2017, "Programmable base editing of A»T to G«C in genomic DNA without DNA cleavage." Nature 551(7681):464-471; Nishida et al. 2016, "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305): aaf8729; Komor et al. "Programmable editing of a target base ingenomic DNA without double-stranded DNA cleavage." Nature 533 (7603) (2016):420-4. A base editing fusion polypeptide may comprise, for example, an active (double strand break creating), partially active (nickase) or deactivated (catalytically inactive) Cas endonuclease and a deaminase (such as, but not limited to, a cytidine deaminase, an adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABEs, or the like). Base edit repair inhibitors and glycosylase inhibitors (e. ., uracil glycosylase inhibitor (to prevent uracil removal)) may be other components of a base editing system, in some examples.EXEMPLIFICATIONExample 1: Design of single guide RNA (sgRNA) chassis

[0308] This Example illustrates a method through which sgRNA chassis can be designed from crRNA and tracrRNA, in accordance with certain embodiments of the present disclosure. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0309] Identified crRNA, tracrRNA, and functional single guide RNA (sgRNA) chassis sequences can be gathered from available sources, including public sources such as literature (e.g., Karvelis et al., Bigelyte et al.). Orthologous RNA sequences and structures can also be gathered from available sources, such as from literature and / or a protein database (e.g., RCSB PDB; resb.org); particularly useful sequences may include, for example, but are not limited to sequences and structures from AsCasl2fl (PDB codes: 8DZJ, 8J12, 8J1J), UnlCasl2fl (PDB codes: 7L49, 74L8), and CnCasl2fl (PDB code: 8HR5).

[0310] Sequences are transformed into 2D contact maps to define proposed secondary structure elements. Orthologous structural elements that share shape and or sequence rules are given common identifiers. The inclusion, placement, number, and distribution of RNA bases within the crRNA, tracrRNA, and linker elements can be chosen using either computational, experimental screening methods, or human design methods. A collection of sgRNA chassis sequences, with a linked crRNA and tracrRNA, are designed with ribonucleotides placed in a number of different locations within the molecule. Additional sgRNA chassis sequences are designed, for example, as follows.

[0311] crRNA and tracrRNA sequences are not well conserved in sequence space across Casl2f polypeptides. However, there are core structural-elements, nucleotide pairing, and positioning of the target DNA interacting region ("spacer") which are conserved. 2D maps can be used to align different Casl2f crRNA and tracrRNA sequences based on these conserved structural elements. These predicted 2D maps can be generated by human design, RNA-folding software (e.g. RNAfold), computational tools, or a combination of approaches.

[0312] Combinations of sequences, secondary structure, lengths of upper and lower stems or bulges, canonical vs non-canonical base-pairing, and potential sgRNA:polypeptide interactions, and / or sgRNA:target DNA are used to inform the positioning, inclusion, or exclusion, of ribonucleotides within the sgRNA chassis. Multiple designs of sgRNA chassis are tested with the understanding that different configurations may support different desired properties (i.e., activity, specificity, stability, etc.). Identity and length of each element, encompassing either individual nucleotides, stem-stem pairs, bulges, loops, or the entire crRNA and tracrRNA, can be individually varied, as well as identity, length, and position of the linker element to generate an sgRNA chassis.

[0313] Table 1 below provides certain exemplary variations that can be applied to particular sgRNA elements in accordance with the present disclosure:Table 1: Exemplary variations of sgRNA elements

[0314] sgRNA chassis sequences are combined with different spacer sequences (see Example 2), to generate a complete sgRNA (aka "Guide RNA"), and evaluated in cells to determine ability to direct SpaCasl2fl to interact with a specific genomic target sequence.Example 2: Design of guiding sequences for guide RNA

[0315] This Example illustrates a method through which guide RNA can be designed from sgRNA chassis and a spacer sequence in accordance with certain embodiments of the present disclosure. Not all of the following steps are required, nor must the order of steps be as presented.

[0316] Sequences of target genes of interest are identified from the human genome assembly hg38 (ncbi.nlm.nih.gov / datasets / genome / GCF_000001405.26 / ). Regions of interest within a target are identified, which can include known transcription and translation regulatory elements, polypeptide interaction regions, open reading frame, etc. Regions of interest can be scanned to identify protospacer adjacent motif (PAM) sequences that match known SpaCasl2fl active sequences ("NTTY", Karvelis et al). Alternatively or additionally, PAMs where SpaCasl2fl is not known to be active, can be identified. The sequence immediately 3' of a putative PAM site can be recorded (for inclusion in a guide RNA as a spacer sequence) and combined with a sgRNA chassis from Example 1 to generate a guide RNA. A spacer will typically be shorter than about 25 nt and longer than about 7 nt. In many cases, a spacer will have a length range of about 17 nt to about 22 nt. In some embodiments, a spacer sequence element may be extended, or positioned adjacent to, one or more residues that is not homologous to the target site. In some embodiments, a spacer sequence can be an exact match to the genomic coordinates; in some embodiments, a spacer may or contain one or a small number of mismatches, insertions, or deletions.

[0317] Guide RNA sequences composed of a sgRNA chassis sequence and spacer sequence can be provided to a commercial manufacturer (e.g., IDT) for synthesis. Alternatively or additionally a DNA template encoding such guideRNA may be provided and expressed, for example via in vitro transcription, to generate the encoded guide RNA (Example 5).

[0318] A guide RNA can be introduced into cells, for example as RNA or as an expressible DNA construct encoding the RNA. In some embodiments, such expressible construct may integrate into the cell genome; in many embodiments, such expressible construct may not integrate. In some embodiments, an RNA is introduced into a cell by transfection of the RNA (e.g., LNP-mediated, lipofectamine, nucleofection). In some embodiments, an RNA is introduced into a cell by administration of an expressible construct (e.g., a plasmid construct, such as a DNA plasmid, or a viral construct, such as an AAV construct). Those skilled in the art will be aware of appropriate transfection, transduction, and / or infection technologies useful for introduction of relevant RNAs, or expressible constructs encoding them, as described herein.Example 3: Design of RNA modifications

[0319] This Example illustrates a method through which guide RNA with chemical modifications to the ribonucleotide backbone chemistry can be designed, in accordance with certain embodiments of the present disclosure. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0320] RNA modifications are chemical changes to the ribonucleotide backbone or bases that change the interaction between a ribonucleotide oligomer and its environment. Examples of interactions that are changed are base-pairing strengths and polypeptide-nucleic acid interactions. As an example, by changing the interaction with polypeptides, RNA modifications can lead to lower cleavage rates when interacting with cellular nucleases. In another example, RNA modifications can also lead to changed interaction kinetics with binding polypeptides, such as CAS nucleases. Incorporating chemical changes to the ribonucleotide guide RNA thus can increase or decrease half-life in a cell, binding interactions with polypeptides and binding interactions with the target DNA, etc.

[0321] Those skilled in the art will be aware of a variety of modifications that may be appropriate for incorporation into certain guide RNAs in accordance with the present disclosure. In some embodiments, a modification is to a base; in some embodiments, a modification is to a sugar; in some embodiments, a modification is to a linkage between residues in a guide RNA.

[0322] In some embodiments, a guide RNA provided by and / or utilized in accordance with the present disclosure may include a single modified residue and / or linkage; in some embodiments, such a guide RNA may include multiple modified residues and / or linkages. In some embodiments, all residues and / or linkages of a particular type (e.g., U residues) are modified. In some embodiments, all residues and / or linkages are modified.

[0323] Certain exemplary modifications include, for example: 2'-O-methyl-modified residues, 2'-O-methoxy-ethyl modified residues, 2' Fluoro bases, locked nucleic acids, and phosphorothioated linkages.

[0324] Identified sgRNA chassis sequences and 2D interaction maps can be captured as in Example 1. Additional information from homologous structures and predicted sites of polypeptide interactions can be labeled onto maps. Multiple designs are generated which include either ribonucleotide base and backbone modifications at locationspredicted to interact with polypeptides, or with uncertain polypeptide interaction potential, or known to not interact with polypeptides, and combinations of all of these.

[0325] Sequences of guide RNAs with annotated modifications can be provided to a commercial manufacturer (e.g. IDT) for synthesis. Guide RNAs containing modification(s) can be prepared, in some embodiments, by chemical or enzymatic (other than transcription) synthesis. Alternatively or additionally, in some embodiments, such modified guide RNAs may be prepared by transcription (e.g., in vitro transcription), for example in a system in which one or more provided NTPs is a modified NTP. Guide RNAs with and without modifications can be transfected into cells (see, for example, Examples 10, 11, 13) to determine activity.Example 4: Production of mRNA

[0326] mRNA for use in cell culture-based activity assays and in vivo activity assays were produced with a plasmid DNA template. In some embodiments, mRNA for use in cell culture-based assays and in vivo activity assays are produced with a PCR generated DNA template.

[0327] mRNA generation using plasmid template started with cloning of a CAS enzyme into an IVT specific vector backbone from Takara Bio (takarabio.com, catalog # 6143). Cloning was performed following the manufacturer's instructions, using primers and PCR amplification to append forward (5' AGAGAACCCGCCACC 3") and reverse (5' CGAGGCTCCAGCTCA 3') sequences necessary for the In-Fusion reaction into linearized vector (specific primers OTB0875 and OTB0876). The PCR reactions were carried out with Q5® High-Fidelity DNA Polymerase following the manufacturer's instructions. Desired sequence was amplified from 1-25 ng plasmid DNA in a final volume of 25 uL under the following conditions: 98C for 30s, then 25 cycles of 15s at 98C, 15s at 64C, 30s at 72C, and a final extension at 72C for 2 min. PCR products were purified by gel extraction and purification by NEB Monarch gel extraction kit or Macherey-Nagel NucleoSpin Gel and PCR Clean-up, and quantified using Qubit. The In-Fusion cloning was performed according to the manufacturer's instructions as follows: 1 uL of the Takara Linearized Template Vector (50 ng / pl), 100 ng of the PCR product of the gene of interest, 2uL of 5x In-Fusion Mastermix, and DI H2O to make up 10 uL of total reaction was combined. This reaction was incubated at 50C for 15min. The InFusion reaction was then transformed into Stellar competent cells following the manufacturer's suggested protocol and plated on LB plates with kanamycin for clonal selection. Clones were picked and sequence verified.

[0328] mRNA was generated from plasmid template using the Takara IVTpro Synthesis System (takarabio.com catalogue number 6144). Plasmid template was digested with Hindlll-HF (NEB, catalog number R3104M), for between 16 and 36hrs at 37C. IVT reactions were carried out according to the manufacturer's protocol at 20uL scale, with 2 uL lOx transcription buffer, 2 uL lOx ATP, 2 uL lOx CTP, 2uL lOx GTP, 2 uL lOx UTP, 1.6 uL CleanCap Reagent AG (Trilink, catalog number N-7113), 2uL lOx Enzyme Mix, 1 ug linearized template DNA, and DI H2O to make up 20uL of total reaction. Reactions were incubated for 2 hours at 37C to produce mRNA. mRNA was cleaned up using Monarch® RNA Cleanup Kit (NEB, catalog number T2050L). mRNA was verified for purity and quantity using the Agilent BioAnalyzer with a RNA 6000 Nano Kit.

[0329] mRNA containing pseudouridine ( was also generated from plasmid template using the same conditions as above, except for the replacement of lOx UTP, with Nl-methylpseudouridine-5'-Triphosphate (Trilink, catalog number N-1081).Example 5: Synthesis of guide RNA

[0330] This Example provides an exemplary description of certain technologies for production and / or characterization of guide RNAs in accordance with the present disclosure, including, for example, certain in vitro, and cell culture activity assays. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0331] Guide RNA can be generated enzymatically with an in vitro transcription (IVT) reaction. Typically, an IVT reaction utilizes a linear DNA template encoding a guide RNA, which template includes a transcription promoter sequence for an RNA polymerase such as a T7 polymerase. In various embodiments, such an IVT template can be generated as a plasmid, as a PCR product, or as a chemically synthesized stretch of double stranded DNA.

[0332] The design of an IVT template is typically based on relevant guide RNA design, for example as described in Example 2. In particular, DNA sequence encoding the relevant guide RNA can be established. At the 5' end of the encoding sequence, a T7 promoter sequence can be added. An example T7 sequence is 5' TAATACGACTCACTATA 3'. In addition to the T7 promoter sequence, a G nucleotide is required to initiate transcription. Between the T7 promoter and the guide RNA-encoding sequence, a single G can be added, if not already present on the 5' terminal end of the guide RNA, or multiple G nucleotides can be added. Different numbers of G nucleotides will result in different efficiencies of RNA production, as well as guide RNA species with different 5' G nucleotides, which could lead to different impacts on activity of the guide.

[0333] Plasmid-based guide RNA IVT templates can be linearized with an enzyme that generates a blunt cut at the 3' end of the guide RNA sequence, or with a 5' overhang at the 3' end. This linearized DNA can be purified with a column or precipitation method.

[0334] PCR-generated guide RNA IVT templates can be generated as a PCR product from a plasmid template, or a dsDNA template, or a ssDNA template. A PCR template can contain the entire guide RNA IVT template sequence, including the T7 promoter through the 3' end of the guide RNA sequence. A PCR template can contain components of the guide RNA IVT template. A PCR template can be broken up into multiple smaller fragments that assemble into a full template either enzymatically or through overlapping homology, which assembles into a full sequence through the cycles denaturing and polymerase extension in a PCR reaction. Such fragments can be dsDNA or ssDNA templates, for example generated through commercial chemical synthesis (e.g., optionally by a commercial provider). Instead of overlapping fragments, a DNA template for PCR can also be completely synthesized through commercial chemical synthesis.

[0335] Guide RNA can be generated by IVT from the plasmid, PCR, or chemically synthesized dsDNA template using commercial T7 polymerase kits. An example commercial kit is the Precision gRNA Synthesis Kit from ThermoFisher (Catalogue # A29377). The reaction that can generate guide RNA is made up of a reaction buffer, equimolar ribose nucleic acids ATP, GTP, CTP and UTP, linear dsDNA template, and T7 enzyme. This reaction can be incubated at 37C for up to 4 hours. The resulting reaction mix can be treated with DNAse I to remove template DNA, purified using a column or precipitation approach, checked for purity on an agarose gel or chromatography system, and quantified using absorbance or fluorescence dye-based measurement systems.Example 6: Production of plasmids for expression of guide RNAs

[0336] This Example exemplifies generation of plasmids for the expression of guide RNAs for testing in cells. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0337] The first step of plasmid construction described in the present Example was in siiico design. Guide RNA sequences were designed as described in Example 2. These sequences were incorporated into a common plasmid backbone based on the pUC18 plasmid. In addition to the elements required for replication in E. coii, the resulting plasmid contained a U6 promoter 5' to the guide to drive expression of the guide RNA in a cell, a "G" initiation nucleotide 24 nt downstream of the U6 TATA box, and a poly T sequence of 6-8 T nucleotides 3' of the guide RNA to stop transcription. In some embodiments, a single G initiation nucleotide was used, in other embodiments an additional G was appended to ensure inclusion of at least one G on the guide RNA transcript, in other embodiments two additional G nucleotides were appended. In some embodiments of guide RNA expression plasmids, an HDV (Hepatitis Delta Virus) ribozyme sequence was included 3' of the spacer sequence, and 5' of the poly T stop signal. This sequence self-cleaves after expression, leaving a precise 3' end as opposed to varied lengths of U's resulting from readthrough of the poly T stop signal (Ferre-D’Amare et al. 1996). Once the in siiico version of the plasmid was confirmed to contain the necessary elements, a cloning strategy was chosen. In one version of cloning, a partial U6 promoter, the entire guide chassis, guiding sequence, ribozyme and poly T sequence was synthesized and cloned into a linearized vector using Gibson assembly (Gibson et al 2009). In another version of cloning, guide RNA sequences are assembled using overlapping oligos and ligated into a plasmid backbone containing a full U6 promoter, ribozyme and poly T site, as is known in the art.

