Compositions and methods for delivering transgenes

Abstract

Disclosed herein are compositions and methods that allow a high efficiency integration of genes of interest into a safe harbor locus in the genome. Such compositions and methods comprise small type V nucleases, guide RNAs, preferably targeting the Albumin locus.

Description

Field The disclosures provide compositions, methods, and systems for targeted delivery of nucleic acids to a target cell such as, e.g., human cell. Some embodiments of the invention relate to compositions, methods, and systems for expressing a transgene in a cell by genome editing. Incorporation by reference of the sequence listing The instant application contains a Sequence Listing, which has been submitted via Online Filing 2.0. The Sequence Listing titled "BHC231038-FC-PCT.xml", which was created on December 16, 2024, is hereby incorporated by reference in its entirety. Background Recent advances in genome sequencing techniques and analytical methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Recently, gene editing using designed site-specific nucleases emerged as a technology for both basic biomedical research and therapeutic development. In the recent years, various platforms based on four major types of endonucleases have been developed for gene editing, namely the meganucleases and their derivatives, the zinc finger nucleases (ZFNs), the transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR) associated endonuclease 9 (Cas9). Each nuclease type is capable of inducing a DNA double-stranded break (DSB) at specific DNA loci, thus triggering two DNA repair pathways. The non-homologous end joining (NHEJ) pathway generates random insertion / deletion (indel) mutations at the DSB, whereas the homology-directed repair (HDR) pathway repairs the DSB with the genetic information carried on a donor nucleic acid. In the context of this invention the term donor nucleic acid is interchangeably used with the term donor template. Therefore, these gene editing platforms can manipulate genes at specific genomic loci in multiple ways, such as disrupting gene function, repairing a mutant gene to normal, and inserting DNA material. However, presently available genome editing techniques can suffer from a number of disadvantages, for example, inaccuracy, unacceptable levels of off-target editing, etc. Accordingly, there remains a need for new genome gene editing platforms that are capable of manipulating genes at specific genomic loci in multiple ways, such as disrupting gene function, repairing a mutant gene to normal, and / or inserting heterologous DNA material at specific loci, such as a safe harbor locus within the genome of a target cell. Cell and gene therapy offers unprecedented opportunities to treat the untreatable. Especially in pedriatric patients standard gene therapy by augmentation of episomal AAV vectors is hampered by organ growth. This leads to dilution of the vectors and consequently lowers therapeutic effects. To mitigate this downside stable gene modification is desired for gene therapeutic applications. Albumin is an attractive target for gene modifying gene therapy, since it is expressed highly liver specific and offers an intronic safe harbor platform for the stable integration and expression of therapeutic transgenes. RNA-programmable nucleases offer a highly dynamic and versatile tool for targeted modification of genomic sequences. Those nucleases enable the framework for integration of templates of choice by cutting open the target site, which is a prerequisite for integration. WO2020 / 082042 discloses methods and reagents based on S.pyogenes CRISPR Cas9 nucleases to integrate transgenes into an albumin locus. WO2020 / 081843 also discloses such systems based on various Cas9 (Type II) CRISPR nucleases. WO2022258753 discloses the type V CRISPR Cas nucleases utilized in this invention, with the proviso that the nuclease named B-Gen.l in this application (SEQ. ID NO: 185) is named B-Gen.2 (SEQ ID NO: 2) in WO2022258753. Until now the high opportunity target albumin was merely approachable with Cas9 from B. pyogenes. However, there is a need for the utilization of this principle with significantly smaller CRISPR Cas effectors, that in further might also have other additional beneficial properties over the systems of the prior art, as e.g. higher nuclease activity, higher selectivity or less complex PAM requirements. The recently developed Type V B-Gen-1 and 2 CRISPR Cas nucleases (WO2022258753) provide small sized effectors with low complex PAM requirements. WO2023 / 220649 disclose the use of certain Type V nucleases for the integration of GAA into exon 1 of the albumin locus. The compositions, systems and methods of the prior art however, are having several disadvantages. a) The nuclease effectors are large enzymes that are difficult or unable to package into certain viral vectors as e.g. AAVs. b) The efficiency of integration of the gene of interest (GOI) is low. c) The efficiency of stable integration of the gene of interest (GOI) is low. d) Integration of the GOI into the genome of a target cell in vivo is not very efficient and / or stable, e) They exhibit too many off-target events that pose a risk for patients. f) They may work for one GOI but not for another GOI. Summary Provided herein are methods and compositions that allow the use of small sized CRISPR Cas nucleases (B-Gen.l (SEQ ID NO:185 or SEQ NO: 186) and polypeptides of at least 95% sequence identity to SEQ ID NOs: 185 and 186, for the integration of genes of interest (GOIs) into a safe harbor locus, preferably into the albumin locus. Another aspect of the compositions and methods according to the invention is that surprisingly high activities of the GOIs can be obtained in vitro and in vivo. One aspect of the invention is a system comprising: (a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; and (b) a guide RNA (gRNA) comprising a spacer sequence from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 58, 59, 60 , or a nucleic acid encoding the gRNA; and (c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof. Another aspect of the invention is a system comprising: (a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; and (b) a guide RNA (gRNA) from any one of SEQ ID NOs: 62 to 122, or nucleic acid encoding the gRNA, or any or any sequence thereof which is 95%, preferably, 98%, more preferably 99% identical to these sequences; and (c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof. Yet another aspect of the invention is a method of editing a genome in a cel I, the method comprising: providing the following to the cell: (a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; and (b) a guide RNA (gRNA) from any one of SEQ ID NOs: 62 to 122, or nucleic acid encoding the gRNA, or any or any sequence thereof which is 95%, preferably, 98%, more preferably 99% identical to these sequences, or A gRNA comprising a spacer sequence from any one of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 58, 59, 60; and (c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof. Provided herein, inter alia, are compositions, methods, and systems for targeted delivery of nucleic acids, including DNA and RNA, to a target cell, e.g., human cell. Also provided are compositions, methods, and systems for expressing a transgene in a cell by integration of the transgene into the genome of the cell in a targeted manner by genomic editing. Certain aspects and embodiments of the disclosure concern compositions, methods, and systems for knocking in a gene-of-interest (GOI) into a specific safe habor location in the genome, in particular to a genomic location within or near an endogenous albumin locus. Further provided are compositions, methods, and systems for treating a subject having or suspected of having a disorder or health condition employing ex vivo and / or in vivo genome editing. In one aspect, provided herein is a guide RNA (gRNA) sequence having a sequence that is complementary to a genomic sequence within or near an endogenous albumin locus. In some embodiments, the gRNA has a sequence selected from those of SEQ ID NOs: 62 to 122, and variants thereof having at least 85%, preferably 90%, more preferably 95%, even more preferably 98% identity to any of those sequences. In another aspect, provided herein is a composition having any of the above-mentioned gRNAs. In one aspect, provided herein is a system including a guide RNA (gRNA), comprising a spacer, as disclosed herein or a nucleic acid encoding the gRNA. In some embodiments, the gRNA of the system has a sequence selected from SEQ ID NOs: 62 to 122 and variants thereof having at least 85%, preferably 90%, more preferably 95%, even more preferably 98% identity to any of those sequences. In some preferred embodiments, the gRNA comprises a guide RNA (gRNA) sequence listed in TABLE 1 for preferred, more preferred, particularly preferred, more particularly preferred, or most preferred gRNA sequences. Brief Description of the Drawings Figurel shows indel activity of 61 gRNA of B-GEn.l targeting the human albumin intron 1 Locus. Experiment was performed in HEK293T cells. Plotted is the mean with standard deviation of two experiments with three technical replicates. Figure2a shows RLU activity of 7 synthetic gRNA of B-GEn.l targeting the human albumin intron 1 locus. Experiment was performed in primary human hepatocytes donor HUM201801 cells. Plotted is the mean with median range of one experiment with three technical replicates. Figure2b shows indel activity of 7 synthetic gRNA of B-GEn.l targeting the human albumin intron 1 locus. Experiment was performed in primary human hepatocytes donor HUM201801 cells. Plotted is the mean with median range of one experiment with three technical replicates. Figure 3 shows indel activity of 53 gRNA of B-GEn.l targeting the mouse albumin intron 1 locus. Experiment was performed in mouse cell line HEPA1-6 Plotted is the mean with standard deviation of one experiment with three technical replicates. Figure 4 shows B-GEn.l gene editing and integration in mice, a) indel activity of 10 gRNA of B-GEn.l targeting the mouse albumin intron 1 locus b) RLU values of the same experiment for the dose lmpk and 0.75 mpk. Plotted is the mean with standard deviation of one experiment with four technical replicates. Detailed Description of the Invention Illustrative embodiments In some embodiments, the system further has one or more of the following: a deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding the DNA endonuclease; and a donor nucleic acid having a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 1 to 61. In some preferred embodiments, the gRNA comprises a spacer sequence listed in TABLE 2 for preferred, more preferred, particularly preferred, more particularly preferred, or most preferred spacer sequences. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 58, 59, 60; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID Nos: 3, 4, 5, 6, 9, 11, 12, 13, 14, 15, 16, 20, 21, 22, 25, 26, 32, 39, 42, 44, 46, 51, 52, 54, 55, 56, 58, 59, 60; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID Nos: 4, 5, 6, 9, 12, 13, 14, 15, 20, 26, 32, 39, 42, 52, 54, 55, 58, 59; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID Nos: 4, 5, 6, 9,15, 20, 42, 52, 59; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID NO 4 or 42; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186; and b) a guide RNA comprising a spacer sequence from any of the SEQ ID NO 4 or 42; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence of the SEQ ID NO 4; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. (a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; and (b) a guide RNA (gRNA) from any one of SEQ ID NOs: 65 or 103, or nucleic acid encoding the gRNA, or any or any sequence thereof which is 95%, preferably, 98%, more preferably 99% identical to these sequences; and (c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof. In some embodiments the system comprises a) a DNA endonuclease according to SEQ ID NO: 185 or 186 or any sequence which is at least 95% identical to these sequences; and b) a guide RNA comprising a spacer sequence of the SEQ ID NO 42; and c) a donor nucleic acid comprising a nucleic acid sequence encoding a gene of interest (GOI) or a functional derivative thereof. In some embodiments, the DNA endonuclease recognizes a protospacer adjacent motif (PAM) having the sequence "DTTN" with "D" representing "A" or "T" or "G"; preferably NGG or NNGG, wherein N is any nucleotide, or a functional derivative thereof, or a functional derivative thereof. In some embodiments, the deoxyribonucleic acid (DNA) endonuclease is a type V nuclease. In some embodiments, the deoxyribonucleic acid (DNA) endonuclease is selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98%, more preferably 99% identical to these sequences. In some embodiments, the nucleic acid encoding the DNA endonuclease is codon optimized for expression in a host cell. In some embodiments, the nucleic acid sequence encoding the gene-of-interest (GOI) is codon optimized for expression in a host cell. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide and a prophylactic polypeptide. In some embodiments, the GOI encodes an alphaglucosidase enzyme (GAA). In some embodiments, the GOI encodes a protein selected from the group consisting of Factor VIII (FVIII) protein, Factor IX protein, alpha-1 -antitrypsin, Factor XIII (FXIII) protein, Factor VII (FVII) protein, Factor X (FX) protein, Protein C, serine protease inhibitor Gl (Serpin Gl), or a functional derivative of any thereof. In some embodiments, the GOI encodes a FVIII protein or a functional derivative thereof. In some embodiments, the GOI encodes a FIX protein or a functional derivative thereof. In some embodiments, the GOI encodes a serpin Gl protein or a functional derivative thereof. In some embodiments, the nucleic acid encoding the DNA endonuclease is a deoxyribonucleic acid (DNA) sequence. In some embodiments, the nucleic acid encoding the DNA endonuclease is a ribonucleic acid (RNA) sequence. In some embodiments, the RNA sequence encoding the DNA endonuclease is linked to the gRNA via a covalent bond. In some embodiments, the composition further has a liposome or lipid nanoparticle. In some embodiments, the donor nucleic acid is encoded in a viral vector. In some embodiments, the donor nucleic acid is encoded in an Adeno Associated Virus (AAV) vector. In some embodiments, the DNA endonuclease is formulated in a liposome or lipid nanoparticle. In some embodiments, the liposome or lipid nanoparticle also includes the gRNA. In some embodiments the nucleic acid encoding the effector protein is an mRNA. In some embodiments the components of the composition (DNA endonuclease, guide RNA, and donor nucleic acid) are encoded by a single viral vector. In some embodiments the components of the composition (DNA endonuclease, guide RNA, and donor nucleic acid) are encoded by separate viral vectors, including embodiments wherein one or two components are encoded by a single viral vector. In some embodiments the components of the system are administered separately. In some embodiments the components of the system are administered simultaneously. In some embodiments, the DNA endonuclease is precomplexed with the gRNA, forming a ribonucleoprotein (RNP) complex. In another aspect, provided herein is a kit having any of the composition described above and further having instructions for use. In another aspect, provided herein is a method of editing a genome in a cell. The method includes providing the following to the cell: (a) any of the gRNA described herein or nucleic acid encoding the gRNA; (b) a deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding the DNA endonuclease; and (c) a donor nucleic acid having a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs:l to 61 listed in TABLE 2. In some embodiments, the gRNA has a sequence selected from those listed in TABLE 1 and variants thereof having at least 85% homology to any of those listed in TABLE 1. In some embodiments, the nucleic acid sequence encoding the gene-of-interest (GOI) is codon optimized. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide and a prophylactic polypeptide. In some embodiments, the nucleic acid sequence encoding the gene-of-interest (GOI) is inserted into a genomic sequence of the cell. In some embodiments, the insertion is at, within, or near the albumin gene or albumin gene regulatory elements in the genome of the cell. In some embodiments, the insertion is in the first intron of the albumin gene. In some embodiments, the insertion is at least 37 bp downstream of the end of the first exon of the human albumin gene in the genome and at least 330 bp upstream of the start of the second exon of the human albumin gene in the genome. In some embodiments, the nucleic acid sequence encoding the gene-of-interest is expressed under the control of the endogenous albumin promoter. In some embodiments, the cell is a hepatocyte. In another aspect, provided herein is a genetically modified cell in which the genome of the cell is edited by any of the method described above. In some embodiments, the nucleic acid sequence encoding the gene-of-interest is inserted into a genomic sequence of the cell. In some embodiments, the insertion is at, within, or near the albumin gene or albumin gene regulatory elements in the genome of the cell. In some embodiments, the insertion is in the first intron of the albumin gene. In some embodiments, the nucleic acid sequence encoding the gene-of-interest is expressed under the control of the endogenous albumin promoter. In some embodiments, the nucleic acid sequence encoding the gene-of-interest is codon optimized. In some embodiments, the cell is a hepatocyte. In another aspect, provided herein is a method of treating a disorder or health condition in a subject. The method includes administering any of the above-mentioned genetically modified cell to the subject. In some embodiments, the genetically modified cell is autologous. In some embodiments, the method further has obtaining a biological sample from the subject wherein the biological sample has a hepatocyte cell and editing the genome of the hepatocyte cell by inserting a nucleic acid sequence encoding the gene-of-interest thereof into a genomic sequence of the cell, thereby producing the genetically modified cell. In another aspect, provided herein is a method of treating a disorder or health condition in a subject. The method has obtaining a biological sample from the subject wherein the biological sample has a hepatocyte cell, providing the following to the hepatocyte cell: (a) any of the gRNA described above or nucleic acid encoding the gRNA; (b) a deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding the DNA endonuclease; and (c) a donor nucleic acid having a nucleic acid sequence encoding a gene-of-interest (GO I) or functional derivative, thereby producing a genetically modified cell, and administering the genetically modified cell to the subject. Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect. The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. Definitions Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings: The terms, "% identical," "% identity," and "percent identity," or grammatical equivalents thereof, refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95). The terms, "amplification" and "amplifying," or grammatical equivalents thereof, as used herein, refers to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof. The term, "base editing enzyme," as used herein, refers to a protein, polypeptide or fragment thereof that is capable of catalyzing the chemical modification of a nucleobase of a deoxyribonucleotide or a ribonucleotide. Such a base editing enzyme, for example, is capable of catalyzing a reaction that modifies a nucleobase that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleobase to a different nucleobase, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). A base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase. The term, "base editor," as used herein, refers to a fusion protein comprising a base editing enzyme fused to an effector protein. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein. Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein. The term, "catalytically inactive effector protein," as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring effector protein may be a wildtype protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein. The term, "cis cleavage," as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by an effector protein complexed with a guide nucleic acid refers to cleavage of a target nucleic acid that is hybridized to a guide nucleic acid, wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide nucleic acid. The terms, "complementary" and "complementarity," as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the "reverse complement" sequence or the "reverse complementary" sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide. The terms, "cleave," "cleaving," and "cleavage," as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double -stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single -stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein. The term, "clustered regularly interspaced short palindromic repeats (CRISPR)," as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen. The terms, "CRISPR RNA" and "crRNA," as used herein, refer to a type of guide nucleic acid, wherein the nucleic acid is RNA comprising a first sequence, often referred to herein as a spacer sequence, that hybridizes to a target sequence of a target nucleic acid, and a second sequence that is capable of connecting a crRNA to an effector protein by either a) hybridizing to a portion of a tracrRNA or b) being non-covalently bound by an effector protein. In some embodiments, the second sequence is referred to as a repeat sequence. In a dual nucleic acid system, where a crRNA and a tracrRNA forms a complex with an effector protein, a crRNA includes the first sequence that hybridizes to the target sequence of the target nucleic acid and the second sequence hybridizes to a portion of the tracrRNA. The term, "donor nucleic acid," as used herein, refers to a nucleic acid that is incorporated into a target nucleic acid or target sequence. The term, "donor nucleotide," as used herein, refers to a single nucleotide that is incorporated into a target nucleic acid. A nucleotide is typically inserted at a site of cleavage by an effector protein. The term, "effector protein," as used herein, refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. A complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid. A non-limiting example of an effector protein modifying a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout. The term, "functional acid alpha glucosidase protein," as used herein, refers to acid alpha glucosidase protein that retains at least some if not all enzymatic activity relative to the wildtype protein. A functional acid alpha glucosidase protein can also include an acid alpha glucosidase protein having enhanced enzymatic activity relative to the wildtype protein. In some instances, the enzymatic activity is degradation of glycogen to glucose. Assays are known and available for detecting and quantifying acid alpha glucosidase protein activity, e.g., by subjecting a labeled substrate that releases a signal (e.g., colorimetric and fluorescent) when the substrate is cleaved. In some instances, the functional acid alpha glucosidase protein is a wildtype human acid alpha glucosidase protein. In some instances, the functional acid alpha glucosidase protein is a functional portion of a wildtype human acid alpha glucosidase protein. The term, "functional domain," as used herein, refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid modification, nucleic acid cleavage, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity. The term, "functional fragment," as used herein, refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity. The terms, "fusion effector protein," "fusion protein," and "fusion polypeptide," as used herein, refer to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein. In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein. The terms, "fusion partner protein" and "fusion partner," as used herein, refer to a protein, polypeptide or peptide that is fused to an effector protein. The fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein. The fusion partner may modify a target nucleic acid, including changing a nucleobase of the target nucleic acid and making a chemical modification to one or more nucleotides of the target nucleic acid. A "genetic disease", as used herein, refers to a disease, disorder, condition, or syndrome caused by one or more mutations in the DNA of an organism. Mutations can be due to several different cellular mechanisms, including, but not limited to, an error in DNA replication, recombination, or repair, or due to environmental factors. A genetic disease comprises, in some embodiments, a single gene disorder, a chromosome disorder, or a multifactorial disorder. The term, "guide nucleic acid," as used herein, refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of connecting an effector protein to the nucleic acid by either a) hybridizing to a portion of an additional nucleic acid that is bound by an effector protein (e.g., a tracrRNA) or b) being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence. In some embodiments, the first sequence is located 5' of the second nucleotide sequence. In some embodiments, the first sequence is located 3' of the second nucleotide sequence. In some embodiments, the first nucleotide sequence is linked to 5' or 3' end of the second nucleotide sequence. In some embodiments, the first nucleotide sequence is linked to the second nucleotide sequence by a linker nucleic acid. In some embodiments, the linker nucleic acid comprises one, two, three, four or five nucleotide bases. In some embodiments, the linker nucleic acid comprises a polynucleotide having two, three, four or five nucleotide bases. The term, "handle sequence," as used herein, in the context of a sgRNA refers to a portion of the sgRNA that is capable of being non-covalently bound by an effector protein. The nucleotide sequence of a handle sequence may contain or be derived from a tracrRNA. For example, in some aspects, a handle sequence can include a portion of a tracrRNA that is capable of being non-covalently bound by an effector protein, but does not include all or a part of the portion of a tracrRNA that hybridizes to a portion of a crRNA as found in a dual nucleic acid system. In some aspects, a handle sequence can include a portion of a tracrRNA as well as a portion of a repeat sequence, which can optionally be connected by a linker. In some aspects, a handle sequence in the context of a sgRNA can also be described as the portion of the sgRNA that does not hybridize to a target sequence in a target nucleic acid (e.g., a spacer sequence). The term, "heterologous," as used herein, means a nucleotide or polypeptide sequence that is not found in a native nucleic acid or protein, respectively. In some embodiments, fusion proteins comprise an effector protein and a fusion partner protein, wherein the fusion partner protein is heterologous to an effector protein. These fusion proteins may be referred to as a "heterologous protein." A protein that is heterologous to the effector protein is a protein that is not covalently linked via an amide bond to the effector protein in nature. In some embodiments, a heterologous protein is not encoded by a species that encodes the effector protein. In some embodiments, the heterologous protein exhibits an activity (e.g., enzymatic activity) when it is fused to the effector protein. In some embodiments, the heterologous protein exhibits increased or reduced activity (e.g., enzymatic activity) when it is fused to the effector protein, relative to when it is not fused to the effector protein. In some embodiments, the heterologous protein exhibits an activity (e.g., enzymatic activity) that it does not exhibit when it is fused to the effector protein. A guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid. The term, "in vitro, " as used herein, is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term, "in vivo," is used to describe an event that takes place in a subject's body. The term, "ex vivo," is used to describe an event that takes place outside of a subject's body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay. The term, "linked amino acids" as used herein, refers to at least two amino acids linked by an amide bond. The term, "linker," as used herein, refers to a bond or molecule that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid. A "peptide linker" comprises at least two amino acids linked by an amide bond. The term, "modified target nucleic acid," as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein. In some cases, the modification is an alteration in the sequence of the target nucleic acid. In some cases, the modified target nucleic acid comprises an insertion, deletion, replacement, or combinations thereof of one or more nucleotides compared to the unmodified target nucleic acid. The term, "mutation associated with a disease," as used herein, refers to the co-occurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation. The terms, "non-naturally occurring" and "engineered," as used herein, are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and / or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a nonlimiting further clarifying example, an effector protein or guide nucleic