[0338] Some plasmids were constructed using Gibson assembly. Linearized plasmid backbone was generated by digesting 4ug of plasmid (pM-GA-0051) in a reaction containing 80 units each of enzymes Agel-HF and KpnI-HF (NEB), 20ul of lOx Cutsmart buffer, in a final volume of 200uL Digestion was carried out at 37C for 48hrs. Linearized plasmid was purified by gel purification: all 200ul were loaded into an E Gel-EX (ThermoFisher, cat# G402022) and run for 10 minutes following manufacturers suggested protocol. The E Gel-EX was opened, the desired band visualized by ambient light and excised with a razor blade, then purified from the gel using a Macherey-Nagel Gel and PCR clean up kit (Macherey-Nagel, cat# 740609). The guide RNA-encoding DNA fragments all shared the same homology ends for Gibson assembly, the 5' end of which was in the U6 promoter, and the 3' end in the pUC18 backbone. dsDNA for each unique guide RNA was ordered from IDT commercial synthesis as an eBlock gene fragment. Upon receipt, the fragments were cloned into the linearized plasmid backbone using Gibson assembly: 0.005pM of linearized backbone was combined in a semi-skirted PCR plate with O.OlpM of the dsDNA eBlock, 5ul of Gibson master mix and DI water added to a final volume of lOul. The Gibson reaction was carried out at 50C deg for Ihr, then placed on ice prior to E. coii transformation. Transformations were carried out using NEB lObeta chemically competent cells following the manufacturer's suggested protocol. Successful clones were selected on LB plates containing carbenicillin, and then sequence verified.Example 7: Production of reagents for LNP production

[0339] This Example illustrates how Casl2f mRNA and guide RNAs can be formulated into lipid nanoparticles (LNPs) for delivery in cell culture or in vivo. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.A: LNPs for delivery in cell culture:

[0340] A total of 18 ug of RNA were formulated in LNPs following the manufacturer's instructions for the Hepato9 kit (Precision Nanosystems, Inc., Canada). Formulated LNPs can be made with RNA comprising either the Casl2f mRNA and guide RNA separately, or co-formulated; in some embodiments, RNAs that are co-formulated will be in a molar ratio within a range of about 1:20 mRNA:guide RNA to about 1: 160 mRNA:guide RNA.

[0341] Briefly, in the present Example, RNA was diluted to 1 ug / uL in nuclease-free water (Thermo Fisher Scientific, USA) and 22 uL of RNA was mixed with 17.6 uL of Formulation Buffer 1. The cartridge for formulating the LNPs was first loaded with 48 uL of Spark Formulation Buffer 2 into Well-1, then 32ul of the RNA into Well-2, and finally 16 uL of Nanoparticle Mix into Well-3. The cartridge was loaded into the NanoAssemblr® Spark™ instrument (Precision Nanosystems, Inc., Canada) and LNPs were formulated using Setting #3. Formulated LNPs were removed from the cartridge and then diluted into 96 ul of Spark Formulation Buffer 2 and then stored at 4C for 1-2 days prior to adding them to cells.B: LNPs for delivery in vivo:

[0342] RNA comprising the Casl2f mRNA and guide RNA, ranging in weight from 0.1 to 2 mg can be formulated in a single-run using the NanoAssemblr® Ignite™ (Precision Nanosystems, Inc., Canada) following the manufacturer's instructions for the GenVoy-ILM kit (Precision Nanosystems, Inc., Canada). Briefly, formulated LNPs can be made with RNA comprising either the Casl2f mRNA and guide RNA separately or co-formulated; in some embodiments, RNAs that are co-formulated will be in a molar ratio within a range of about 1:20 mRNA:guide RNA to about 1: 160 mRNA:guide RNA.

[0343] Briefly, in some embodiments, RNA can be diluted to a concentration within a range of, for example, about 0.09 to about 0.17 mg / mL in PNI Formulation Buffer, which encompass to N:P ratios of positively-chargeable polymer amine (N) groups to negatively-charged nucleic acid phosphate (P) of 4: 1 to 8: 1.

[0344] An amount of diluted RNA, for example within a range of about 1.5mL to about lOmL can be loaded into the instrument, e.g., using an appropriately sized syringe (e.g., of a size within a range of about 3mL to about lOmL) (Becton, Dickinson and Company, USA). GenVoy-ILM lipids, for example at a concentration of about 12.5 mM and in a volume for example within a range of about 0.5 mL to about 3.33mL can be loaded into the instrument, for example using an appropriately sized syringe (e.g., of a size within a range of about ImL to about 5mL) (Becton, Dickinson and Company, USA). In some embodiments, LNPs can be formulated using the following settings on the instrument: a flow rate ratio (RNA:lipid) of 3: 1, a total flow rate of 12 mL / min, start waste volumes within a range of about 0.45 mL to about 1 mL, and an end waste volume of 0 mL. In some embodiments, formulated LNPs can be diluted 40-fold with PBS (Thermo Fisher Scientific, USA) and then concentrated down to a desired volume, for example within a range of about 0.25 mL to about 1.5 mL, for example using centrifugal lOkDa MWCO filter concentrators (Sigma-Millipore, USA) through repeated spins at 2,000xg for 30 min at 4C. Concentrated LNPs can bepassed through a 0.22um Acrodisc syringe filters (Cytivia, USA) and then stored at 4C for up to 8 days prior to delivering them in vivo.Example 8: Production of AAV containing certain guide RNAs of the present disclosure

[0345] This Example illustrates how guide RNA can be produced to be expressed from AAV virus particles for measuring activity in cell culture or in vivo. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0346] AAV particles are polypeptides encapsulating a ssDNA genome. Those skilled in the art will appreciate that AAV capsids will package ssDNA, typically having a length between about 3kb and about 5kb, that is flanked by inverted terminal repeat (ITR) sequences, such as ITRs from the AAV2 subtype (ITR2). Functional AAV particle production is typically accomplished by, for example, transient transfection of a plasmid(s) expressing the necessary polypeptides, as well as DNA containing the desired ssDNA genome flanked by two ITRs. AAV particles containing guide RNAs were generated by first constructing a plasmid with guide RNA expression cassette of the appropriate size flanked by ITR2s.

[0347] AAV guide RNA expression cassettes utilized in the present Example were designed in siiico using standard computational molecular biology tools (Geneious). The design included certain expression cassette features as described in Example 3, with a human U6 promoter driving expression of a guide chassis with a specific target spacer, with or without an HDV ribozyme, and a poly T stop signal. In one design, the expression cassette contained multiple other elements including, 2 copies of ITR2, CAG promoter driving the expression of a SpaCasl2fl polypeptide with an N-terminal protein tag eGFP, a wPRE expression element, and a bGH poly A signal sequence. In another design, the guide RNA expression cassette was oriented opposite to a fluorescence gene expression cassette, with a CAG promoter driving tagBFP2, followed by a wPRE sequence and bGH poly A, all between two ITR2 sequences. Each design was validated to contain all necessary elements for packaging into AAV particles, and confirmed to fit the size requirements for AAV (less than 4.8Kb). Then the design was provided to a gene synthesis company (Genscript), where everything between the two ITRs sequences was chemically synthesized and cloned into a plasmid with ITRs already present. Plasmid sequence and ITR accuracy was validated with Sanger sequencing.

[0348] Production of the AAV particles was achieved by providing the ITR containing plasmid to an AAV production company (Packgene).Example 9: Production of lentivirus vector containing certain guide RNAs of the present disclosure

[0349] This Example illustrates how guide RNA can be produced to be expressed from lentivirus viral vector (LVV) for measuring activity in cell culture or in vivo. For clarity, the present Example provides exemplary procedures. In some embodiments, as a skilled person reading the present disclosure will appreciate, one or more steps may be left out, and / or certain steps may be performed in a different order.

[0350] Lentivirus (LV) is an enveloped virus with a glycoprotein envelope encapsulating an RNA genome. Those skilled in the art will appreciate that the enclosed RNA is expressed from a DNA template and contains specific elements to be correctly packaged into the virus particle, while lacking elements detrimental to full RNA packaging. Packaging and envelope components are frequently expressed from separate DNA plasmids, and the intended RNAgenome is expressed from a transfer plasmid. The RNA genome expressing transfer plasmid contains Long Terminal Repeats (LTRs) flanking expression cassettes, which contain elements necessary for transcription and translation of the desired polypeptide or RNA.

[0351] SpaCasl2fl polypeptide and guide RNA components (e.g., "transgenes") can be encoded into a transfer plasmid. First, the desired elements can be designed in silico, with necessary promoters and termination signals. An example polypeptide expression element is a CMV promoter upstream of an open reading frame, followed by a WPRE untranslated region and a polyA termination sequence. An example RNA expression element is a U6 promoter, the desired RNA with an appropriate 5' G (see Example 5), followed by a poly T termination element. As the Lentiviral genome is packaged as an RNA molecule, care must be taken to ensure expression of the full genome during lentivirus production. Other elements required to be encoded between the LTRs include the HIV-1 packaging signal (UJ), the Rev Response Element (RRE), and a polypurine tract (cPPT). Frequently a drug selection marker is also encoded to enable selection of cells with an integrated LVV genome. Designs can be generated as plasmids by various cloning methods, including providing the entire sequence to a gene synthesis company (e.g., Genscript) for synthesis and sequence validation.

[0352] Modifications can be made to the lentivirus to lower the risk associated with integration and constitutive expression. These include modifications to the int gene to make the virus integrase deficient (ie. IDLV). Other modifications include "safety-switches", such as including self-targeting sequences in the viral genome to disrupt CAS or guide RNA transgene expression in parallel with disrupting the host genomic target locus. Additional examples include regulating expression of the transgene(s) through endogenous or exogenous signals or regulating polypeptide or guide stability through endogenous or exogenous signals.

[0353] To generate lentiviral particles, a transfer plasmid can be provided to a viral production company (e.g., Cellecta).Example 10: Delivery of plasmids to cells via transfection

[0354] This Example illustrates transfection of HEK293T cells with plasmids to evaluate the ability of guide RNAs to direct a Casl2f enzyme to genomic target sites for double stranded DNA cleavage and error-prone repair.

[0355] Lenti-X HEK293T cells (Takara Bio, USA), passaged fewer than 10 times, were cultured in media comprising high-glucose DMEM (Thermo Fisher Scientific, USA) and 10% heat-inactivated fetal bovine serum (Sigma, USA), in collagen-coated T-175 flasks (ZenBio, USA), in Steri-Cycle-il60 Incubators (Thermo Fisher Scientific, USA) set to 37C and 5% C02. Cells were detached and replated 18-26 hours prior to transfection. To detach the cells, culture media was aspirated from the flask and 15 mL of TrypLE Express Enzyme (Thermo Fisher Scientific, USA) was added and incubated for 5 minutes at 37C. The flask was knocked to further detach the cells and then detached cells were transferred to a 50 mL conical containing 30 mL of 37C pre-warmed culture media. Cells were pelleted at 420xg for 4 minutes at room-temperature, resuspended in 5 mL of culture media and then counted using a Vi-Cell XR (Beckman Coulter, USA). 2.5xl04cells per 100 ul were seeded in individual wells of a 96-well collagen-coated plate (StemCell Technologies, Canada), allowed to settle for 3-5 minutes at room temperature, and then moved to the incubator. 18- 26 hours post-seeding, when HEK293T cells reached confluences of 70-90%, transfection mix was prepared.

[0356] Transfection mixture was then generated following the manufacturer's suggested protocol. Briefly, 0.6uL of Lipofectamine3000 (Thermo Fisher Scientific, USA) was diluted in lOuL of Opti-MEM and vortexed for 5-10 seconds to mix. Each of the Casl2f expressing and guide RNA expressing plasmids were diluted to 50ng DNA per uL in nuclease-free water (Thermo Fisher Scientific, USA). 2.36uL of Casl2f plasmid and 1.64uL of guide plasmid, equaling 118ng of Casl2f plasmid and 82ng of guide plasmid respectively, were diluted in 8uL of Opti-MEM. 0.4uL of P3000 reagent was added to the diluted Casl2f and guide plasmid and vortexed for 5-10 seconds to mix. The entire volume of diluted Casl2f plasmid and guide plasmid in P3000 was added to the diluted Lipofectamine3000, vortexed for 5-10 seconds to mix, and incubated for 12 minutes at room temperature.

[0357] During the incubation of transfection mixture, HEK293Ts were removed from the incubator (typically at 10.5 minutes), and at the 12 minute mark the entire volume of DNA: lipid mixture was added to the cells. The plate was vigorously shuffled side to side to disperse the transfection mixture in the wells and then returned to the incubator. 72 hours post-transfection genomic DNA was harvested, and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 7).Example 11: Delivery of mRNA / gRNA to primary hepatocytes via transfection

[0358] The present Example illustrates transfection of primary human hepatocytes with a guide RNA and an mRNA expressing a Casl2f. Such transfection permits, among other things, demonstration and / or evaluation of one or more features of a guide RNA's ability to direct a Casl2f enzyme to genomic target sites, and / or to achieve double stranded DNA cleavage and error-prone repair at such sites.

[0359] Transfection was performed following the manufacturer's suggestion for MessengerMax (Thermo Fisher Scientific, USA). Briefly, cryopreserved-plateable primary human hepatocytes (Lonza, USA) were thawed in 50 mL of 37C pre-warmed hepatocyte thawing media (Lonza, USA) in a 50mL conical and then pelleted at lOOxg for 8 minutes. Hepatocytes were resuspended in 3mL of hepatocyte plating media (Lonza, USA), diluted 1: 1 in Trypan-blue (Thermo Fisher Scientific, USA), and then counted using a MilliCell disposable hemacytometer (Millipore-Sigma, USA). 4xl04cells in 50ul were plated in 96-well collagen-coated plates (StemCell Technologies, Canada) containing 50ul of plating media pre-warmed to 37C, allowed to settle for 2-3 minutes at room temperature, and then moved to Steri- Cycle-il60 Incubators (Thermo Fisher Scientific, USA) set to 37C and 5% CO2. 4-6 hours post-plating, plating media was removed and lOOul of 37C pre-warmed hepatocyte maintenance media (Lonza, USA) was added to the cells and the cells were returned to the incubator.16-24h post-seeding, dead hepatocytes were removed by removing the media and flushing dead cells with 200ul of room-temperature PBS (Thermo Fisher Scientific, USA). PBS was promptly removed and lOOul of fresh 37C pre-warmed hepatocyte maintenance media was added to the cells.

[0360] Transfection mix was prepared. Lipofectamine MessengerMax (Thermo Fisher Scientific, USA) was vortexed for 5-10s, and 8.8uL was diluted in 243uL of Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific, USA) in a ImL deep 96-well plate (Thermo Fisher Scientific, USA). The plate was sealed with adhesive film (Thermo Fisher Scientific, USA), vortexed for 5-10s to mix, and then incubated at room-temperature for 10 minutes. Casl2f mRNA (as described in Example 4) and a chemically synthesized guide RNA (as described in Example 19) were diluted to 150ng RNA / uL and 200ng RNA / uL respectively in nuclease-free water (Thermo Fisher Scientific, USA). 3.2uL of the diluted mRNA and 21.9uL of the diluted guide RNA were added to 233uL of Opti-MEM in a second ImL deep 96-well plate, which was then sealed with adhesive film and vortexed for 5-10s to mix. Then, 252 ul of the MessengerMaxdiluted in Opti-MEM was added to the plate containing diluted mRNA and guide RNA, the plate was sealed with adhesive film, vortexed for 5-10s to mix, and then incubated at room-temperature for 5 minutes.

[0361] Following the incubation of transfection mix, hepatocytes were removed from the incubator and 85uL of transfection mix was added to each well of hepatocytes, corresponding to 80ng of Casl2f mRNA and 730 ng of guide RNA. The plate was vigorously shuffled side to side to disperse the transfection mixture in the wells and then returned to the incubator. Hepatocytes received complete media changes with lOOul of 37C pre-warmed hepatocyte maintenance media at 16h-24 hours post-transfection. 72 hours post-transfection genomic DNA was harvested and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 16).Example 12: Delivery of mRNA / gRNA to T cells via electroporation

[0362] The following Example illustrates the use of T cell electroporation to evaluate the ability of guide RNAs to direct a Casl2f enzyme to genomic target sites, for example for double stranded DNA cleavage and / or error-prone repair.A. Isolation of human T cells:

[0363] The following protocol describes isolation of T cells from peripheral blood mononuclear cells (PBMC) using negative-selection following the manufacturer's suggested protocol with the EasySep Human T Cell Isolation Kit (StemCell Technologies, Canada, Cat# 17951). Freshly isolated human PBMCs (AllCells, USA) were transferred to a 250mL bottle (Corning, USA) and then counted using a Vi-Cell XR (Beckman Coulter, USA). 2.25xl09cells were distributed evenly to four 50mL conicals and then pelleted at 300xg for 5 minutes. Pellets were resuspended in a total of 45mL of EasySep Buffer (StemCell Technologies, Canada, Cat# 10010049) in a 50mL conical. 2.25mL of isolation cocktail (StemCell Technologies, Canada Cat # 17951C) was added to the cells, the solution pipetted up and down five times with a 25mL serological pipet to mix, and then incubated for 10 minutes at room temperature. Rapidspheres (StemCell Technologies, USA, Cat # 50103) were vortexed for 10 seconds, 1.8mL was added to the cells, and then mixed up and down two to three times gently, with a 25 mL serological pipet. The 50mL conical containing the cells was placed on an EasySep Magnet (StemCell Technologies, Canada) at room-temperature for 5 minutes to capture non-T cells. The supernatant that contains T cells was transferred out of the conical using a 50 mL serological pipet in a single-motion into a new 50 mL conical. Purified T cells were counted, pelleted at 300xg for 5min, resuspended to 5xl07cells / mL in ice-cold CS10 Cell Freezing media (StemCell Technologies, Canada), ImL of cells were transferred to 1.8mL cryovials (Thermo Fisher Scientific, USA) and then cryopreserved in a CoolCell Containers (Corning, USA) at -80C prior to being stored long-term at -160C.B. Thawing and activation of human T cells:

[0364] The following protocol describes thawing and activating human T cells. 5xl07cryopreserved T cells were thawed in of 37C pre-warmed culture media consisting of in Optimizer T-Cell Expansion Medium (Thermo Fisher Scientific, USA) supplemented with 2% CTS Immune Cell SR (Thermo Fisher Scientific, USA), 1% Glutamax (Thermo Fisher Scientific, USA), and 100 lU / mL of recombinant IL-2 protein (PreproTech, USA). T cells were pelleted at 300xg for 5 minutes, resuspended in lOmL of 37C pre-warmed culture media, counted using a Vi-Cell XR (Beckman Coulter, USA), transferred to T-175 flasks at IxlO6cells / mL, and then moved to Steri-Cycle-i 160 Incubators (Thermo Fisher Scientific, USA) set to 37C and 5% CO2 for 2-4 hours. Following incubation, T cells were mixed with CD3 / CD28Dynabeads (Thermo Fisher Scientific, USA) at 1: 1 cell number: bead number ratio and then returned to the incubator to activate the T cells.C. Transfection of T cells with Casl2f mRNA and guide RNA:

[0365] Casl2f mRNA (as described in Example 4) and a chemically synthesized guide RNA (as described in Example 19) were transfected into activated T cells using the 4D-Nudeofector™ 96-well Unit (Lonza, USA) using the following protocol. 8pmol of Casl2f mRNA and 279pmol of a chemically synthesized guide RNA were combined and diluted to a final volume of 4.7uL of nuclease-free water (Thermo Fisher Scientific, USA) and then transferred to individual wells of a 96-well Nucleocuvette® Plate (Lonza, USA). After 72 hours of activation, T cells were washed and added to the wells containing RNA. Briefly, T cells were transferred to 50 mL conical tubes, which were placed on a Dynamag Magnet (Thermo Fisher Scientific, USA) for 1 minute to allow removal of CD3 / CD28 DynaBeads. De-beaded T cells were counted as previously described and 5.5xl07cells were transferred to a 50mL conical and pelleted at 90xg for lOmin. Cells were resuspended in 20mL of PBS and then pelleted at 90xg for lOmin. Cells were then resuspended to a density of 5xl07cells / mL in P3 Nucleofection Buffer (Lonza, USA). 20ul of the cell suspension was added to individual wells containing 4.7uL of Casl2f mRNA and guide RNA. The plate was loaded onto the 4D-Nucleofector™ 96-well Unit (Lonza, USA) and the cells were electroporated using the DN-130 nucleofection program (Lonza, USA). Post-electroporation, 80ul of 37C pre-warmed complete culture media consisting of Optimizer T-Cell Expansion Medium (Thermo Fisher Scientific, USA) supplemented with 2% CTS Immune Cell SR (Thermo Fisher Scientific, USA), 1% Glutamax (Thermo Fisher Scientific, USA), and 100 lU / mL of recombinant IL-2 protein (PreproTech, USA) was added to each well, and the plate incubated for 15 minutes at 37C and 5% CO2. After incubation, 80ul of the cell suspension was transferred to a 96-well cell culture plate containing 120ul of 37C pre-warmed complete culture media. The plate was then incubated for 72 hours at 37C and 5%CO2, cells were then counted as previously described, diluted to cell density of IxlO5cells / mL with complete culture media, and 200ul of cells were transferred to a 96-well cell culture plate. The plate was then incubated for 48h-72h and then genomic DNA was harvested and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 16).Example 13: Delivery of AAV to HEK293T cells and primary human hepatocytes

[0366] The following Example demonstrates transfection of HEK293T cells and primary human hepatocytes with AAV (e.g., which, in some embodiments, may deliver Casl2f mRNA and / or a guide RNA as provided and / or otherwise described herein).

[0367] HEK293T cells were plated at 15,000 cells per well in 96 well collagen I-coated microplates (Stem Cell Technologies, USA) in DMEM (Thermo Fisher Scientific, USA) containing 10% FBS (Thermo Fisher Scientific, USA) 24 hours before AAV transduction. It was estimated that cells doubled in the 24 hours, thus AAV genome copy per cell was calculated based on 30,000 cells per well. Transduction virus mix was prepared by diluting desired amount of AAV preparation in lOOul of DMEM containing 0.3% FBS (Thermo Fisher Scientific, USA). The medium was completely removed and lOOul of the virus dilution was added. 24 hours after transduction, the virus-containing medium as removed and fresh DMEM containing 10% FBS was added, in order to remove the bulk of the untransduced virus. Cells were incubated for 48 hours at 37C and 5% CO2, before being harvested for next generation sequencing to detect genome editing events (Example 16).

[0368] Primary human hepatocytes were plated 24 hours before transduction. Cryopreserved primary human hepatocytes (Lonza, USA) were thawed in a 37C water bath resuspended in 50 ml of Hepatocyte Thawing Medium (Lonza, USA) per cryovial, and centrifuged at 100 x g for 8 minutes at room-temperature. Cells were resuspended in Plating Medium (Lonza, USA), plated at 40,000 cells per well in 100 ul medium volume in 96 well collagen I-coated microplates and incubated at 37C and 5%CO2. 4 hours after plating, the medium was replaced with Hepatocyte Complete Medium (HCM, Lonza, USA) and incubated at 37C and 5%CO2 for 20 hours. Immediately before transduction, dead cells were removed by first removing the original medium, adding 200ul of fresh HCM, and replacing the dead cell-containing medium with lOOul of fresh HCM. To prepare for transduction, desired amount of AAV preparation was diluted in lOOul of HCM. The medium in the microplates was then replaced with lOOul of AAV- containing medium to achieve transduction. Cells were incubated at 37C and 5%CO2 for 24 hours, before media change to remove the untransduced AAV in the medium. Cells were incubated for 48 hours at 37C and 5%CO2, before being harvested for next generation sequencing to detect genome editing events (Example 16).Example 14: Delivery of LNP to primary Human Hepatocytes in vitro

[0369] The following Example illustrates the transfection of primary human hepatocytes with mRNA expressing a Casl2f and guide RNA formulated in lipid nanoparticles (LNPs), as described in Example 7, for example to direct a Casl2f enzyme to genomic target sites for double stranded DNA cleavage and error-prone repair.

[0370] Cryopreserved-plateable primary human hepatocytes (Lonza, USA) were thawed in 50mL of 37C prewarmed hepatocyte thawing media (Lonza, USA) in a 50mL conical and then pelleted at lOOxg for 8 minutes. Hepatocytes were resuspended in 3mL of hepatocyte plating media (Lonza, USA), then diluted 1: 1 in Trypan-blue (Thermo Fisher Scientific, USA), and then counted using a MilliCell disposable hemacytometer (Millipore-Sigma, USA). 4x104 cells in 50ul were plated in 96-well collagen-coated plates (StemCell Technologies, Canada) containing 50ul of 37C warmed plating media, allowed to settle for 2-3 minutes at room temperature, and then moved to Steri-Cycle- il60 Incubators (Thermo Fisher Scientific, USA) set to 37C and 5% CO2. 4-6 hours post-plating, plating media was removed and lOOul of 37C pre-warmed hepatocyte maintenance media (Lonza, USA) was added to the cells and the cells were returned to the incubator. 16-24h post-seeding, dead hepatocytes were removed by removing the media and flushing dead cells with 200ul of room-temperature PBS (Thermo Fisher Scientific, USA). The PBS was immediately removed and lOOul of fresh 37C pre-warmed hepatocyte maintenance media was added to the cells.

[0371] LNPs containing Casl2f mRNA and guide RNA were diluted in 1 ug / mL of recombinant human ApoE4 (PeproTech, USA)-supplemented hepatocyte maintenance media at molar ratios within a range of about 1:20 mRNA:guide RNA to about 1: 160 mRNA:guide RNA, pipeted up and down 10 times to mix, and then warmed to 37C for 5-20 min in an incubator set to 37C and 5% CO2. The plate containing primary human hepatocytes was removed from the incubator, media in the wells were aspirated, and then lOOul of LNPs were added to the cells in increasing concentrations within a range from about 2.5 ng to about 160ng of Casl2f mRNA and about 6 ng to about 1460 ng of the guide RNA per lOOuL. Hepatocytes received medium changes with lOOul of 37C pre-warmed hepatocyte maintenance media at 16h-24 hours post-transfection. 72 hours post-transfection, genomic DNA was harvested and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 16).Example 15: Delivery for in vivo genome engineering in a mammal

[0372] The following Example illustrates how Casl2f and guide RNA can be administered to mice, for example to direct a Casl2f enzyme to genomic target sites for double stranded DNA cleavage and error-prone repair. Delivery can be achieved using either formulated lipid nanoparticles (LNPs) with mRNA expressing a Casl2f and guide RNA(s), or AAVs that encode for both or either Casl2f and guide RNA. Delivery can be done, for example, in the following mice: WT CD-I mice, WT-C57BL / 6 mice. Alternatively or additionally, in some embodiments, mice such as strains with humanized genes including "C57BL / 6-Pcsk9tml(PCSK9) / Bcgen" (Biocytogen catalogue # 110928) could be utilized.A. Delivery of LNPs for in vivo genome engineering in mice:

[0373] LNPs formulated as described in Example 7 can be dosed into mice as previously described by Finn et al. 2018 (DOI 10.1016 / j.celrep.2018.02.014), which is incorporated herein by reference in its entirety. Briefly, LNPs can be dosed via the lateral tail vein in a volume of 0.2mL in phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, USA), and a mass of about 0.3mg to about 3mg of total RNA per kg of mouse total body weight. Animals can be euthanized at various time points via cardiac puncture under isoflurane anesthesia and tissue can be collected from each animal for DNA extraction and analysis.B. Delivery of AA Vs for in vivo genome engineering in mice:

[0374] AAVs produced as described in Example 8 can be dosed into mice as previously described by Ran et al. 2015 (DOI 10.1038 / naturel4299) and Li et al. 2021 (DOI 10.1016 / j.omtm.2021.02.005), both of which are incorporated herein by reference in their entirety. Briefly, AAVs can be dosed intravenously via injection into the lateral tail vein of male C57 / BL6 mice. About 0.5X1011to about 4xlOnvector genomes of the AAV can be adjusted to 0.1ml or 0.2ml in phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, USA) before injection. Animals can be euthanized at 7, 14, 21, or 28 days post-injection via cardiac puncture under isoflurane anesthesia and tissue can be collected from each animal for DNA extraction and analysis.C. Delivery of L W for in vivo genome engineering in mice:

[0375] LVVs as produced as described in Example 9 can be dosed into mice as previously described by Follenzi et al. 2002 (DOI 10.1089 / 10430340252769770) and Follenzi et al. 2004 (DOI 10.1182 / blood-2003-09-3217) which are incorporated herein by reference in their entirety. Briefly, LWs can be dosed intravenously via injection into the lateral tail vein of C57 / BL6 mice. About 0.2 to 1.0xl0A9 transducing units (TUs) of LWs can be adjusted to 0.2 to 0.5 ml in in phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, USA) before injection. Animals can be euthanized at 7, 14, 21, or 28 days post-injection via cardiac puncture under isoflurane anesthesia and tissue can be collected from each animal for DNA extraction and analysis.Example 16: Detection of genome modifications by sequencingA: Eukaryotic Genomic DNA Extraction

[0376] Genomic DNA was isolated from mammalian cell culture following the manufacturer's suggested protocol using the Quick Extract™ DNA Extraction Solution (LGC Biosearch Technologies, Hoddesdon, UK). Briefly, cells in a 96 well plate were pelleted and washed, before being resuspended with 50ul Quick Extract solutions. When using PHH cells, dead cells can be removed prior to resuspension with light acutase treatment. Cells in Quick Extractsolution were incubated on an orbital shaker set at 800 rpm for 15min at 37C. Following this incubation, the reaction was mixed and moved to a PCR plate, where it was incubated at 65C for 6min and then 98C for 2min. The resulting genomic DNA was then quantified, for instance with a Quant-iT™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA) and then stored at -80C until further processing.B: PCR Amplification of target region

[0377] Next-Gen Sequencing library preparation typically begins with PCR amplifying the isolated genomic DNA using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA.) at IX concentration in a 25ul reaction, forward and reverse primers at 0.5uM final concentration each, and input genomic DNA equivalent to 8000 genomic copies. PCR primers were designed to amplify a region of genomic DNA around the target site, 180-240bp in length. PCR primers contained a tag sequence at the 5' end of each primer, where the tag sequence is used as the primer binding site for adding unique barcodes and sequencer-specific sequences. Such a reaction was run at 98C for 30 seconds followed by 30 cycles of 98C for 10 seconds, 68C for 30 seconds, and 72C for 30 seconds. A final extension was carried out at 72C for 2 minutes.C: PCR Amplification to add unique barcodes

[0378] PCR products are typically diluted 1: 100 in water and used for a barcoding PCR to attach unique dual indexes (i5 and i7) and Illumina overhangs containing a sequencing binding site to facilitate multiplex sequencing. Such barcoding PCR was performed using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA.) at IX concentration in a 25ul reaction, forward and reverse primers at 0.5uM final concentration each, and input of 2. Sul of 1: 100 diluted first PCR. The reaction is run at 98C for 30 seconds followed by 10 cycles of 98C for 10 seconds, 60C for 30 seconds, and 72C for 30 seconds. A final extension is carried out at 72C for 2 minutes.D: SPRIseiect bead-based purification

[0379] Barcoded PCR reaction products are then typically pooled into a single microcentrifuge tube and mixed with 0.8x volume of SPRIseiect beads, and incubated at room temperature for lOminutes. Samples with beads were placed on a magnetic rack and the supernatant was discarded. Beads underwent two 80% ethanol washes and were left to dry at room temperature for 3-5 minutes. Once the ethanol was fully dried from the beads, the DNA was eluted in water. Samples were then checked for size distribution and concentration using a gel electrophoresis instrument similar to an 4200 Agilent TapeStation (Agilent, Santa Clara, CA).E: Sequencing and target sequence modification analysis

[0380] Depending on the amplicon size, the correct Illumina sequencing kit was used. Samples were diluted down to 950pM and phiX control was spiked into the sample at a final 10-30% of mass to increase diversity during sequencing. A NextSeq 1000 (illumina, San Diego, CA.) is used to sequence the sample, with output BCL files used for analysis. The BCL files were converted to Fastq files using the on-board DragonTM computing structure (Illumina, San Diego, CA.). The Fastq files were used as input to the CRISPResso (crispresso2.pinellolab.org) pipeline for indel detection using the GRCh38 reference genome.Example 17: Detection of gene expression changes by flow cytometry

[0381] The following Example illustrates an exemplary method by which protein expression changes can be measured using flow cytometry.