acid that is "natural," "naturally-occurring," or "found in nature" includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man. The term, "nucleic acid expression vector," as used herein, refers to a plasmid that can be used to express a nucleic acid of interest. The term, "nuclear localization signal," as used herein, refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment. The term, "nuclease activity," as used herein, refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids; the term "endonuclease activity" refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bond within a polynucleotide chain. An enzyme with nuclease activity may be referred to as a "nuclease." The term "pharmaceutically acceptable excipient, carrier, or diluent" refers to any substance that is formulated alongside the active ingredient in a pharmaceutical composition, allowing the active ingredient to maintain its biological activity while being non-reactive with the subject's immune system. Such substances may be included to achieve long-term stabilization, to bulk up solid formulations containing potent active ingredients in small quantities, or to enhance the therapeutic properties of the active ingredient in the final dosage form, such as improving absorption, reducing viscosity, or increasing solubility. The choice of suitable substances can depend on factors such as the route of administration, dosage form, active ingredient, and other considerations. Compositions containing these substances can be prepared using conventional methods known in the field (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy, 21st Ed. Mack Publishing, 2005). The term "protospacer adjacent motif (PAM)" refers to a nucleotide sequence located in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific site. A PAM sequence may be necessary for the complex formed by an effector protein and a guide nucleic acid to hybridize with and modify the target nucleic acid. However, certain effector proteins may not require the presence of a PAM sequence in the target nucleic acid to effect modification. The term "recombinant," when applied to proteins, polypeptides, peptides, and nucleic acids, refers to products resulting from various combinations of cloning, restriction, and / or ligation processes that yield a construct with a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Typically, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers or from a series of synthetic oligonucleotides, creating a synthetic nucleic acid that can be expressed from a recombinant transcriptional unit within a cell or a cell-free transcription and translation system. Such sequences may be presented as an open reading frame that is uninterrupted by internal non-translated sequences or introns, which are commonly found in eukaryotic genes. Genomic DNA containing the relevant sequences may also be utilized to create a recombinant gene or transcriptional unit. Nontranslated DNA sequences may be present 5' or 3' from the open reading frame, provided that these sequences do not interfere with the manipulation or expression of the coding regions and may modulate the production of the desired product through various mechanisms. Thus, the term "recombinant polynucleotide" or "recombinant nucleic acid" refers to one that is artificially created, meaning it is formed by the combination of two otherwise separate sequence segments through human intervention. This artificial combination is typically achieved through chemical synthesis or by the manipulation of isolated nucleic acid segments using genetic engineering techniques. This often involves replacing a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a recognition site. Alternatively, it may involve joining nucleic acid segments with desired functions to create a desired combination of functions. Similarly, the term "recombinant polypeptide" or "recombinant protein" pertains to one that is not naturally occurring, formed through the artificial combination of two otherwise separated segments of amino acid sequences via human intervention. For instance, a polypeptide that contains a heterologous amino acid sequence would be classified as a recombinant polypeptide. The term "trans-activating RNA (tracrRNA)" refers to a nucleic acid that includes a first sequence capable of forming a non-covalent bond with an effector protein. TracrRNAs may also contain a second sequence that hybridizes to a segment of a crRNA, often referred to as a repeat hybridization sequence. In certain embodiments, tracrRNAs are covalently attached to a crRNA. The terms "treatment" and "treating" pertain to a pharmaceutical or other intervention regimen aimed at achieving beneficial or desired outcomes for the recipient. Such beneficial outcomes may encompass therapeutic and / or prophylactic benefits. A therapeutic benefit typically involves the eradication or alleviation of symptoms, or the underlying disorder being addressed. Additionally, therapeutic benefits can manifest as the reduction or improvement of one or more physiological symptoms associated with the underlying disorder, even if the subject continues to have the disorder itself. Prophylactic effects may include delaying, preventing, or eliminating the onset of a disease or condition, as well as delaying or eliminating the emergence of symptoms, slowing, halting, or reversing the progression of a disease, or any combination of these outcomes. For prophylactic benefits, individuals at risk of developing a specific disease or those exhibiting one or more physiological symptoms of a disease may receive treatment, even if a formal diagnosis has not yet been established. The term "viral vector" refers to a nucleic acid intended for delivery into a host cell via a recombinantly produced virus or viral particle. This nucleic acid can be single-stranded or double-stranded, linear or circular, segmented or non-segmented, and may consist of DNA, RNA, or a combination of both. Examples of viruses or viral particles that can serve as carriers for a viral vector include retroviruses (such as lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses, and poxviruses. A viral vector delivered by these viruses or viral particles may be identified by the type of virus used for delivery (for instance, an AAV viral vector indicates that the delivery is facilitated by an adeno-associated virus). A viral vector named after the type of virus used for delivery can include viral elements (such as nucleotide sequences) essential for the viral vector's packaging into the virus or viral particle, replication of the virus, or other desired viral functions. A virus containing a viral vector may be replication competent, replication deficient, or replication defective. Disclosed herein are compositions, systems, and methods comprising at least one of: (a) a polypeptide (e.g., programmable nuclease) or a nucleic acid encoding the polypeptide; and (b) a guide nucleic acid or a nucleic acid encoding the guide nucleic acid. In some embodiments, compositions, systems, and methods comprise a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide is an effector protein, also referred to as a programmable nuclease or programmable nickase. Effector proteins and programmable nucleases / nickases are described herein and throughout. In general, programmable nucleases (e.g., effector proteins) are proteins that bind nucleic acid in a sequence-specific manner. In some embodiments, programmable nucleases (e.g., effector proteins) are proteins that bind and cleave nucleic acids in a sequence-specific manner. A programmable nuclease (e.g., effector proteins) may bind a target region of a nucleic acid and cleave the nucleic acid within the target region or at a position adjacent to the target region. In some embodiments, a programmable nuclease (e.g., effector proteins) is activated when it binds a target region of a nucleic acid to cleave regions of the nucleic acid that are near, but not adjacent to the target region. A programmable nuclease (e.g., effector proteins), such as a CRISPR-associated (Cas) protein, may be coupled to a guide nucleic acid that imparts activity or sequence selectivity to the programmable nuclease (e.g., effector proteins). A programmable nuclease (e.g., effector proteins) described herein comprises an effector protein. In general, guide nucleic acids comprise a CRISPR RNA (crRNA) or a single guide RNA (sgRNA) that is at least partially complementary to a target nucleic acid. Accordingly, in some embodiments, the effector protein and guide nucleic acid may form a complex that recognizes a target sequence acid and cleaves the nucleic acid within the target sequence, at a position adjacent to the target sequence, or at a position near to the target sequence. In some cases, a composition comprising effector proteins and guide nucleic acids further comprise a trans-activating crRNA (tracrRNA), at least a portion of which interacts with the programmable nuclease (e.g., effector proteins). In some embodiments, compositions, systems, and methods comprising effector proteins and guide nucleic acids further comprise an intermediary RNA at least a portion of which interacts with the programmable nuclease (e.g., effector proteins). In some cases, a tracrRNA or intermediary RNA is provided separately from the guide nucleic acid. The tracrRNA may hybridize to a portion of the guide nucleic acid that does not hybridize to the target nucleic acid. Programmable nucleases (e.g., effector proteins) may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single -stranded DNA (ssDNA), or a combination thereof. Programmable nucleases (e.g., effector proteins) may provide binding activity, cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide RNA (crRNA or sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guideRNA. Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide RNA. Trans cleavage activity is triggered by the hybridization of guide RNA to the target nucleic acid. Nickase activity is the selective cleavage of one strand of a dsDNA molecule. Programmable CRISPR-associated (Cas) nucleases, through their ability to cleave DNA at a precise target location in the genome of a wide variety of cells and organisms, allow for precise and efficient editing of DNA sequences of interest. In some embodiments, Cas comprises single -stranded DNA binding (SSB) protein and double-stranded DNA binding (DSB) protein. SSBs and DSBs are an effective way to disrupt a gene of interest, generate DNA or RNA modifications, and to treat genetic disease through gene correction. The compositions, systems, and methods described herein are non-naturally occurring. In some embodiments, compositions, methods and systems comprise at least one of an engineered effector protein and an engineered guide nucleic acid, which may simply be referred to herein as an effector protein and a guide nucleic acid, respectively. In some embodiments, compositions, systems, and methods comprise an effector protein or a use thereof. In some embodiments, compositions, systems and methods comprise an isolated effector protein or a use thereof. In general, an effector protein and a guide nucleic acid refer to an effector protein and a guide nucleic acid, respectively, that are not found in nature. In some embodiments, systems, methods and compositions described herein comprise at least one non-naturally occurring component. For example, disclosed compositions, methods, and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some embodiments, compositions, systems, and methods comprise at least two components that do not naturally occur together. For example, disclosed compositions, systems, and methods may comprise a guide nucleic acid comprising a repeat region and a spacer region, which do not naturally occur together and / or are heterologous to each other. Also, by way of non-limiting example, disclosed compositions, systems, and methods may comprise a guide nucleic acid and an effector protein that do not naturally occur together. Likewise, by way of non-limiting example, disclosed compositions, systems, and methods may comprise a ribonucleotide-protein (RNP) complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. Conversely, and for clarity, an effector protein or guide nucleic acid that is "natural," "naturally-occurring," or "found in nature" includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine. Alternatively, in some embodiments, compositions and systems comprise at least two components that do not occur together in nature, wherein the at least two components comprise at least one of an effector protein, a fusion partner and a guide nucleic acid.

[146] In some embodiments, the guide nucleic acid comprises a non-natural nucleotide sequence. In some embodiments, the non-natural nucleotide sequence is a nucleotide sequence that is not found in nature. The non-natural nucleotide sequence may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some embodiments, the non-naturally occurring sequence is generated by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. In some embodiments, the guide nucleic acid comprises two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, compositions and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, a guide nucleic acid may comprise a sequence of a naturally-occurring repeat region and a spacer region that is complementary to a naturally-occurring eukaryotic sequence. The guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism. A guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3' or 5 ' end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. For example, a guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA sequence coupled by a linker sequence (e.g., a sgRNA). In some embodiments, the guide nucleic acid comprises two heterologous sequences arranged in an order or proximity that is not observed in nature. Therefore, compositions and systems described herein are not naturally occurring. In some embodiments, compositions, systems, and methods described herein comprise an effector protein that is similar to a naturally occurring effector protein. The effector protein may lack a portion of the naturally occurring effector protein. The effector protein may comprise a mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature. The effector protein may also comprise at least one additional amino acid relative to the naturally-occurring effector protein. For example, the effector protein may comprise an addition of a nuclear localization signal relative to the natural occurring effector protein. In some embodiments, a nucleotide sequence encoding the effector protein is codon optimized (e.g., for expression, in a eukaryotic cell) relative to the naturally occurring sequence. I. Polypeptide Systems Provided herein are compositions, systems and methods comprising a polypeptide or polypeptide system, wherein the polypeptide or polypeptide system described herein comprises one or more effector proteins or variants thereof, one or more effector partners or variants thereof, one or more linkers for peptides, or combinations thereof. In some embodiments, a variant is a form or version of a protein that differs from a naturally occurring protein or wild-type protein. For example, a variant may have one or more amino acid substitutions, insertions, or deletions relative to a wildtype protein. In some embodiments, a variant is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a respective wildtype protein. A variant may have a different function or activity relative to the naturally occurring or wild-type protein. A polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more engineered modifications, or both, relative to a naturally occurring or wildtype protein. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some instances, when a heterologous peptide, such as a fusion partner protein, protein tag or nuclear localization signal (NLS), is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein may be removed or absent. Effector Proteins Provided herein, in certain embodiments, are compositions, systems, and methods that comprise one or more effector proteins, or nucleotide sequences encoding the one or more effector proteins. In some embodiments, an effector protein is a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that interacts with a target nucleic acid. An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary with a target sequence in the target nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein. Modification activity of an effector protein or an engineered protein described herein may be cleavage activity, binding activity, insertion activity, substitution activity, and the like. Modification activity of an effector protein may result in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or any combination thereof. In some embodiments, modification of a target nucleic acid comprises introducing or removing epigenetic modification(s). In some embodiments, an ability of an effector protein to edit a target nucleic acid may depend upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or combinations thereof. A target nucleic acid comprises a target strand and a non-target strand. Accordingly, in some embodiments, the effector protein may edit a target strand and / or a nontarget strand of a target nucleic acid. An effector protein may modify a nucleic acid by cis cleavage or trans cleavage. The modification of the target nucleic acid generated by an effector protein may, as a non-limiting example, result in expression of a protein that is encoded by a donor nucleic acid. The modification of the target nucleic acid generated by an effector protein may, as a nonlimiting example, result in modulation of the expression of the target nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g., inactivation of a protein binding to an RNA molecule or hybridization). Accordingly, in some embodiments, provided herein are methods of editing a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Also provided herein are methods of modulating expression of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Further provided herein are methods of modulating the activity of a translation product of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. In some embodiments, a given effector protein may not require a PAM sequence being present in a target nucleic acid for the effector protein to modify the target nucleic acid. Accordingly, in some embodiments, an effector protein may also recognize a sequence that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides away from 5' or 3' terminus of a PAM sequence present in the target nucleic acid, which may direct the modification activity of the effector protein. In some embodiments, effector proteins disclosed herein may provide catalytic activity (e.g., cleavage activity, nickase activity, nuclease activity, other activity, or combinations thereof) similar to that of a naturally-occurring effector protein, such as, for example, a naturally-occurring effector protein with reduced cleavage activity including cis cleavage activity, trans cleavage activity, or combinations thereof. In some embodiments, effector proteins disclosed herein may be fused to effector partners or fusion proteins wherein the effector partners or fusion proteins are capable of some function or activity not provided by an effector protein. An effector protein may be a CRISPR-associated ("Cas") protein. An effector protein may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). An effector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g., modifying a target nucleic acid). In some embodiments, the effector protein or a multiprotein complex thereof binds to the guide nucleic acid by a non-covalent interaction. Nonlimiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. In