[0382] Flow cytometry can be used to measure gene knock-outs resulting from CAS induced double strand breaks and the resulting disruptive repair, as well as CAS based activation or repression of gene expression.A. Flow Cytometry with HEK293T cells

[0383] HEK293T cells were seeded at 1.5 x 104in 96 well microplates in lOOul DMEM supplemented with 10% FBS and incubated at 37C in 5% C02 for 24 hours. Cells were transfected as in Example 10, using 60ug of guide plasmid and 80ug of a Casl2fl expression plasmid, sometimes with an additional 70ug of an MCP expression construct, using FuGene XD (Fugene, USA). In one variation of this assay, the target gene is CXCR4. CXCR4 is normally expressed at a very low level in HEK293T cells. When measuring CRISPRa or CRISPRi, cells were harvested 48 hours after plasmid co-transfection. When measuring gene knockout resulting from DSB induced INDELs, cells were harvested after 72 hours. Cells were harvested by first incubating with 0.25% Trypsin-EDTA (Thermo Fisher Scientific, USA, cat# 25200072) for 5 minutes at 37C. The digestion was neutralized with DMEM with 10% FBS, then cells were transferred to U-bottom assay microplates, pelleted down and washed once with ice-cold PBS. Cells were stained with LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher, USA, cat# L34964) that was diluted 1: 1000 in PBS for 30 minutes on ice, washed twice with ice-cold Flow Cytometry Staining Buffer (Thermo Fisher Scientific, USA cat#00-4222-26) and blocked with the Staining buffer for 10 minutes on ice.

[0384] When measuring CXCR4 gene modulation, antibody staining was conducted by incubating the cells with a PE-conjugated anti-CXCR4 antibody (Biolegend, USA, cat#306506) diluted 1:25 in the staining buffer for 30 minutes on ice. Finally the cells were washed twice with the staining buffer before subjected to measurement using the Attune flow cytometer (Thermo Fisher Scientific, USA). The flow cytometric analysis was done by first gating for cells based on FSC-A x SSC-A scatter plot, followed by single cells based on FSC-H x FSC-A, and then live cells defined by the absence of LIVE / DEAD Violet dye staining. CXCR4 expression was measured by emission on the PE channel on this live, single cell population.B. Flow Cytometry with primary T cells.

[0385] Five hundred thousand primary T cells were retrieved from cryopreservation, activated, then electroporated with 4ug of in vitro-transcribed SpaCasl2fl mRNA and 16ug of chemically synthesized sgRNA as described in Example 12. Cells were processed for antibody staining the same manner as the above example for HEK293T cells on 3 and 6 days post-electroporation except for the omission of Trypsin-EDTA treatment. When measuring B2M expression, the cells were stained with a PE-conjugated anti-B2M antibody (Biolegend, USA, cat#316306) at 1:200 dilution and measured for B2M expression by Attune flow cytometry on a live single cell gate as described above.Example 18: Identification and screening of circularly permuted Casl2f guide RNA

[0386] The following Example describes design and evaluation of circularly permuted guide RNAs to be used with Casl2f CRISPR systems. The reference to exemplary Figures and SEQ ID NOs is not intended to be limiting. Those skilled in the art, reading the present disclosure, will appreciate, for example, that, including as exemplified in Tables 2-9 and the associated SEQ ID NOs and exemplary Figures, provided circularly permuted Casl2f guide RNAs (e.g.,for use with Casl2f CRISPR systems) can include RNAs that differ from one another, for example, in start and / or end junctions, positions of tracr and crispr RNAs, and / or positions of spacer sequences.A: Design of circularly permuted SpaCasl2fl guide RNA

[0387] Circular permutation is defined as changing the 5' end of the sgRNA chassis by either the removal of sequences including linkers, the addition of new sequences, or the combination of both, which results in a guide that maintains essential secondary structures and tertiary orientation for active SpaCasl2fl ribonuclease polypeptide complexes while rearranging the primary sequence of ribonucleic acids.

[0388] SpaCasl2fl has a predicted secondary structure that loops back on itself such that the 5' end of the tracr is one half of a stem pairing, with the tracr 3' end forming the other half. Upstream of the 3' end of the tracr is an additional stem-loop, and further upstream is a sequence with homology to the crispr RNA, potentially forming the native tracr-crispr stem pairing.

[0389] SpaCasl2fl-MS13 guide chassis was chosen as a starting point for circular permutation. As stated in Example 2, Casl2f single guide RNA sequences were arranged in new 2D structures to identify potential arrangements of stem and loop elements, an example numbering scheme is shown in Figure 1. First, the "GGAA" linker inserted between the tracr and crispr RNA in MS13 was removed. Next, the tracr was opened at the 3' end of the tracr-crispr stem pairing. Then the crispr RNA was truncated to just the ribonucleotides predicted to base pair with the tracrRNA. A new "GAAA" linker was inserted between the newly truncated tracr and crispr sequences, connecting them in a single strand. Finally, another "GUUA" linker was added between the original 3' and 5' ends of the tracrRNA. These modifications resulted in a sgRNA chassis which has a new 5' end upstream of stem-loop -1, instead of in the middle of stem-loop 0. These modifications also modify the base ordering so that instead of the tracr 5' and 3' ends forming stem-loop 0 being very distant in primary sequence, they are now immediately adjacent with only a 4nt linker sequence separating them (AGNT35, Figure 1).B: Truncation of guide after circular permutation

[0390] Starting with AGNT35, multiple truncations were generated. In one series, the guide RNA was truncated back from the new 5' end, removing up to both stem-loop-1 and stem-loop 0 (AGNT39, AGNT43, AGNT47). In another series, the guide RNA was truncated at the crispr-tracr stem-loop (AGNT36, AGNT37, AGNT38). Then these truncations were tested in combination (AGNT36 through AGNT50). To assess activity, each guide was encoded into a plasmid using exemplary Spacers in Table 4 (FANCF, DNTM1, WTAP) (Example 6), transfected along with another plasmid encoding SpaCasl2fl (WT) into HEK cells (Example 10), and the activity determined by NGS (Example 16). The results are shown in Figure 11, with 5' end truncations being more active than AGNT246 or the starting circularly permuted guide AGNT35, and crispr-tracr truncations were less active. The top performing guide chassis were AGNT39, AGNT43, and AGNT47 (Figure 3).

[0391] Re-introduction of SpaCasl2fl tracr self-hybridization between stem-loop 0 and the tracr-crispr stem-loop was neutral, but did not increase activity (Figure 2, AGNT126, AGNT127, AGNT161, AGNT162, AGNT163) (Figure 14). Thus, this circularization is not essential for highly active guides.

[0392] When the top performing truncations were tested without the circular permutation (AGNT182, AGNT183, AGNT184, AGNT53, AGNT54) (Figure 4), they were much less active, showing that the permutation was essential to achieve highly active truncation variants (Figure 11, Figure 15).C: Evaluation of circularly permuted and truncated guides when chemically synthesized

[0393] Circularly permuted guide RNAs AGNT35through AGNT50were designed and provided to a commercial synthesis company (IDT). Each guide was synthesized with phosphorothioated 2' O-methyl bases at the 3 terminal nucleotides on both 5' and 3' ends (i.e. end protection). They were then evaluated for activity in a T cell electroporation cleavage assay (Example 12, Figure 16). In general, the same patterns emerged in T cells versus when evaluated in HEK293Ts as plasmid generation (Figure 11). Additionally, the top performing guide chassis from plasmid assays were tested against two more targets (Figure 16B). AGNT47 was much less active as a chemically synthesized guide with end protection in T cells than other circularly permuted deletions.

[0394] Chemically synthesize guide AGNT47 activity was rescued by modifying the 5' end bases. Three uracil nucleotides were added to the 5' end, and those bases used for end protection (AGNT1003, AGNT1001). This new guide chassis was synthesized with two different spacers, and both were more active than the original AGNT47 end protected guide (AGNT946, AGNT941, Figure 21). The 2'-0Me modification of the 5' nucleotides of AGNT47 were not the cause of loss of activity, as adding 2'-0Me modification to those bases on AGNT39 (AGNT1002, AGNT1004), which are internal in this guide, did not decrease activity of the guide, and may have improved its activity (Figure 21).

[0395] Those skilled in the art, reading the present disclosure, will appreciate, for example, that additional circularization variants, with or without truncations, can be designed and evaluated. For example, following design principles of connecting the crispr RNA to the tracrRNA through a linker, the 5' end of the linker can be attached to any 3' end of a new break point, where the other end is a new 5' end of the guide RNA. Free 5' and 3' ends can then be linked until only one of each remains. In another example of design principles, two sets of 5' and 3' ends can remain, resulting in a new design of a separate crispr and tracr RNA that must contain some complementarity or means of hybridizing with each other. Another example of modifications includes different linker sequences, such as that used to form stem-loop 0 in the circularly permuted variants, where variations of "GAAA" can be converted to contain combinations of U's instead of A's (AGNT233, AGNT234, AGNT235).Example 19: Certain chemical modifications of provided guide RNAs

[0396] The following Example describes design and evaluation of chemically modified guide RNAs to be used with Casl2f CRISPR systems. The reference to exemplary figures and SEQ IDs is not intendent to be limiting in anyway and it is understood by one of skill in the art that, based on disclosures in Tables 2-9 and the associated SEQ ID Nos and exemplary figures, Casl2f guide RNAs for use with Casl2f CRISPR systems can be designed and synthesized to have one or more modifications to the chemical backbone and bases in the RNA sequence.A: Identification of domains for modifications

[0397] In addition to phosphorothioate with or without 2'-0-methyl end protection, which inhibits nuclease activity, other backbone modifications can be incorporated in chemical synthesis to protect the internal residues from nuclease attack, for example as described in Example 3.

[0398] Using a 2D diagram of likely stem-loops along with public structures of other Casl2f enzyme:guide complexes (Uni, As, Cn, referenced in first design example), multiple domains of the SpaCasl2fl MS13 guide RNA were chosen for 2'-0-methyl modifications, in combination with phosphorothioate 2'-O-methyl protection of the three terminal nucleotides on both ends (i.e. end protection). Each design was combined with one of two spacer sequences, provided to a commercial synthesis provider (IDT), and tested along with a SpaCasl2fl mRNA through co-lipofection into Primary Human Hepatocytes (PHHs) (Example 11, Figure 9, Figure 17). Some top performing guide RNAs modifications (AGNT978, AGNT979, AGNT980) were also tested by encapsulation into LNP and transfection into PHHs, where they maintained 2-to-3-fold higher activity than with only end protection modifications (Figure 18). These beneficial modifications cover stem-loop -1, stem-loop 0, stem-loop 4, and the tracr:crispr stemloop. Inhibitory modifications were within stem-loop 2, near the putative homologous stem shown to interact with Casl2f in crystal-structures, as well as in stem-loop 1, which contains bases likely to interact with the 3' end of the crispr RNA. Other regions were either neutral or only slightly decreased activity.B. Identification of additional domains for modifications

[0399] Following the same strategy as used with SpaCasl2f, modifications were designed and applied to the truncated AsCasl2fl guide (AGNT170) that was shown to be active with SpaCasl2fl (see Example 22). Again, relying on published Protein-nucleic acid complex structures, nucleotides were identified as likely to be amenable to 2'-O-methyl modifications. Designs containing modifications were then combined with two spacer sequences and ordered from a commercial provider (IDT) (AGNT1083 through AGNT1091, and AGNT1109 through AGNT1117). Controls with modifications predicted to disrupt activity were also included (AGNT1092, AGNT1118). Chemically synthesized guides were tested in PHH cells (see Example 11), where the majority showed improved activity over the three terminal nucleotide modification variants (AGNT1070 and AGNT1072) (Figure 23). In total, 39nt out of the lOlnt AGNT170 chassis were modified in the set of guides that showed increased activity, and approximately lOnt were modified with neutral impact on activity (AGNT1083, AGNT1084, AGNT1109, AGNT1110).C: Additional strategies

[0400] Those skilled in the art, reading the present disclosure, will appreciate, for example, that design strategies described herein, and / or documented successful or neutral modifications can be combined in additional guide RNA structures. The present disclosure appreciates that modifications which are neutral in one context may provide an impact in another (e.g., in a different cellular or delivery contexts). The present disclosure provides guidance sufficient to undertake relevant assessments so that useful guide RNAs not specifically exemplified are nonetheless within the scope of the disclosure, and useful application of and context for such additional guide RNAs (and / or for exemplified guide RNAs) can be established by following teachings provided herein. Certain contemplated modifications include, for example, additive or combinatorial changes to modifications that improve activity, reduction of certain modifications to fewer base-pairs, or single nucleotides in a pair.Example 20: Certain provided guide RNAs

[0401] The present Example describes certain provided guide RNAs, for example including internalized spacer sequences, alternative linkages, and / or alternative circularization strategies.A: Design and testing guide RNAs with internalized spacer seguences

[0402] Previously characterized Casl2f guide RNAs are oriented with the spacer sequence, at the 3' end of the guide. Using secondary contact maps and homologous guide RNA structures as described in Example 1, we designed guide RNA sequences with additions to the 3' end of the spacer. Resulting guide RNAs have an internalized spacer sequence. Internalized spacer sequences can enable, for example, unique positioning of additional sequences for interacting with polypeptides (Example 23) or other nucleic acids (AGNT312 through AGNT321) as well as circular guide RNA sequences. Circular guide RNA sequences don't have any exposed ends and are thus protected from cellular exonuclease activity (Litke et al 2019).

[0403] Starting with MS13 (AGNT246), and similar to certain circular permutations described herein (see, for example, Example 18), provided guide RNAs were designed with different linkages and end points, with the spacer linked into the tracrRNA at different stem-loops.

[0404] In one example design tested, the 3' end of the spacer was connected to the original 5' end at stem-loop 0 (AGNT55), and the 5' end was placed at the end of the crispr RNA sequence (Figure 7). In another example, this connection was expanded with a new linker sequence "UUAUU" (AGNT56). In another series of examples, the attachment was made onto a series of truncations on stem-loop 0, with additional modifications onto the new 5' and 3' ends (AGNT57 through AGNT84). In another series of examples longer linkers as well as extensions to stem-loop 0 were designed (AGNT185, AGNT186, AGNT187, AGNT188). Each of these designs were constructed as in Example 6, and evaluated for activity with 2 plasmid transfection into HEK293T cells as described in Example 10. Certain of these guides showed activity as measured by INDELS (Example 16) in HEK293T (AGNT60, AGNT64, AGNT79) (Figure 12, Figure 15). The most active guides evaluated were those with either longer linkers and / or stem-loop 0 sequence.B: Examples Unking to other stem-loops.

[0405] Certain guide RNAs were designed with spacers linked to other elements in the tracrRNA sequence. For example, instead of connecting the spacer to stem-loop 0, other stem-loops can be linked to the spacer, with or without additional linker sequences. In AGNT189, the spacer was linked to the 5' of stem-loop -1, which is the new 5' resulting from circular permutation AGNT35 (Example 18). AGNT191contains a link between the spacer and stemloop 1, AGNT190 contains a link between the spacer and stem-loop 2, and AGNT192 contains a link between the spacer and stem-loop 3 (Figure 15).C: Design and testing with terminal ribozymes

[0406] Building from the active sequences with internalized spacers, and to achieve fully circularized guides, another set of sequences was designed and tested. To achieve circularization, we appended Twister p3 (AGNT7) and p5 ribozymes (AGNT8) to the 5' and 3' ends of the guide RNA, respectively. These ribozyme sequences self-cleave, and the products combine to form a new hairpin that can be ligated with cellular RNA ligases such as RctB (Litke et al 2019) to form a covalent circle.