some embodiments, an effector protein, when functioning in a multiprotein complex, may have differing and / or complementary functional activity to other effector proteins in the multiprotein complex. In some embodiments, the complementary functional activity of effector proteins comprising modified or artificial base pairs can be based on other types of hydrogen bonding and / or hydrophobicity of bases and / or shape complementarity between bases. Multimeric complexes, and functions thereof, are described in further detail below. In some embodiments, an effector protein may be a modified effector protein having different modification activity and / or substrate binding activity (e.g., substrate selectivity, specificity, and / or affinity) relative to an unmodified effector protein. For example, in some embodiments, an effector protein may be a modified effector protein having reduced modification activity (e.g., a catalytically defective effector protein) or no modification activity (e.g., a catalytically inactive effector protein). Accordingly, an effector protein as used herein encompasses a modified or programmable nuclease that does not have nuclease activity. In certain embodiments, the effector proteins described herein may include one or more functional domains. These functional domains can encompass a protospacer adjacent motif (PAM)-interacting domain, an oligonucleotide-interacting domain, various recognition domains, a non-target strand interacting domain, and a RuvC domain. A PAM-interacting domain may be classified as a target strand PAM interacting domain (TPID) or a non-target strand PAM interacting domain (NTPID). In some instances, a PAM interacting domain, such as a TPID or NTPID, refers to a segment of a polypeptide that engages with the target nucleic acid. The effector proteins may also contain a RuvC domain, which is often situated near the C-terminus of the protein. This RuvC domain is capable of cleaving a target nucleic acid and, in some cases, processing a pre-crRNA. A single RuvC domain can comprise several subdomains, such as RuvC-l, RuvC-ll, and RuvC-lll. In some embodiments, the RuvC domain may also include a RuvC-like domain. Various RuvC-like domains are well-documented and can be identified using online resources like InterPro (https: / / www.ebi.ac.uk / interpro / ). For instance, a RuvC-like domain may exhibit homology to regions found in TnpB proteins of the IS605 and related transposon families, as detailed in review articles like those by Shmakov et al. (Nature Reviews Microbiology volume 15, pages 169-182 (2017)) and Koonin E.V. and Makarova K.S. (2019, Phil. Trans. R. Soc., B 374:20180087). In certain cases, the RuvC domain may possess substrate binding activity, catalytic activity, or both. It can be defined by a single contiguous sequence or by a collection of RuvC subdomains that are not contiguous in the primary amino acid sequence. An effector protein may feature multiple RuvC subdomains that work together to form a RuvC domain with substrate binding or catalytic capabilities. For example, an effector protein could incorporate three RuvC subdomains (RuvC-l, RuvC-ll, and RuvC-lll) that are not contiguous in the primary sequence but assemble into a functional RuvC domain upon protein production and folding. Often, effector proteins include a recognition domain (REC domain) that binds to either a guide nucleic acid or a guide nucleic acid-target nucleic acid heteroduplex. In some embodiments, the REC domain may consist of an a-helical recognition region or lobe. A CRISPR / Cas protein may contain two REC domains (RECI and REC2), which typically assist in accommodating and stabilizing the hybrid of the guide nucleic acid and target nucleic acid. An effector protein may contain at least one REC domain (such as RECI or REC2) and may also include a zinc finger domain. In some instances, the effector protein does not possess an HNH domain. Effector proteins may be relatively small, which can be advantageous for nucleic acid detection or editing applications (for instance, their smaller size may reduce the likelihood of adsorption to surfaces or other biological entities). The compact nature of these effector proteins may facilitate more efficient packaging and delivery in the context of genome editing, as well as their incorporation as reagents in assays. In certain embodiments, the length of the effector protein is at least 400 linked amino acid residues. In other cases, the length may be less than 500 linked amino acid residues. Additionally, the length may range from about 400 to about 500 linked amino acid residues, or from about 450 to about 550, or within specific ranges such as about 400 to about 420, about 420 to about 440, about 440 to about 460, about 460 to about 480, about 480 to about 500, about 500 to about 520, about 520 to about 540, about 540 to about 560, about 560 to about 580, about 580 to about 600, about 600 to about 620, about 620 to about 640, about 640 to about 660, about 660 to about 680, about 680 to about 700 linked amino acids. In some cases, an effector protein may be capable of recognizing various PAMs as described herein. Additionally, the effector proteins may generate blunt or short staggered ends. Blunt cutting can be advantageous compared to staggered cutting provided by other effector proteins, as it reduces the likelihood of spontaneous (or perfect) repair, potentially enhancing the success rate of target nucleic acid editing and / or donor nucleic acid insertion. In certain embodiments, the effector proteins function as endonucleases that catalyze cleavage within a target nucleic acid. They may also be capable of catalyzing non-sequence-specific cleavage of a singlestranded nucleic acid. In some instances, the effector proteins (e.g., those with any of the amino acid sequences listed in TABLE 1) are activated to perform trans cleavage activity following the binding of a guide nucleic acid to a target nucleic acid. This trans cleavage activity may also be referred to as "collateral" or "transcollateral" cleavage, and it may involve non-specific cleavage of nearby single stranded nucleic acids by the activated effector protein, such as the trans cleavage of detector nucleic acids containing a detection moiety. The effector proteins described herein may act as endonucleases that facilitate cleavage at a specific site (for example, at a particular nucleotide within a nucleic acid sequence) in a designated target nucleic acid. The target nucleic acid can be single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA). In certain embodiments, the target nucleic acid is singlestranded DNA, while in others, it is single-stranded RNA. The effector proteins may exhibit cis cleavage activity, trans cleavage activity, nickase activity, or a combination of these functionalities. Cis cleavage activity refers to the cleavage of a target nucleic acid that is hybridized to a guide RNA (such as in a dual guide nucleic acid system or a single guide RNA, sgRNA), where the cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide RNA. Trans cleavage activity, also known as transcollateral cleavage, involves the cleavage of ssDNA or ssRNA that is nearby but not hybridized to the guide RNA. This trans cleavage activity is initiated by the hybridization of the guide nucleic acid to the target nucleic acid. In some instances, trans cleavage refers to the hydrolysis of one or more nucleic acids by an effector protein that is complexed with both a guide nucleic acid and a target nucleic acid. These nucleic acids may include the target as well as nontarget nucleic acids. Trans cleavage may occur in proximity to, but not within or directly adjacent to, the region of the target nucleic acid that is hybridized to the guide nucleic acid. Nickase activity involves the selective cleavage of one strand of a double-stranded DNA (dsDNA). TABLE 1 presents an illustrative amino acid sequence for effector proteins that are applicable in the compositions, systems, and methods discussed herein. In certain embodiments, an effector protein exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even 100% identity to any of the amino acid sequences listed in TABLE 1. Additionally, the nucleic acid encoding the effector protein may be operably linked to a promoter that is functional in either a eukaryotic or prokaryotic cell. The promoter may be one or more of the following: a constitutive promoter, an inducible promoter, a cell type-specific promoter, or a tissue-specific promoter. In some cases, the promoter is effective in various cell types, including plant cells, fungal cells, animal cells, invertebrate cells, fly cells, vertebrate cells, mammalian cells, primate cells, non-human primate cells, and human cells. Furthermore, in certain embodiments, the nucleic acid may be part of a nucleic acid expression vector as described herein. In certain embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200 contiguous amino acids, at least about 220 contiguous amino acids, at least about 240 contiguous amino acids, at least about 260 contiguous amino acids, at least about 280 contiguous amino acids, at least about 300 contiguous amino acids, at least about 320 contiguous amino acids, at least about 340, at least about 360, at least about 380 contiguous amino acids, at least about 400, at least about 420 contiguous amino acids, at least about 440 contiguous amino acids, at least about 460 contiguous amino acids, at least about 480 contiguous amino acids, at least about 500 contiguous amino acids, at least about 520 contiguous amino acids, at least about 540 contiguous amino acids, at least about 560 contiguous amino acids, at least about 580 contiguous amino acids, at least about 600 contiguous amino acids, at least about 620 contiguous amino acids, at least about 640 contiguous amino acids, at least about 660 contiguous amino acids, at least