[0407] In one example, Twister Ribozymes were added to the 5' and 3' end of a guide sequence with the spacer linked to stem-loop 0 (Figure 8, AGNT196). For this guide RNA, the spacer was again linked to the original 5' end of the SpaCasl2fl tracrRNA with a "UUAUU" linker, though with an extension of the stem-loop 0 and 5' tail relative to MS13, where more of the wild-type sequence was included. A plasmid expressing this guide was constructed as in Example 6 and evaluated as above (Figure 15). This guide also showed INDEL activity in HEK, demonstrating thatCasl2f guides can be functional when circularized. Other designs were built and evaluated as above, with the spacer linked to stem-loop 2 (AGNT198), or with both ends of the crispr RNA linked to stem-loop 0 with the twister sequences fused to new 5' and 3' ends both at an opened stem-loop 2 (AGNT200). Neither AGNT198 nor AGNT200 were active in this assay (Figure 15).D: Additional circularization strategies

[0408] Design strategies and data provided herein teach those skilled in the art about additional and / or alternative circularization strategies. For example, additional sequences linking crispr-tracr sequences, and introducing new start and stop sites either at loops or stems, can be designed, for example (AGNT193, AGNT194, AGNT195, AGNT197, AGNT199). Inclusion of non-structured linkers, such as"UUAUU", additional Casl2f guide sequences, and ribozymes are possible modifications, though not required. The present disclosure teaches, among other things, that, in many embodiments, the sequence is desirably contiguous from the 5' to 3' and includes the spacer in a 5' to 3' orientation.Example 21: Reducing guide RNA size

[0409] The present Example demonstrates reduced-size guide RNAs designed and / or tested in accordance with the present disclosure.A: Design and testing of reduced-size, circularly permuted guide RNA

[0410] Additional truncations were explored to identify smaller guide RNAs, and critical sequence components. Following Example 1, a minimal structure was identified as containing stem-loop 1, stem-loop 2, and a tracr:crispr stem-loop. These minimal elements have been confirmed by two groups for AsCasl2f guide (Hino et al, Wu et al), and are consistent with other Casl2f secondary structure homology.B: Design and testing of stem-loop 3 and stem-loop 4 modifications

[0411] To generate reduced-size guide RNAs (e.g., smallest feasible size guide RNAs), further truncations were built and tested starting from a circularly permuted guide with truncations at the 5' terminal loops, AGNT35, AGNT39, AGNT43, and AGNT47. Designs of different truncated elements were all designed as in Example 1, built to be expressed off of plasmids as in Example 6, and tested for INDEL activity in HEK293T cells as in Example 10 and 16. Modifications (insertions and truncations) to stem-loop 4 (AGNT97 through AGNT102) were all detrimental (Figure 13). Modifications to stem-loop 3 on the other hand were well tolerated, though not more active than the original stem-loop 3 sequence (Figure 13, AGNT103 through AGNT109). Loss of activity was observed to occur when all of stem-loop 3 base pairs are removed (AGNT109), however just one base-pair in stem-loop 3 was shown to be sufficient (AGNT107) (Figure 13:).C: Combination stem-loop removal

[0412] Starting with either MS13, or circularly permuted guides AGNT35 or AGNT39, additional guide RNAs were designed with removal of stem-loop 3 and stem-loop 4. Solved structures of UnlCasl2f and AsCasl2f guide:polypeptide complexes (Example 1) demonstrate that there is a conserved orientation of stem-loop 2 and the spacer RNA. In order to maintain this orientation, multiple variations of linkages between stem-loop 2 and the crispr:tracr were designed and tested (AGNT116, AGNT117, AGNT118, AGNT119, AGNT120, AGNT121, AGNT129, AGNT130), all showed minimal to no activity (Figure 14). Activity of a short guide without both stem-loop 3 andstem-loop 4 (AGNT116) was greatly improved when the design included additional homology between the loop of stem-loop 1 and the 3' end of the crispr RNA sequence (AGNT164, Example 25).D: Additional strategies

[0413] The present disclosure teaches, for example, that design strategies provided by Examples included herein (e.g., Example 29) can achieve shorter guide chassis with increased activity in cell-based assays. For example, starting with a stem-loop 3,4 deletion variant AGNT164, guides can be designed that modify either or both the position of the tracncrispr stem-loop, or the crispr 3' end, relative to stem-loops 1 and 2 (e.g. AGNT267 through AGNT285).Example 22: Cross species insights on guide RNA structure

[0414] The present Example provides exemplary guide RNAs with features informed by insights from cross-species analyses.A: Design and testing of cross-species guides

[0415] As described, for example, in Example 1, the present disclosure appreciates that there are highly conserved structures in the published Casl2f:Guide complex structures (Uni, As, Cn). The present disclosure furthermore provides an insight that shape of guide chassis may be more important than the sequence. The ability of single guide RNAs to function with SpaCasl2fl was tested by using a spacer sequence downstream of a SpaCasl2fl PAM (Example 2), but with the rest of the guide sequence from the source organism (Example 1). These guides were constructed to be expressed from plasmids (Example 6), and tested for activity in HEK293T cells (Example 10, Example 16, Figure 14).

[0416] We evaluated guide chassis from UnlCasl2f, AsCasl2f, OsCasl2f and RhCasl2f at one or two SpaCasl2fl target sites (AGNT323, AGNT322). AsCasl2f single guide chassis was able to function well with SpaCasl2fl, resulting in approximately 50% of the activity of SpaCasl2fl optimized MS13. The RhCasl2f single guide chassis was also compatible with SpaCasl2fl. UnlCasl2f and OsCasl2f guide chassis did not show activity with SpaCasl2fl mRNA (Figure 14).

[0417] In addition to full length AsCasl2f guide chassis we evaluated two truncated variants, AsCasl2f_sgRNA_DS3-5_v7 (AGNT169, Hino et al 2023) and AsCasl2f_sgRNA-v2 (AGNT170, Wu et al 2023). Both resulted in increased activity over the wild type guide chassis (AGNT248) when evaluated using two plasmid transfections into HEK293T (Figure 15). Additionally, these truncated chassis were evaluated with chemically modified ends against two targets in PHH cells using RNA delivery (FIGURE 17B). AGNT170 guide chassis were more active (AGNT1070 and AGNT1072), than AGNT169 guides, and near or equal to the activity using MS13 chassis with both internal modifications and end-protections (Figure 17B, AGNT978 and AGNT1038).B: Design and testing of chimeric guides

[0418] Expanding on the idea that the function of the guide is driven by structure vs specific sequence, chimeric guides were built which mixed stem-loops from different species (AGNT132 through AGNT160). While all previously- identified Casl2f guides have the core stem-loops stem-loop 1, stem-loop 2, and the tracr:crispr stem-loop, they each have a unique combination of additional stem-loops. For example, UnlCasl2f does not have stem-loops 3 and 4between the stem-loop 2 and tracr:crispr stem-loops, while neither AsCasl2f, OsCasl2f nor RhCasl2f have stemloops upstream of stem-loop 1. SpaCasl2fl on the other hand has both stem-loops upstream and downstream of the core-stem-loops. To make a truncated guide, we replaced one or more stem-loops from either AsCasl2f or UnlCasl2f, as well as truncated the stem-loops -1, 0, 3, or 4 which are not universally conserved. When changing the tracncrispr stem-loop, care was taken to keep the homology between the loop 1 and 3' crispr RNA tail sequence (Example 25). These chimeric and truncated guides were built as plasmids and tested in HEK cells against two targets (Figure 14).

[0419] The most active chimeric guides contained only a replacement of SpaCasl2fl tracr:crispr stem-loop with the tracr:crispr stem-loop from AsCasl2f or UnlCasl2f (AGNT132, AGNT146), the AsCasl2f based chimera being much more active. In both of these designs the loop 1 homology at the 3' end of the crispr sequence was changed to match the SpaCasl2fl sequence (e.g., the SpaCasl2fl loopl sequence). Of the other stem-loop chimeras and deletions, AGNT134 with stem-loop2 from AsCasl2f was the next most active, with others showing little to no activity. Together with the previous data, AsCasl2f and SpaCasl2fl guides have the best cross-compatibility for use with the SpaCasl2fl enzyme.C: Species-adjusted stem-loops 1 and 2

[0420] The present disclosure observes, among other things, that UnlCasl2f and AsCasl2f guide chassis sequences show high similarity in stem-loop 2, a region shown to interact with their respective polypeptides. This sequence homology is not conserved in SpaCasl2fl stem-loop 2. Additional features shared or unique to AsCasl2f and L)nlCasl2f guides around the core stem-loops were probed, including length of base-pair stretches in the stemloops, or the spacing between the stem-loops. We designed and tested SpaCasl2fl guide chassis with different single and multiple base-pair changes to mimic the conserved sequences. These designs were built as plasmids and tested against two targets in HEK293T as described in Examples 6,9 and 16.

[0421] Starting with AGNT35guide chassis, changes were introduced at stem-loop 2, either proximal or distal to stem-loopl (AGNT122, AGNT123), at the bulge in the middle of stem-loop 2 (AGNT124), as well as at the base of stem-loop 1 (AGNT131). These changes were well tolerated (Figure 14). The addition of a nucleotide opposite the GAA bulge (AGNT124) mimicking UnlCasl2f guide chassis, was most disruptive (Figure 14). Greater modifications were introduced to make stem-loop 2 more like, but not completely identical to, AsCasl2f and UnlCasl2f (AGNT125). This variant was completely inactive (Figure 14).D: Improvements to AsCasl2f truncated guide chassis

[0422] The present disclosure observes that the shortest tested guide with strong activity was derived from AsCasl2f (AGNT170), and that there are other design insights from SpaCasl2fl guide chassis that can be combined to lead to an even more active guide. Further, differences between the Casl2f chassis were exploited to make AsCasl2f specific modifications. For example, stem-loop 2 in AsCasl2f guide is much longer than SpaCasl2fl and was trimmed back (AGNT292 through AGNT296), or modified with SpaCasl2f loops and base-pair swaps (AGNT257, AGNT300). Additionally, complete removal of the 5' end upstream of stem-loop 1 was shown to cause large hits to activity in SpaCasl2fl complex activity when delivered as RNA (Example 18, AGNT47). Thus, we designed AsCasl2f chassis variants that added back stem-loops to the 5' end (AGNT297, AGNT298). Also, extended cr:tr loops wereshown to improve activity of SpaCasl2fl variants (Example 21), and variants of AsCasl2f cr:tr stem-loop were designed (AGNT303 through AGNT306).

[0423] These modified AsCasl2f designs were appended to spacers targeting B2M and PCSK9 (AGNT325, AGNT336), ordered as chemically synthesized guides (AGNT1074 through AGNT1125), and tested for ability to form indels in PHH cells as described in Example 11 (Figure 23). The most active guides were based on chassis AGNT292 (AGNT1078, AGNT1104) and AGNT298 (AGNT1095, AGNT1121). AGNT292 is a 3nt deletion in the loop of AsCasl2f stem-loop 2, and further deletions were detrimental to activity. AGNT298 is an addition of partial SpaCasl2fl stemloop 0 to the 5' end of AGNT170. Addition of more base-pairs from stem-loop 0 were less active (AGNT297), as well as deletion of 5' G nucleotides from AGNT170 (AGNT307, AGNT308). Taken together, AsCasl2f minimal guide can be improved by the lessons from SpaCasl2f engineering as taught by this disclosure.E: Additional designs

[0424] Design strategies and / or data taught herein provide those skilled in the art with other guide sequences, for example based on homologues or a chimera between homologues, that may be shorter and / or more active than SpaCasl2fl or AsCasl2f guide RNAs. For example, guides can be designed starting from the AsCasl2f truncated guide chassis (AGNT169 through AGNT170), with base-pairs in stem-loops 1 and 2, and the crispr-tracr, that are swapped to the equivalent sequence from SpaCasl2fl loops 1 and 2 (e.g. AGNT251 through AGNT266). See, for example, Examples 28 and 33. Along with further truncations, modifications to AsCasl2f guides can include addition of stem-loops, such as those shown to increase activity on SpaCasl2fl. These modifications can also be combined.Example 23: Addition of functional elements to guide RNAs

[0425] The present Example describes provided RNAs into which additional functions have been engineered, and strategies for design and / or testing of such RNAs.A: Design and testing of MS2 sequence insertions

[0426] The present disclosure teaches, among other things, that one or more modifications to the guide chassis can be made to introduce new functions. An example for adding new functions to the guide chassis is introducing hairpins that can interact with polypeptides, thus recruiting additional polypeptides to the SpaCasl2fl complex, and / or to the genomic target site. Introductions of hairpins can generate multiple outcomes including disrupting guide expression and stability, interaction with SpaCasl2fl, interaction with the target DNA, and functional or nonfunctional positioning of the recruited polypeptide.

[0427] We designed and built multiple SpaCasl2fl guide chassis with the MS2 hairpin (AGNT9), which recruits the MS2 phage coat protein (i.e. MCP, AGNT34). Designs started from the MS13 chassis. MS2 hairpin insertions sites included loops (e.g. loop -1, AGNT204) and the 5' end (AGNT202). Some designs inserted one MS2 hairpin, while other designs inserted multiple MS2 hairpins (e.g. AGNT206, AGNT207, ANG209). The guides were constructed for plasmid-based expression using guides for two targets at the CXCR4 gene identified to work as CRISPR activation targets. Each guide was tested with two polypeptide conditions; a cleavage deficient dSpaCasl2fl-VPR fusion (AGNT23) to determine if the guide was still able to recruit SpaCasl2fl to the target site, or with a cleavage deficient dSpaCasl2fl (AGNT24) and a separate MCP-VPR fusion (AGNT26), along with MS13 as a control (Figure 19).

[0428] All designed and tested guides were able to maintain target engagement, as measured by CXCR4 gene activation with dSpaCasl2f-VPR fusion. Some demonstrated reduced activation as compared to MS13 (AGNT202, AGNT206), and some were even higher. Guides with multiple insertions of MS2 were equally functional to single insertions in recruitment of dSpaCasl2fl-VPR. Therefore, none of these insertion points broke the ability of the guide chassis to interact with either dSpaCasl2fl or the target site at the genome.

[0429] Not all designs were equivalent in their ability to recruit MCP-VPR. One guide was completely deficient, AGNT202, which was an insertion at the 5' end of MS13, at the end of stem-loop 0. Other guides demonstrated increased gene activation relative to the polypeptide based VPR recruitment control, including AGNT203. AGNT203 has an M2 insertion in the crispr RNA sequence upstream of the tracr homology region, immediately downstream of the tetra loop "AAGG". The ranked activity in ability to recruit MCP-VPR for CXCR4 gene activation was AGNT203 > AGNT204 > AGNT207 > AGNT205 > AGNT209 > AGNT210 > AGNT208 > AGNT206 > AGNT202. AGNT203 AND AGNT204 are on different sides of the secondary structure diagram (Figure 10) but are both 4nt upstream of the tracr: crispr hairpin, identifying a region of the guide with an optimal geometry for recruiting activation domains. AGNT205 AND AGNT208 are both on the large loop at the end of stem-loop 2, predicted to be projecting out of the SpaCasl2fl enzyme on the opposite side from PAM recognition, however AGNT205 was more active at recruiting MCP-VPR than AGNT208, demonstrating that small positional changes can impact the outcome of gene activation through this method of recruitment.B: SpaCasl2fl guide chassis additions that can be used for tempiated insertion

[0430] Guide RNA sequences can also include a nucleic acid template and make use of a polymerase (endogenous or exogenous) to incorporate a genetic alteration through replacement of an endogenous DNA strand at the target site (for example see Liang et al 2024, D01: 10.1038 / s41587-023-02095-x). The nucleic acid template can be fused to the gRNA sequence at the 5' or 3' end, or internalized at various locations, and can optionally comprise a linker at the fusion junctions (AGNT312 through AGNT321), with template sequence denoted as Poly X, Figure 22). The linker can be, for example, a non-structured sequence of various length (e.g. "UUAUU"), or can be a structured hairpin (e.g. MS2), or a combination of both.

[0431] Guide and template fusions can be constructed starting from the MS13 chassis by adding template fusions at stem-loops -1, 0, and 2. Or, template sequences can be incorporated into linker loops, such as between stem-loop 0 and the crispr-tracr hairpin, or between the crispr-tracr hairpin and stem-loop -1. Other guide and template fusions can be constructed starting from circularly permuted or minimal guide chassis, such as AsCasl2f (AGNT170, Figure 2). The template can again be fused internally such as within the stem-loop 2 or the crispr-tracr stem-loop, or terminally at the 5' and 3' ends.

[0432] The template sequence can be designed to encode a replacement strand with homology to the DNA strand at the target site. In one embodiment, the template sequence can serve as a primer for extending the non-target strand with a desired edit incorporated. In other embodiments, the fused templates can be used to extend the target strand with a desired edit incorporated. In still other embodiments, two templates can be fused to the same guide RNA and designed to extend the non-target and target strand simultaneously. Additionally, fused templates can be generated with different homology lengths to the target site and incorporate desired edits at any position in the template. The template sequences can contain separate homology and priming regions. In various embodiments, thehomology region may range from about 8 to about 20 nucleotides upstream of the desired edit and about 10 to about 50 nucleotides downstream of the desired edit. In still other embodiments, the desired edit(s) can be contiguous or non-contiguous within the template and can vary in length and sequence composition.Example 24: Addition of elements to the 3' end of the guide RNA

[0433] The present Example describes guide RNAs with certain 3' sequence modifications.A: Design and testing of 3' seguence

[0434] The Casl2f variable spacer sequence is located on the 3' end of the guide. We have demonstrated that this 3' end of the spacer can be internalized in the primary guide sequence when seeking to generate a circular guide (Example 21). Other sequences can be added onto the 3' end to modify activity, for example the UnlCasl2f guide was shown to be more active with a poly U4RU4 sequence (Kim et al 2022). The group that identified and published the MS13 guide RNA also evaluated LUAUgon the 3' end of the original full-length guide and did not observe any difference (Wang et al 2022). The present disclosure provides an insight of additional features worthy of exploration, including for example varied lengths of polyll sequences, and / or inclusion of additional stem-loops 3' of the spacer; without wishing to be bound by any particular theory, the present disclosure provides an insight that such 3' feature(s) may protect the guide RNA from exonuclease or other degrading factors in cells.

[0435] We built a series of SpaCasl2fl guides with different poly U sequences, and different combinations of Histone Like (HSL) stem-loops (AGNT5). In order to achieve a specific poly U sequence from a Pol III U6 expression system, which relies on a poly Ug-s as a stop sequence, plasmids were constructed in the following order of elements: U6 promoter, SpaCasl2fl guide chassis, spacer, poly U test sequence with or without HSL hairpins, HDV ribozyme, poly U8 stop. The HDV ribozyme cleaves off the poly U read through on the stop signal and leaves a precise tail as designed. These plasmids were tested in a two-plasmid transfection (Example 10) at one target site (Figure 20). The presence of different poly U sequences did not greatly impact editing either positively or negatively. Additionally, inclusions of one or more Histone Stem-Loops after the poly U sequence (HSLs) was well tolerated. There are some poly U sequences that are potentially higher activity than MS13 alone, though the improvement was not significant in this experiment. These results are consistent with the ability to append modifications 3' of the spacer and not eliminate activity (Example 20).B: Additional designs

[0436] Poly U sequences, and additional hairpins, could interact with the spacer sequence, the target sequence, or both. Modifications to the guide chassis, truncations or sequence, could also interact differently with the poly U sequence, or factors in the cell, to change the turnover dynamics. Thus, the present disclosure teaches that one or more characteristics of each particular guide RNA chassis and spacer combination may be impacted by poly U sequence (e.g., poly U sequence length and / or composition), with or without hairpins. For example, the present disclosure contemplates guide RNAs with a selected chassis and spacer combination and varied poly U elements - e.g., poly U sequences that are just poly Ui <, or that contain combinations of U1-4 stretches from 4 to lOnt, or even greater than lOnt in length.Example 25: Internal guide homology modificationA: Design and testing of variants with different stem-ioopl:crispr 3' end homologies

[0437] The present disclosure observes that SpaCasl2fl has a patch of nucleotides present in the loop of stemloop 1, 5' rGrUrll 3', which could base pair with the 3' end of the crispr RNA immediately upstream of the spacer sequence, 5' rArArC 3' (also referred to as 5'AAC 3', also referred to as a SAM). Such pairing would form an additional linkage generating a pseudoknot between the tracr and crispr guides, immediately upstream of the spacer sequence, and may participate in (and / or be required for) positioning of the spacer relative to the enzyme: guide complex. The present disclosure observes that other Casl2f guides contain more complimentary sequences between loop 1 and crispr 3' end, 5bp in AsCasl2f and 6bp in UnlCasl2f. Additionally, two independent optimization efforts for AsCasl2f guide (Hino et al and Wu et al) extended the pairing to 6bp.

[0438] We designed a series of SpaCasl2fl guides to optimize the pairing between loop 1 and the crispr 3' end. Some of these modifications were based on MS13, others were based on circularly permuted and truncated variants (Example 18). Some of these designs changed the identity of the base pairs (e.g. AGNT51, Figure 6), some of these designs extended the base-pairing from 3 to 5 base pairs (e.g. AGNT51), some of these designs extended the basepairing from 3 to 6 base pairs (AGNT128). For designs with extended base pairing, the minimum change required is to insert or modify nucleotides upstream of the 5' AAC 3' tail (e.g. AGNT51). Replacing nucleotides may lead to more optimal spacing of the spacer, for example in design AGNT52 (Figure 6). AGNT51 and AGNT52 SpaCasl2fl guides were designed to be more similar to the AsCasl2f loopl:crispr 3' complimentary sequence, 5' GUUCA 3' and 5' UGAAC 3' respectively. When constructed as plasmids and tested against 3 separate targets (as described in Examples 6, 9, and 16), neither AGNT51 or AGNT52 generated clear editing activity above background (Figure 11).

[0439] One insertion design had better overall activity than others, AGNT128. This design inserted three nucleotides, 5' CCU 3', upstream of the crispr 3' end to increase the complement with the existing loop 1 sequence to 6bp, more closely mimicking the natural complementation length of AsCasl2f and UnlCasl2f. When constructed as plasmids and tested against two separate targets (as described in Examples 6, 9, and 16), AGNT128 guide chassis variants generated around 65% of the activity of the parent guide, AGNT35 (Figure 14). Thus, the insertion was well tolerated though normal guide activity was still disrupted.

[0440] Instead of insertions at the crispr 3' end, designs AGNT110 through AGNT113 have modifications to the identity of bases to modify interaction strength. All were built onto the AGNT47 circularly permuted and truncated guide (Example 19). AGNT110 was designed with a single nucleotide change in the crispr 3' end one U:A bp modified to U:G, AGNT111 with both A's to G's to make both a U:G pair. AGNT112 was designed with a complementary change in addition to the modification in AGNT110, so that the new base pair is C:G from the original U:A. Finally AGNT113 swapped both U:A base pairs to C:G. When constructed as plasmids and tested against 3 separate targets (as described in Examples 6, 9, and 16), only AGNT110 had measurable activity, though it was only at 10% of the activity of AGNT47 (Figure 13). Together these data demonstrate that the position, identity, and homology length of the loopl:crispr 3' homology stretch are highly sensitive to modifications and subtle changes can greatly attenuate activity of the guide sequence.B: Rescue of minima! seguence with ioopl:cr addition

[0441] While designing minimal SpaCasl2fl sequences (Example 21), additional designs were generated that incorporated longer loopl:crispr 3' homologies, based on AGNT128. The design principle employed was to obtain optimal position of the spacer, which is disrupted after removal of bases between stem-loop 2 and the end of the crispr RNA, such as in deletion of stem-loops 3 and or 4. Initial designs for deleting stem-loops 3 and 4 did not result in functional guides (Example 21). AGNT116 was one guide designed with deletions in loop 3 and 4, which had no detectable activity when tested against two targets in HEK293T (Figure 14). However, introduction of the crispr 3' extension from design AGNT128 onto design AGNT116, which became design AGNT164, rescued activity, as demonstrated by greater than 15% INDEL activity at two targets in HEK293T cells (Figure 14). Without wishing to be bound by any particular theory, we propose that this introduction of 3' sequence (from AGNT128) may facilitate proper pseudo knot folding, useful and / or necessary for guide activity. In fact, activity rescued activity documented here may be analogous to that observed in Example 29, when deletion of loop 1 binding site sequences between stem-loops 3 and 4 disrupted activity, which was then restored by re-introduction of those sequences into the 3' SAM; again without wishing to be bound by any particular theory, we propose that this re-introduction may also facilitate proper pseudo knot folding.C: Additional internal homology

[0442] In addition to extending the homology between loop 1 and the crispr 3' end, designs were generated that add homology between new domains. In one set of designs, the homology was meant to mimic the predicted covalent connection between stem-loop -1 and the crispr:tracr region, which was eliminated in the circularly permuted guides (Example 18). In these design based on AGNT35, complimentary bases to a loop -1 sequence were added to the 5' end of the crispr: tracr stem-loop (AGNT126, AGNT127). When tested as plasmids against two targets in HEK293T (Examples 6, 9, 15), these guide chassis changes were mostly neutral (Figure 14). Another set of guides were designed to mimic the covalent link between the crispr and tracr sequences in the MS13 and other single guide designs. Starting with AGNT35, complimentary bases to the stem-loop 0 linker were inserted at the crispr: tracr stem-loop 5' end (AGNT161, AGNT162). AGNT163 was designed with crispr:tracr insertions complimentary to both loops 0 and -1. Again, when tested as plasmids transfected into HEK293T (Examples 6, 9, 16), all modifications were generally neutral (Figure 14).D: Additional designs

[0443] Design strategies and / or data taught herein provide those skilled in the art with additional designs that, for example, may add or improve internal homologies, thus increasing stability or activity of a guide RNA. For example, a design can be generated to change the stem-loop 0 linker sequence added in circ permutations (Example 18) to not be identical to loop 1 (GUUA) and thus prevent competition with the loop l:crispr 3' homology (e.g. AGNT286).Example 26: Delivery of mRNA / gRNA to HEK293T cells via transfection

[0444] The present Example illustrates transfection of HEK293T cells with RNA to evaluate the ability of guide RNAs to direct a Casl2f enzyme to genomic target sites for double stranded DNA cleavage and error-prone repair.

[0445] Lenti-X HEK293T cells (Takara Bio, USA), passaged fewer than 10 times, were cultured in media comprising high-glucose DMEM (Thermo Fisher Scientific, USA) and 10% heat-inactivated fetal bovine serum (Sigma, USA), in collagen-coated T-175 flasks (ZenBio, USA), in Steri-Cycle-il60 Incubators (Thermo Fisher Scientific, USA) set to 37Cand 5% C02. Cells were detached and replated 18-26 hours prior to transfection. To detach the cells, culture media was aspirated from the flask and 15 mL of TrypLE Express Enzyme (Thermo Fisher Scientific, USA) was added and incubated for 5 minutes at 37C. The flask was knocked to further detach the cells and then detached cells were transferred to a 50 mL conical containing 30 mL of 37C pre-warmed culture media. Cells were pelleted at 420xg for 4 minutes at room-temperature, resuspended in 5 mL of culture media and then counted using a Vi-Cell XR (Beckman Coulter, USA). 2.0x104 cells per 100 ul were seeded in individual wells of a 96-well collagen-coated plate (StemCell Technologies, Canada), allowed to settle for 3-5 minutes at room temperature, and then moved to the incubator. 18- 26 hours post-seeding, when HEK293T cells reached confluences of 70-90%, transfection mix was prepared.

[0446] Briefly, 0.43ul of MessengerMax (Thermo Fisher Scientific, USA) was diluted in 18.65 uL of Opti-MEM, vortexed for 10 seconds to mix, and incubated for 10 minutes at room temperature. Casl2f mRNA was diluted to 50ng RNA per uL and gRNA was resuspended to 6500ng per uL in nuclease-free water (Thermo Fisher Scientific, USA). 0.8uL of Casl2f mRNA and 0.013 uL of gRNA, equaling 20ng of Casl2f mRNA and 86.3ng of gRNA respectively, were diluted in 17.73 uL of OptiMEM and mixed. The entire volume of diluted Casl2f mRNA and gRNA in Opti-MEM was added to the diluted MessengerMax, mixed and incubated for 5 minutes.

[0447] During incubation of transfection mixture, HEK293Ts were removed from the incubator (typically at 3.5 minutes), and at the 5 minute mark the entire volume of RNA: lipid mixture was added to the cells. The plate was vigorously shuffled side to side to disperse the transfection mixture in the wells and then returned to the incubator. 72 hours post-transfection genomic DNA was harvested, and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 16).A. Protocol for high-throughput guide chassis screening using a Viafiow96

[0448] Cells were prepared as above. Transfection mixture was generated following the manufacturer's suggested protocol. Briefly, 714.7uL of Lipofectamine MessengerMAX reagent (Thermo Fisher Scientific, USA) was diluted in 8.8mL of Opti-MEM (Thermo Fisher Scientific, USA) and vortexed for 10 seconds to mix. 76uL per well of the MessengerMAX reagent diluted in Opti-MEM was dispensed into a 96-well 500uL plate (Axygen® Deep Well and Assay Plates, Corning), sealed and incubated for 10 minutes at room temperature. 17uL of mRNA at a concentration of 1.5ug per uL was diluted in 11.4mL of Opti-MEM and vortexed for 10 seconds to mix. 87uL / well of the mRNA diluted in Opti-MEM was dispensed into a 96-well 500uL plate (Axygen® Deep Well and Assay Plates, Corning) and sealed. The gRNA was diluted to 200ng per uL in nuclease-free water (Thermo Fisher Scientific, USA) and 8uL per well was dispensed into the plate containing mRNA diluted in Opti-MEM. The entire volume in the wells was mixed 15 times using the Viaflo96 with a 300uL pipetting dispenser head (Integra). Using a custom program on the Viaflo96, 76uL per well of the mRNA / gRNA mix in OptiMEM was stamped into the plate containing the MessengerMAX reagent diluted in Opti-MEM and mixed 15 times. The plate was then sealed and incubated for 5 minutes at room temperature.

[0449] During the incubation of transfection mixture, HEK293TS were removed from the incubator (typically at 3.5 minutes), and at the 5 minute mark, 38uL per well of DNA: lipid mixture was added to the cells. The plate was vigorously shuffled side to side to disperse the transfection mixture in the wells and then returned to the incubator. 72 hours post-transfection genomic DNA was harvested, and DNA-cleavage activity was evaluated through targeted amplicon sequencing (as described in Example 16).Example 27: Identification of multiple improvements to a truncated Casl2f guide

[0450] The present Example describes design and evaluation of sequence permuted guide RNAs to be used with Casl2f CRISPR systems. Reference(s) to exemplary Figures and SEQ ID NOs is not intended to be limiting. For example, those skilled in the art, reading the present disclosure, will appreciate that it provides not only specific exemplified RNAs and sequences, but structural features relevant to performance, and will furthermore appreciate, for example, that, provided Casl2f guide RNAs (e.g., for use with Casl2f CRISPR systems) can include RNAs that may include certain differences relative to those exemplified herein. For example, in some embodiments, one or more nucleotides (or binding pairs thereof) may be substituted or modified relative to a provided exemplified RNA (and the present specification includes RNAs that differ from one another in the identity of a substitution or modification at a particular one or more residues). In some embodiments, one or more nucleotides may be added to or removed from one or more stem-loops, for example so that a particular stem present in one RNA (e.g., an exemplified RNA) is lengthened, or shortened, or interrupted as the case may be, and / or a particular loop present in one RNA (e.g., an exemplified RNA) is shrunk or extended; alternatively or additionally, in some embodiments, one or more spacers may be lengthened or shortened, or may have altered position relative to that in a reference RNA (e.g., in an exemplified RNA), in each case in accordance with guidance provided herein, as will be appreciated by a skilled person reading the present disclosure.

[0451] The AsCasl2f minimal guide (AGNT170, Wu et al 2023) contains 3 stem loops, Figure IB, with alternate arrangements observed for the nucleotide pairings within stem-loop 2 between two of the published structures (PDB structures 7WJU and 8J12). The predicted secondary structure of SpaCasl2f (AGNT246) (Figure 24D) was compared to these AsCasl2f structures to identify base-pairs that were conserved or different between the two chassis. Starting with AGNT170, AsCasl2f nucleotides were switched for SpaCasl2f nucleotides at positions within stem-loop 1 and stem-loop 2 in chassis AGNT251 - AGNT266.

[0452] To assess activity, each chassis was encoded into two expression plasmids, each with an exemplary spacer (AGNT322 and AGNT323), see Examples 2 and 6. The guide plasmids were co-transfected into HEK cells with a SpaCasl2fl expressing plasmid (AGNT18) (Example 10), and INDEL activity quantified (Example 16). As shown in Figure 25A-25B, multiple chassis variants are improved over both the starting guide (AGNT170), as well as a SpaCasl2fl guide AGNT246, with one or both spacers.

[0453] In stem-loop 1, there are 6 base-pairs in each guide chassis, with 4 base-pair differences between AsCasl2f and SpaCasl2f. Changing each individually was either neutral or positive (AGNT262 - AGNT265) and changing all at the same time, making the stem a perfect match to SpaCasl2fl stem 1, was the most active (AGNT266, Figure 25A).

[0454] Stem-loop 2 is more variable than stem-loop 1 between the homologues, in both sequence and length, as well as potentially existing in multiple states within a given homologue (Figure 24B, 24C). The stem-loop 1 proximal residues in stem-loop 2 in AGNT170 were also changed to match residues from SpaCasl2f predicted in accordance with the present disclosure to be homologous (Figure 24D, AGNT258 - AGNT260), including breaking of the most proximal base pair (AGNT261). Many of these changes again led to improvements, with only AGNT258 being detrimental. Even breaking the stem-loop 1 proximal base-pair in AGNT261 was tolerated when used with one of the two spacers. Overall, changes in AGNT259 and AGNT260 were the most improved in this set.

[0455] Additional stem-loop 2 changes distal to stem-loop 1 were tested. Base pairs immediately adjacent to the central bulge, of which there is discrepancy between the secondary interactions in the solved structures, were generally not tolerated, with changes such as AGNT251 completely eliminating activity, highlight an important role of these nucleotides near the bulge. However other changes further away from the bulge (e.g., distal to stem-loop 1) and towards the loop were again beneficial to activity, with AGNT254 - AGNT256 showing improved activity with both spacers, and the larger change of swapping the stem-loop approximately 6 base-pairs downstream of the bulge with the A / G rich loop from SpaCasl2f also showing improvement at one of the spacers (AGNT257, Figure 24A).

[0456] Stem-loop 2 is longer in the AsCasl2f chassis than in the SpaCasl2fl chassis. Truncations were built and evaluated starting from the loop and moving towards the bulge of the stem-loop (AGNT292 - AGNT296). When evaluated using two plasmid transfections, the truncations showed improvement down to a certain length (Figure 24A). AGNT294 and AGNT257 have identical stem lengths, with different terminal loop length and sequence, and both are improved over AGNT170, with spacer dependent preferences.

[0457] Further to the improvement achieved with the variety of chassis changes described above, the present specification documents that many combinations of these changes may improve activity even further. For example, combining sequence permutations found in AGNT256, AGNT259, AGNT266, AGNT292, and AGNT1151 in different regions of the guide, and / or making pairs, triplets, quadruple, or a complete combination of these changes, can generate enhancing and / or otherwise useful combinations (AGNT1159 - AGNT1166, AGNT1185 - AGNT1194). Alternatively or additionally, other permutations (e.g., one or more other permutations shown to also increase activity) can be tested with this combination, such as AGNT260 (e.g., AGNT1181), and / or certain permutations (e.g., one or more permutations shown to be neutral) can be removed, such as AGNT265 (e.g., AGNT1183).

[0458] The present disclosure provides an insight that modifications which impact the length of AGNT170 and lead to improved activity can be explained by the structure of the SpaCasl2f polypeptide and its guide chassis. Furthermore, the present specification provides additional insights from such structural observations, which insights include certain additional modifications that may improve function. AGNT1151 is a chassis with a 5' addition of a stem-loop, i.e. stem-loop 0, and the SpaCasl2f polypeptide structure shows contacts with this stem-loop. Removal of this stem-loop completely from SpaCasl2f was tolerated but not an improvement over chassis with more limited deletions (AGNT47). Thus, adding back chassis stem-loops that fill in the binding surface of SpaCasl2f can be beneficial to the activity of the complex. Another example is the guide chassis:PID interactions (see Example 30).

[0459] Those skilled in the art, reading the present disclosure, will appreciate, for example, that additional basepair or nucleotide permutations to make a chassis more like SpaCasl2f can be designed and evaluated. For example, the present specification (see, for example, Example 28) provides insights relating to predicted pairings and nucleotide changes that may achieve improved guide(s) relative to an AsCasl2f reference guide (e.g., to AGNT170). In some embodiments, the present specification provides insights that more than one state of pairings can be observed in molecular structures. In some embodiments, SpaCasl2f structures may identify additional pairings other than those described herein; the present disclosure provides insights that can be applied to such additional pairings and therefore provides further modified RNAs - e.g., new AsCasl2f derived guides that match the SpaCasl2f observation(s). Alternatively or additionally, changes can be made to the sequence that are not complementary to a putative SpaCasl2fl secondary map, but instead strengthen or weaken the interactions of the guide with itself orother parts of the complex in such a way as to make the Casl2f complex more active or more stable, and these changes can also be utilized in provided Casl2f guide RNAs, for example in combination with one or more modifications exemplified or otherwise taught herein. The present disclosure appreciates that permutations which are neutral in one context may provide an impact in another (e.g., in different cellular or delivery contexts).Example 28: Additional AsCasl2f permuted sequences to improve Casl2f complex activity

[0460] The present Example describes certain sequence permuted guide RNAs designed in accordance with the present disclosure as useful with (at least) one or more Casl2f CRISPR systems. Reference to exemplary Figures and SEQ ID NOs herein is not intended to be limiting. For example, those skilled in the art, reading the present disclosure, will appreciate that it provides not only specific exemplified RNAs and sequences, but structural features relevant to performance, and will furthermore appreciate, for example, that, provided Casl2f guide RNAs (e.g., for use with Casl2f CRISPR systems) can include RNAs that may include certain differences relative to those exemplified herein. For example, in some embodiments, one or more nucleotides (or binding pairs thereof) may be substituted or modified relative to a provided exemplified RNA (and the present specification includes RNAs that differ from one another in the identity of a substitution or modification at a particular one or more residues). In some embodiments, one or more nucleotides may be added to or removed from one or more stem-loops, for example so that a particular stem present in one RNA (e.g., an exemplified RNA) is lengthened, or shortened, or interrupted as the case may be, and / or a particular loop present in one RNA (e.g., an exemplified RNA) is shrunk or extended; alternatively or additionally, in some embodiments, one or more spacers may be lengthened or shortened, or may have altered position relative to that in a reference RNA (e.g., in an exemplified RNA), in each case in accordance with guidance provided herein, as will be appreciated by a skilled person reading the present disclosure.

[0461] A wild-type SpaCasl2f structure (with wild-type polypeptide and single chain guide chassis, see Bigelyte et al 2021) was obtained in which the SpaCasl2f guide chassis secondary structure differed from a previously predicted structure. In this secondary structure map, stem-loop 2 has a different base-pairing structure, resulting in fewer bases linking stem-loops 2 and 3 (Figure 26). This new mapping identifies a different possible pairing, however, as seen with AsCasl2f complex structures, it's not the only possible mapping. With this new mapping different stemloop 2 base-pairs of SpaCasl2f are predicted to be in the equivalent positions of the AsCasl2f chassis than designed and tested in Example 27.

[0462] Additional guide chassis were designed relative to AGNT170. In some guide chassis, the region of stem-loop 2 most proximal to stem-loop 1 was modified (AGNT1152 - AGNT1156). Among these changes, some pairings were broken, such as AGNT1152, which introduces a conserved C:ll in place of A:ll in AGNT170. Additional modifications include inserting an additional uracil, which is flipped out of the RNA duplex in the SpaCasl2fl structure and could possibly form a unique to SpaCasl2fl polypeptide: guide contact (e.g., AGNT1153). In other guide chassis designs, the region more distal to stem-loop 1 was modified even more than that described in Example 27 (AGNT1158). In other examples, these additional modifications were combined with modifications shown to increase activity of the complex in Example 27 (e.g., AGNT1157, AGNT1176 - AGNT1178, AGNT1180, AGNT1182).

[0463] Along with modifications described herein to potentially increase affinity of the AsCasl2f guide chassis (AGNT170) with SpaCasl2f polypeptide, other sequence permutations can be made that may improve activity. For example, poly U sequences have been shown to increase the activity in multiple Cas systems, including withUnlCasl2f (Xu et al 2021, Kim et al 2022). AGNT1171 through AGNT1174 were designed with variable UnRUnlengths, which may, for example, improve stability of the guide.

[0464] Those skilled in the art, reading the present disclosure, will appreciate, for example, that additional sequences can be added or removed, and that doing so may achieve additional activities and / or other benefits from these guide chassis. For example, in some embodiments, a hairpin such as an MS2 hairpin can be introduced at the ends or at loop regions, such as stem-loop 2 (AGNT1179) without significant impact on ability of the complex to interact with target DNA, while providing additional functionality by acting as a binding surface for other RNA binding polypeptides. Alternatively or additionally, other structures can be generated (either solved using techniques such as cryo-EM or crystallography, or predicted from primary sequence, or some combination of approaches) that suggest different secondary maps and corresponding nucleotide permutations which mimic those interactions. As another example, while these changes are predicted to improve function through direct SpaCasl2fl-guide chassis interactions, they may be beneficial for other Casl2f guide polypeptide complexes (see Example 31).Example 29: Additional truncated SpaCasl2f guide chassis sequences

[0465] The present Example describes the design and evaluation of truncated and permuted guide RNAs to be used with Casl2f CRISPR systems. Reference(s) to exemplary Figures and SEQ ID NOs are not intended to be limiting. For example, those skilled in the art, reading the present disclosure, will appreciate that it provides not only specific exemplified RNAs and sequences, but structural features relevant to performance, and will furthermore appreciate, for example, that, provided Casl2f guide RNAs (e.g., for use with Casl2f CRISPR systems) can include RNAs that may include certain differences relative to those exemplified herein. For example, in some embodiments, one or more nucleotides may be removed relative to a provided exemplified RNA, or placed adjacent in primary sequence relative to a desired RNA, in order to achieve a desired tertiary structure for complex activity, in each case in accordance with guidance provided herein, as will be appreciated by a skilled person reading the present disclosure.

[0466] As described herein, the SpaCasl2fl chassis contains multiple stem-loops that are not present in other Casl2f homologues, for example the UnlCasl2f chassis (AGNT247) and the engineered AsCasl2f chassis (AGNT170). Two of these stem-loops present in SpaCasl2f but not in the engineered AsCasl2f or UnlCasl2f, stemloops 3 and 4, cannot be individually removed without substantial hits to activity. One designed chassis with stem loop 3 and 4 deletion was active only with inclusion of extended cr 3': loop 1 homology to 6nt (AGNT164, Figure 2B). To further improve activity, additional modifications to AGNT164 were designed and evaluated, specifically removing nucleotides in the linker between stem-loop 2 and the cr:tr stem-loop, to optimize the spacing of the key elements (AGNT267 - AGNT285, AGNT287 - AGNT291). In each of these designs, stem-loop -1 was also removed to achieve a more minimal sequence. Each chassis was cloned into two expression plasmids, each with an exemplary spacer (AGNT322 and AGNT323), see Examples 2 and 6. The guide expression plasmids were co transfected into HEK cells with a SpaCasl2fl expressing plasmid (AGNT18) (Example 10), and INDEL activity quantified (Example 16).

[0467] As seen in Figure 25B, the majority of these new minimal SpaCasl2f chassis were not functional, including the design of AGNT164 without stem-loop -1 (AGNT267). This result shows that the 5' stem-loops play a larger role in chassis without stem-loops 3 and 4 than with, as removal of this stem-loop -1 increases the activity of the full length and circularly permuted chassis (AGNT35 vs AGNT39, Figure 25B). AGNT286, AGNT309 and AGNT310 alsoshow the sensitivity of stem-loop 0 in the circular permutations to the choice of synthetic linker, as subtle changes to the linker sequence makeup decreased activity relative to the original designs AGNT39 and AGNT43.

[0468] Another learning from these designs was to identify the optimal spacing between stem-loop 2 and the 3' cr loop 1 homology domain (i.e., the SAM). AGNT269 was the most active, identifying a spacing that is closest to optimal. Other design variables explored were the addition of a nucleotide between the cr:tr stem-loop and the 3' cr homology domain (AGNT275, AGNT277 - AGNT 79), however these also included large deletions in the stem-2 / 3 linker that were not tolerated. Other modifications which rescued the activity of AGNT267 included adding sequence to the end of the cr:tr stem-loop which was homologous to the stem 0 loop (AGNT281), or just inserting stem-loop - 1 and an original cr hairpin into the truncated cr:tr hairpin (AGNT280). Chassis where the cr:tr stem-loop was replaced with the equivalent loop (plus variants) in the AsCasl2f minimal guide (AGNT170) were not active (AGNT282 - AGNT284). Also, a benefit of the full 6nt 3' cr: homology domain was confirmed with chassis AGNT287 - AGNT291, where 1, 2, or 3 nt were replaced with non-homologous bases, or deleted: all were inactive.

[0469] Additional SpaCasl2fl guide chassis were designed which eliminate stem-loop 3 and 4. The following observations supported these designs: (1) There is a conserved physical distance between the unpaired nucleotides immediately downstream of stem-loop 2, and an unpaired nucleotide immediately upstream of the cr 3':loop 1 homology domain. (2) The domain complementary to loop 1 in the SpaCasl2fl chassis is split in primary sequence between the cr 3' end (i.e., the SAM) and the linker between stem-loop 3 and 4. This observation is similar to the AsCasl2f guide (AGNT248), where a single nucleotide (a cytosine) between stem-loop 3 and 4 is base-paired with loop 1, and was placed into the cr 3' homology region in the design of the minimal guide (AGNT170), also with an upstream unpaired adenine nucleotide. Combining these insights with other observations described herein (e.g., AGNT269 as closest to the most active spacing) generated additional designs (e.g., AGNT1196, Figure 26) that vary the position of the 6nt cr 3' homology domain relative to stem-loop 2. These designs rely on the design principles described herein to both maintain a specific distance pattern of core stem-loops, and also utilize circular permutation, in order to keep the 3' terminal stem-loop in the right orientation for spacer placement.

[0470] The present disclosure further describes permutations including: spacing between stem-loop 2 and the terminal stem-loop, spacing between the cr 3': loop 1 homology and the 3' terminal stem-loop, the sequence make-up of the 3' terminal stem-loop, and truncations at the 5' end (see, for example, AGNT1196 - AGNT1207, AGNT1211 - AGNT1213). In another example, a stem-loop 3 and 4 truncation maintaining the split loop 1 homology regions was designed (AGNT1214). Other chassis designs include extended hairpins within the 3' terminal stem-loop or 5' terminal stem loop, and conserved changes between loop 1 and its homology to change the binding energy.

[0471] Other SpaCasl2fl truncations were generated which maintain unique interactions between stem-loops and the polypeptide (see also Example 28 and Example 30). For example, adenine ribonucleotides in the loop of stemloop 4 can form contacts with the polypeptide. A truncation that connects the two homology loops into a single strand (e.g., AGNT1196) would remove both stem-loop 4 and cr:tr stem-loop. Instead of eliminating these loops entirely, guide chassis were designed which only trim back the cr:tr stem-loop to a minimal length (AGNT1209), and then the links between stem-loop 4 and the cr:loop 1 homology sequence were strengthened by changing A:U base pairs to C:G (AGNT1210, AGNT1217).

[0472] The crispr:tracr stem-loop is amenable to truncations, extensions and base swaps, across multiple Casl2f homologues (Hino and Wu et al). Through analysis of the structures of Casl2f homologues, there are conserved pockets in each polypeptide capable of forming contacts with either stem-loop 3 or the crispntracr stem-loop, with subtle differences in electrostatic charge, or ability to accept a flipped out base, between the homologues. With truncated guides where everything post stem-loop 2 is reduced to a single stem-loop, the new 3' terminal loop can fill either the pocket left by the absence of stem-loop 3 or 4, or the pocket left by the crispr:tracr stem-loop. In addition to truncated designs that eliminate the SpaCasl2f crispntracr stem-loop and leave stem-loop 3(e.g., AGNT1196 - AGNT1199), or maintain the variations of the original crispr:tracr stem-loop (e.g., AGNT1204 - AGNT1206), we also designed variants that include variations of chimeric stem-loops from AsCasl2f sequence, with differing positions of unpaired bases and base identities, to optimize the interaction of the guide with the stem-loop 3 binding pocket of SpaCasl2f (AGNT1218 - AGNT1221).

[0473] Similar to the polypeptide pockets left vacant on SpaCasl2f with the 3' stem-loop truncated guide(s), there are pockets that are present for stem-loops 0 and -1 that are left vacant in the circularly permuted 5' truncations. To optimize the binding of the polypeptide and guide in these domains, chassis were designed with different loops, lengths, and linkers between the 5' end and loop 0 (AGNT1222 - AGNT1227).Example 30: Additional chassis modifications to increase activity with SpaCasl2fl

[0474] The present Example identifies unique guide chassis characteristics that can be exploited to improve activity of a SpaCasl2fl-guide complex as described herein. Reference(s) to exemplary Figures and SEQ ID NOs is not intended to be limiting. Those skilled in the art, reading the present disclosure, will appreciate, for example, that, provided Casl2f guide chassis can incorporate the identified critical contacts with a different starting scaffold sequence, or can be achieved with other nucleotides, or can be achieved with a combination of polypeptide and nucleotide changes to modify the specified binding interactions.A: PID interacting chassis domain

[0475] Casl2f polypeptides are homodimers and have an asymmetric interaction with the guide and target DNA. A consequence of this unique complex formation is that, while one PAM interacting domain (PID) from one monomer will be performing roles in, for example, DNA engagement and R-loop formation, the PID in the second monomer cannot be occupying that same DNA. A prediction is that this PID domain will be occupied by a section of the guide chassis, which was confirmed by a cryo-EM structure. A poly-A stretch in loop 2 is adjacent to the PID as well as downstream polypeptide residues that can be critical for creating or stabilizing the R-loop (Figure 27). The PID residues are contacting a mixture of RNA backbone as well as ribonucleotide bases, which are the complement of the T rich PAMs preferred by many Casl2f polypeptides, including SpaCasl2fl. However, each Casl2f has a unique PID, thus it may be likely that when swapping guide chassis between Casl2f polypeptides, this loop will need to be optimized. Additionally, changing the PID to modify the targeting space of a Casl2f may require that the guide chassis also be modified to optimally interact with the new PID.

[0476] We have demonstrated the ability of the AsCasl2f guide chassis to be modified to contain the SpaCasl2fl stem 2 loop poly-A's (AGNT257, Figure 25A), where with at least one spacer the activity was improved. This modification was done along with additional modifications to stem-loop 2 that may be non-optimal for this chassis.Another example of a modification to the AsCasl2f guide chassis which results in poly A in the same relative position as SpaCasl2fl is the chassis with a large deletion in stem-loop 2, AGNT294. Compared to AGNT292, with a deletion of 3nt in the loop, AGNT294 is a deletion of 13nt, resulting in a new stem-loop 2 with a poly A approximately the same distance away from the stem-loop 2 central bulge as the SpaCasl2fl sequence. AGNT294 was an improved chassis over AGNT246 and the previous top performing AsCasl2f guide AGNT170 (Figure 25A). Further modifications of this type, such as AGNT1195, may be both compatible with the AsCasl2f chassis and be more optimal for SpaCasl2fl. It will be desirable to ensure that the adenines in this PID contacting stretch which are flipped out of the RNA duplex are properly positioned relative to the other stem-loop 2 landmarks, so that the same SpaCasl2f residues are positioned to interact with those bases (e.g., N88, N92). An example of this design would be to remove base-pairs from the stem-loop 2 distal region in AGNT257 or AGNT294 (see AGNT1215 and AGNT1216).B: flipped-out guide chassis bases as polypeptide contacts

[0477] Casl2f guide chassis have conserved nucleotides that are flipped-out (oriented away from the center of the RNA helix). A SpaCasl2fl structure confirmed that this remains true for SpaCasl2fl guide chassis as well. These bases include a guanine in the central bulge of stem-loop 2, and an adenine in the linker between stem-loops 1 and 2. An additional nucleotide that is flipped out of the RNA duplex backbone in the SpaCasl2fl guide chassis, is a uracil two nucleotides downstream of the guanine in the central bulge. This nucleotide is adjacent to positive residues in the polypeptide and may help strengthen the interaction of the guide chassis and the polypeptide (for instance by contacts with residues R37 and R107). As this base is not present in the AsCasl2f chassis, adding it may improve function, both alone and with other modifications (e.g., AGNT1157, AGNT1182).Example 31: Casl2f target engagement optimizations

[0478] The following Example identifies certain characteristics of the Casl2f-guide-target complex which can be exploited, for example to improve the activity of the complex or achieve other benefits. Reference(s) to exemplary tables or figures are not intended to be limiting. For example, those skilled in the art, reading the present disclosure, will appreciate that it provides not only specific exemplified RNAs and sequences, but structural features relevant to performance, and will furthermore appreciate, for example, that, provided Casl2f guide RNAs (e.g., for use with Casl2f CRISPR systems) can include RNAs that may include certain differences relative to those exemplified herein. For example, in some embodiments, one or more residues forming contacts with certain bases, such as those in solved structures, may be oriented to form non-base specific contacts in other contexts, and vice-versa: residues adjacent to but not forming base specific contacts with one spacer may form those contacts in another spacer context, in each case in accordance with guidance provided herein, as will be appreciated by a skilled person reading the present disclosure.

[0479] Cas polypeptides frequently make use of positive residues to interact with RNA and / or DNA backbones, increasing affinity of the complex when engaged with the DNA target. SpaCasl2fl, along with other Casl2f polypeptides, have some amino acids positioned to achieve this within, for instance, multiple alpha-helical segments in the REC domain, which is frequently a target of improvement via targeted mutagenesis (Xu et al 2021, Hino et al 2023, Wu et al 2023, Kong et al 2023). There are however multiple alpha-helixes (including the REC domain) that are positioned near the groove of the DNA-RNA duplex, suggesting that the polypeptide can form base-specific contacts.Thus, in addition to non-specific nucleic acid backbone contacts that can be modified to improve Casl2f function, a guide spacer sequence itself can modify function through affinity with the polypeptide.Table 2: Exemplary Polypeptide contacts

[0480] When choosing a spacer (see Example 2), the Cas polypeptide sequence defines the DNA sequence space most likely to be active. A primary factor is the PAM-interacting domain, which imparts a preference for certain base pair sequences immediately upstream of the spacer sequence. Within the spacer sequence there are other polypeptide contacts that lead to preferences, either mandating correct base-pairing by dis-allowing mismatches (for instance through steric clashes), or by direct polypeptide pairing. By identifying specific contacts, then measuring preferences through screening many guides with differences at those positions, a preference table for specific bases at specific positions in the spacer will be generated (as an example, see Table 2). Using this preference table, spacers can be identified that are predicted to be more active prior to functional testing.Example 32: Casl2f chassis optimizations across homologues

[0481] The following Example provides examples of how the information gained from optimizing a guide chassis for use with SpaCasl2f can be applied to optimize chassis for use with other Casl2f polypeptides. Those skilled in the art, reading the present disclosure, will appreciate, for example, that, while large portions of Casl2f guide chassis are conserved amongst homologues, there are important polypeptide differences that relate to variations both in domain organization and in the electrostatic surface potential that will necessitate customized engineering solutions between species.A: optimizing for use with SpaCasl2fl polypeptide derivatives

[0482] Various Examples included herein describe modifications to guide chassis and spacer use for optimizing activity with SpaCasl2fl. As a DNA targeting enzyme with cleavage domains, SpaCasl2f is suitable as an enzyme for many uses targeting DNA, including uses with an inactive RuvC domain. The present disclosure appreciates that particular contexts (e.g., particular uses, particular target-specific modifications, for example, as described inExample 31) may benefit from one or more (e.g., a particular set of) polypeptide modifications to achieve optimal activity. Furthermore, Casl2f is an asymmetric dimer where changes to improve the function of one monomer may decrease activity in the other monomer. Thus, the present specification appreciates that...

Claims

CLAIMS1. An RNA including sequence elements selected from a group comprising: a. a crispr sequence element, b. a tracr sequence element, c. a spacer sequence element, and combinations thereof wherein the RNA optionally includes one or more stem-loop sequence elements selected from the group consisting of: stem-loop -1, stem-loop 0, stem-loop 1, stem-loop 2, stem-loop 3, stem-loop 4, crispr tracr stem-loop and is a variant of a reference sgRNA selected from a group comprising:(i) reference sgRNA AGNT248,(ii) reference sgRNA AGNT246, or(iii) the combination thereof, wherein the arrangement of sequence elements in the variant differs from that found in reference sgRNAs, and wherein the variant optionally includes one or more of the following changes to the reference sgRNA sequence element relative to that found in the reference:(i) removal of ribonucleic sequences;(ii) addition of new ribonucleic sequences; or(iii) the combination thereof, wherein the variant maintains essential secondary structures and tertiary orientation for active ribonuclease polypeptide complexes while rearranging the primary sequence of ribonucleic acids.

2. An RNA that is a variant of a reference sgRNA (AGNT246 or AGNT248) in that:(a) arrangement of one or more of a crispr sequence element, a tracr sequence element, a spacer sequence element, and a stem-loop element is structurally permuted relative to its arrangement relative to other of the sequence elements in the reference sgRNA; and optionally:(b) one or more of the sequence elements is altered relative to that found in the reference.

3. The RNA of claim 1, wherein the one or more sequence elements that is altered is selected from the group consisting of:(i) stem-loop -1, which is altered in that it is appended to a reference sequence lacking this stem-loop, its hairpin is extended, it includes one or more non-canonical residues, or is modified in length and sequence composition, or it is eliminated;(ii) stem-loop 0, which is altered in that it is appended to a reference sequence lacking this stem-loop, its hairpin is extended, a linking loop is added, it includes one or more non-canonical residues, or is modified in length and sequence composition, or it is eliminated;(iii) stem-loop 1, which includes one or more base pair substitutions that match base pairs present within other Casl2f homologues, or is a unique base pair, and / or contains one or more residue substitution with a non- canonical residue;(iv) stem-loop 2, which is extended or truncated, or which contains one or more base pair or residue substitution that match nucleotides present within other Casl2f homologues, or contains 1, 2, or 3 unique single residue or base pair changes, and / or contains one or more residue substitution with a non-canonical residue;(v) stem-loop 3, which is extended or truncated and / or contains one or more residue substitutions with a canonical or non-canonical residue, or is eliminated;(vi) stem-loop 4, which is truncated and / or contains one or more non-canonical residues, or is eliminated;(vii) a tracncrispr stem that is truncated or extended, or which contains one or more base pair or residue substitution that match nucleotides present within other Casl2f homologues, or contains a unique base pair or residue substitution, and / or that includes one or more base pair substitutions with canonical or non-canonical residues;(viii) one or more loops is longer or shorter and / or is altered to be complementary with a single-stranded section elsewhere in the RNA;(ix) loop 1-crispr 3' end homology that is extended and / or that includes one or more residue insertions or substitutions with canonical or non-canonical residues; and(x) one or more unpaired sequences between stem-loops that is truncated or extended and / or that includes one or more nucleotide substitutions with canonical or non-canonical residues; and(xi) combinations thereof.

4. The RNA of claim 1 or claim 2, wherein the arrangement that is structurally permuted is selected from the group consisting of:(i) stem-loop -1 is positioned within the tracr sequence or at the 5' end of the RNA;(ii) stem-loop 0 is internalized or artificially linked;(iii) an intra-RNA covalent linkage is created by opening a loop and generating a new bond to a site elsewhere in the RNA;(iv) spacing between or among sequence elements is altered; and(v) combinations thereof.

5. The RNA of claim 1 or claim 2, which RNA includes a stem-loop 1 sequence element whose sequence is unaltered.

6. The RNA of claim 5, wherein the stem-loop 1 sequence is selected from the following:(i) 5' - AGGGCAGUUAGGUGCCCU - 3', or(ii) 5' - UCGUCGGUUCAGCGACGA - 3'.

7. The RNA of any one of the preceding claims, which RNA is characterized in that it complexes with a Casl2f polypeptide.

8. The RNA of claim 7, which RNA is characterized in that, when a complex of the RNA and the Casl2f polypeptide is present in a cell, editing is detectable at a site targeted by the RNA's crispr sequence element.

9. The RNA of claim 8, wherein the detectable editing is greater than about 1% INDEL.

10. The RNA of claim 9, which RNA is characterized in that it complexes with a plurality of different Casl2f polypeptides.

11. The RNA of claim 10, wherein the plurality of different Casl2f polypeptides includes at least two Casl2f polypeptides selected from the group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

12. A complex comprising the RNA of claim 1 with a Cas polypeptide.

13. The complex of claim 12, wherein the Cas polypeptide is a Casl2f polypeptide.

14. The complex of claim 13, wherein the Casl2f polypeptide is selected from the group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

15. An in vitro system comprising a complex of claim 14 and a target nucleic acid targeted by the RNA's crispr sequence element.

16. A mammalian cell engineered to contain or express the complex of claim 15.

17. A plant cell engineered to contain or express the complex of claim 15.

18. A nucleic acid that encodes the RNA of any one of claims 1-11.

19. The nucleic acid of claim 18, which nucleic acid also encodes a Casl2f polypeptide.

20. The nucleic acid of claim 19, wherein the nucleic acid is a template whose transcription produces the RNA.

21. The nucleic acid of any one of claims 18-20, which nucleic acid is a plasmid.

22. The nucleic acid of any one of claims 18-20, which nucleic acid is a vector.

23. A viral vector that encodes the RNA of any one of claims 1-11.

24. A vector system comprising a first vector that encodes the RNA of any one of claims 1-11 and a second vector that encodes a Casl2f polypeptide.

25. A pharmaceutical composition comprising the RNA of any one of claims 1-11 and a pharmaceutically acceptable excipient.

26. A method of making RNA of any one of claims 1-11 comprising: incubating the nucleic acid of claim 18 with: i. an RNA polymerase; and ii. ribonucleotides.

27. The method of claim 26, wherein the ribonucleotides comprise one or more naturally occurring ribonucleotides.

28. The method of claim 26 or 27, wherein the ribonucleotides comprise one or more modified ribonucleotides.

29. The method of any one of claims 26-28, wherein the polymerase is T7 RNA polymerase.

30. A method of making the complex of claim 12, the method comprising a step of: contacting an RNA of any one of claims 1-11 with a Cas polypeptide, wherein the contacting is in vitro.

31. A method of making the complex of claim 12, the method comprising a step of: contacting an RNA of any one of claims 1-11 with a Cas polypeptide, wherein the contacting is in vivo.

32. The method of claim 30 or 31, wherein the contacting comprises expressing the RNA in a cell that contains the polypeptide.

33. The method of claim 30 or 31, wherein the contacting comprises expressing the Cas polypeptide in a cell that contains the RNA.

34. The method of any one of claims 30-33, wherein the Cas polypeptide is a Casl2 polypeptide.

35. The method of claim 34, wherein the Casl2 polypeptide is a Casl2f polypeptide.

36. The method of claim 35, wherein the Casl2f polypeptide is selected from the group consisting of: Casl2fl, Casl2f2, Casl2f3, AsCasl2fl, AuCasl2f2, CnCasl2fl, MilCasl2f2, Mi2Casl2f2, PtCasl2fl, RuCasl2fl, SpaCasl2fl, UnlCasl2fl, Un2Casl2fl, RhCasl2fl, OsCasl2fl, or a functional portion thereof.

37. A method of making a pharmaceutical composition, the method comprising a step of: combining an RNA of any one of claims 1-11 or a complex of any one of claims 12-14, or a nucleic acid encoding an RNA of any one of claims 1-11, or a cell expressing the vector system of claim 24, with one or more pharmaceutically acceptable carriers or excipients.

38. A method of treating a disease, disorder, or condition, associated with a target sequence, the method comprising a step of modifying the target genetic sequence by contacting it with a complex of any one of claims 12- 14, wherein the RNA's crispr sequence element is substantially complementary to the target genetic sequence.

39. A method of modifying a target sequence with a complex of any one of claims 12-14, wherein the target is in vitro.

40. A method of modifying a target sequence with a complex of any one of claims 12-14, wherein the target is in vivo.

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