about 680 contiguous amino acids, or at least about 700 contiguous amino acids or more of any one of the amino acid sequences recited in TABLE 1. In certain embodiments, compositions, systems, and methods as described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200 contiguous amino acids or more of any one of the amino acid sequences recited in TABLE 1. In certain embodiments, compositions, systems, and methods as described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 300 contiguous amino acids or more of any one of the amino acid sequences recited TABLE 1. In certain embodiments, compositions, systems, and methods as described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 400 contiguous amino acids or more of any one of the amino acid sequences recited TABLE 1. In some embodiments, compositions, systems and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1. In some embodiments, the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1, wherein the portion does not comprise at least the first 10 amino acids, at least the first 20 amino acids, at least the first 40 amino acids, at least the first 60 amino acids, at least the first 80 amino acids, at least the first 100 amino acids, at least the first 120 amino acids, at least the first 140 amino acids, at least the first 160 amino acids, at least the first 180 amino acids, or at least the first 200 amino acids of any one of the amino acid sequences recited in TABLE 1. In some embodiments, the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1, wherein the portion does not comprise the last 10 amino acids, the last 20 amino acids, the last 40 amino acids, the last 60 amino acids, the last 80 amino acids, the last 100 amino acids, the last 120 amino acids, the last 140 amino acids, the last 160 amino acids, the last 180 amino acids, or the last 200 amino acids of any one of the amino acid sequences recited in TABLE 1. In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 65% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 70% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 75% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% identical to anyone of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is identical to any one of the amino acid sequences as set forth in TABLE 1 In certain embodiments, the compositions, systems, and methods described herein may include a variant of a reference effector protein (wild-type effector protein). These embodiments may feature an effector protein or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even 100% similarity to any of the amino acid sequences listed in TABLE 1. The similarity of an amino acid sequence to a reference sequence is determined by calculating a value that is derived from dividing a similarity score by the length of the alignment. The similarity between two amino acid sequences can be assessed using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89: 10915-10919 (1992)), which is modified such that any score greater than 1 is replaced with +1, and any score less than 0 is replaced with 0. For instance, a substitution from His (H) to Leu (L) is scored at +2.0 using the BLOSUM62 matrix, which translates to +1 in the modified matrix. This transformation facilitates the calculation of percent similarity rather than merely a similarity score. Alternatively, when comparing two full protein sequences, pairwise alignment can be performed using MUSCLE alignment, allowing the percent similarity to be determined for each residue and divided by the total length of the alignment. For assessing percent similarity over a specific protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) may be employed to evaluate the conservation of each domain or motif. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, if a substitution is considered conservative among any of the amino acids at that position in the multilevel consensus sequence, +1 point is awarded. For example, given the multilevel consensus sequences RLG and YCK, the test sequence QIQ would receive three points based on the following scores from the transformed BLOSUM62 matrix: Q-R: +1; Q-Y: +0; l-L: +1; l-C: +0; Q-G: +0; Q-K: +1. The highest score for each position is used when calculating the overall similarity. Percent similarity can also be computed using commercially available software, such as Geneious Prime, with parameters set to use the BLOSUM62 matrix and a threshold greater than 1. Therefore, percent similarity is defined as the value obtained by dividing a similarity score by the length of the alignment. In some embodiments, the compositions, systems, and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar to the sequence recited in TABLE 1. Specifically, an effector protein may have an amino acid sequence that is at least 65% similar to any of the sequences presented in TABLE 1. Similarly, additional embodiments may specify that the effector protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even 100% similar to the sequences outlined in TABLE 1. Such an effector protein may contain one or more modifications, which can be classified as either conservative or non-conservative alterations (e.g., conservative or non-conservative substitutions). In certain embodiments, these modifications can independently consist of one or more conservative substitutions, one or more non-conservative substitutions, or a combination of both. A conservative alteration (e.g., conservative substitution) refers to the replacement of one amino acid with another from a family of amino acids that share similar side chain characteristics. Conversely, a nonconservative alteration (e.g., non-conservative substitution) involves substituting one amino acid residue with another that does not belong to the same side chain family. Genetically encoded amino acids can be categorized into four families based on their related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar further divided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Amino acids can also be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) sometimes categorized separately as aliphatic-hydroxyl. Aromatic side chains include: Phe (F), Tyr (Y), Trp (W). Amide side chains consist of: Asn (N), Gin (Q), while sulfur-containing side chains include: Cys (C) and Met (M). In certain instances, an effector protein that contains one or more amino acid modifications is regarded as a variant of the effector protein described herein. It is important to note that any reference to an effector protein also includes its variants as outlined. Modifications may involve the deletion of an amino acid or the insertion of an amino acid. Furthermore, an amino acid alteration may comprise a non-conservative substitution. In some cases, these modifications may consist of a combination of one or more conservative amino acid substitutions along with one or more non-conservative amino acid substitutions. The effector protein or the nucleic acid that encodes it may exhibit one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid alterations compared to any of the amino acids specified in TR. In certain embodiments, the compositions, systems, and methods described herein may include an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein contains one or more substitutions compared to any of the amino acid sequences listed in TABLE 1. These substitutions may range from at least one to twenty, or more, and can be categorized into various ranges such as one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty substitutions relative to any of the sequences in TABLE 1. The substitutions may consist of one or more conservative substitutions, one or more nonconservative substitutions, or a combination of both. In some cases, the effector protein or nucleic acid encoding it may include one or more conservative substitutions compared to any of the amino acid sequences listed in TABLE 1. These conservative substitutions can also fall within the same range of one to twenty or more, with various specified ranges as previously mentioned. Similarly, the effector protein may also comprise one or more non-conservative substitutions relative to the sequences in TABLE 1, with the same potential ranges for the number of substitutions. In some instances, the amino acid alterations may lead to changes in the activity of the effector protein compared to its naturally occurring counterpart. For example, these alterations might enhance or reduce the catalytic activity of the effector protein or affect its binding activity relative to the naturally occurring version. In certain embodiments, the alterations may result in a catalytically inactive variant of the effector protein. Moreover, the effector proteins described herein can perform enzymatic reactions similar to those of the wild-type (WT) effector protein. Variants of the WT effector protein may include modifications that confer beneficial characteristics, such as increased activity (e.g., improved indel activity, catalytic activity, specificity, selectivity, or affinity for substrates like target nucleic acids or guide nucleic acids). In some cases, the activity of the effector proteins may be equal to or greater than that of the WT effector protein, meaning they exhibit one or more activities that are the same as or higher than those of the effector protein without any variant at the same amino acid positions. For instance, variants may show increased activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or even 200% compared to the WT effector protein. The activity of the effector proteins or their variants can be assessed in relation to the WT effector protein through a cleavage assay. In some embodiments, the effector proteins may include one or more amino acid substitutions compared to any of the sequences in TABLE 1, with the remaining amino acid sequence being at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the reference amino acid sequence from TABLE 1. Additionally, the remaining amino acid sequence of the variant may also exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the corresponding reference sequence. In some cases, the substitutions may involve one or more positively charged amino acid residues, such as Lys (K), Arg (R), His (H), or combinations thereof. Modification of effector protein includes modification of one or more nucleic acid residues of a nucleotide sequence that encodes the effector protein, or one or more amino acid residue of an amino acid sequence of the effector protein. In some embodiments, the modification includes chemical modification of one or more nucleobases; or chemical modifications to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to an effector protein amino acid sequence or nucleotide sequence encoding the effector protein. Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the modification(s) may be located at any position(s) of the nucleotide sequence or amino acid sequence. In some embodiments, the modification may substantially change the function of the effector protein, composition or system. In some embodiments, the modification may substantially decrease the function of the effector protein, composition, or system. In some embodiments, the modification may substantially increase the function of the effector protein, composition, or system. In some embodiments, the modification may substantially change the structural stability of the effector protein. The modified effector protein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in w / ro-transcription. cloning, enzymatic, or chemical cleavage, etc. In certain embodiments, the compositions, systems, and methods described herein may include an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein contains one or more substitutions compared to any of the amino acid sequences listed in TABLE 1. These substitutions can range from at least one to twenty or more, and may be categorized into various ranges such as one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty substitutions relative to the sequences in TABLE 1. The substitutions may include one or more conservative substitutions, one or more non-conservative substitutions, or a combination of both. In some instances, the effector protein or the nucleic acid encoding it may comprise one or more conservative substitutions compared to any of the amino acid sequences in TABLE 1, with the number of substitutions again falling within the previously mentioned ranges. Similarly, the effector protein may also include one or more non-conservative substitutions relative to any of the sequences in TABLE 1, with the substitutions potentially ranging from one to twenty or more. In some cases, the amino acid alterations may lead to changes in the activity of the effector protein compared to its naturally occurring counterpart. For instance, these modifications may enhance or reduce the catalytic activity or binding activity of the effector protein. In certain embodiments, the alterations may result in a catalytically inactive variant of the effector protein. Moreover, the effector proteins described herein can perform enzymatic reactions similar to those of the wild-type (WT) effector protein. Variants of the WT effector protein may contain modifications that confer beneficial characteristics, such as increased activity (e.g., improved indel activity, catalytic activity, specificity, selectivity, or affinity for substrates like target nucleic acids or guide nucleic acids). In some cases, the activity of the effector proteins may be equal to or greater than that of the WT effector protein, indicating that they exhibit one or more activities that are the same as or higher than those of the effector protein without any variant at the same amino acid positions. For example, variants can demonstrate increased activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or even 200% compared to the WT effector protein. The activity of the effector proteins or their variants can be assessed relative to the WT effector protein using a cleavage assay. In some embodiments, the effector proteins may feature one or more amino acid substitutions compared to any of the sequences in TABLE 1, with the remaining amino acid sequence being at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the reference amino acid sequence from TABLE 1. Additionally, the remaining amino acid sequence of the variant may also exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the corresponding reference sequence. In some cases, the substitutions may involve one or more positively charged amino acid residues, such as Lys (K), Arg (R), His (H), or combinations thereof. In some embodiments, the term "in vitro" refers to processes occurring outside of a living organism, typically in a controlled environment such as a test tube or culture dish. Effector proteins described herein may include both coded and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids, and proteins with altered peptide backbones. These effector proteins may contain one or more mutations, engineered modifications, or both. It is understood that the coding sequences for these effector proteins do not necessarily need to include a codon for an N-terminal Methionine (M) or Valine (V). A skilled individual in the field would recognize that a start codon can be substituted with one that encodes an amino acid residue sufficient for initiating translation in a host cell. Additionally, the compositions, systems, and methods may include heterologous peptides or polypeptides, which can be fusion proteins comprising an effector protein along with one or more fusion partner proteins. The fusion partner proteins can be located at the N-terminus of the effector protein. In such cases, the start codon for the heterologous peptide or polypeptide may also serve as the start codon for the effector protein, allowing for the removal or absence of the natural start codon that typically encodes an amino acid residue necessary for translation initiation. Heterologous polypeptides may consist of at least two different polypeptide sequences that are not found together in nature. A heterologous system may include at least one component that does not naturally occur in conjunction with the other components. In some embodiments, a heterologous peptide or polypeptide may contain a subcellular localization signal, such as a nuclear localization signal (NLS) that facilitates the targeting of a nucleic acid, protein, or small molecule to the nucleus within a cell. Other types of localization signals may include nuclear export signals (NES), signals for retaining an effector protein in the cytoplasm, mitochondrial localization signals, chloroplast localization signals, endoplasmic reticulum retention signals, and others. In certain cases, an effector protein may not include a subcellular localization signal to prevent targeting to the nucleus, which can be beneficial when the target nucleic acid is an RNA present in the cytosol. In some embodiments, the heterologous polypeptide may be an endosomal escape peptide (EEP), designed to disrupt the endosome quickly to minimize the time a delivered molecule, such as an effector protein, remains in the endosomal environment, thus avoiding entrapment in endosomal vesicles and subsequent degradation in the lysosomal compartment. Additionally, a heterologous polypeptide may be a cell-penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD), which facilitates the traversal of lipid bilayers, cell membranes, organelle membranes, or vesicle membranes. In some instances, a heterologous peptide or polypeptide may include a protein tag, which can serve as a purification tag or a fluorescent protein. Such tags may be detectable for the purpose of identifying or purifying the effector protein. Various protein tags can be utilized depending on the intended application, including but not limited to fluorescent proteins, histidine tags (e.g., 6XHis), hemagglutinin (HA) tags, FLAG tags, Myc tags, and maltose binding proteins (MBP). Examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato. A heterologous polypeptide may be positioned at or near the amino terminus (N-terminus) or the carboxy terminus (C-terminus) of the effector protein. In some cases, the heterologous polypeptide may be located internally within the effector protein at a suitable insertion site. II. Guide Nucleic Acids The compositions, systems, and methods of the present disclosure may include a guide nucleic acid or its use. These may also encompass compositions, systems, and methods that feature one or more guide nucleic acids and DNA molecules encoding these guide nucleic acids. A person skilled in the art understands that a DNA molecule that "encodes" a nucleic acid, such as a guide nucleic acid, refers to a DNA molecule with a nucleotide sequence that produces an RNA molecule (e.g., a guide nucleic acid) upon transcription. It is understood that mention of a guide nucleic acid also includes a DNA molecule encoding that guide nucleic acid. A guide nucleic acid, along with its components (e.g., spacer sequence, repeat sequence, linker nucleotide sequence, handle sequence, intermediary RNA, etc.), may consist of one or more deoxyribonucleotides (DNA), ribonucleotides (RNA), or a combination of both (e.g., RNA containing a thymine base), as well as biochemically or chemically modified nucleotides. Such nucleotide sequences can be described as either DNA or RNA; however, regardless of the form, it is understood that these sequences can be adapted to be RNA or DNA as required for describing a sequence within a guide nucleic acid or the sequence that encodes it, such as a nucleotide sequence for a vector. The disclosure of the nucleotide sequences also includes their complementary sequences, reverse sequences, and reverse complement sequences, any of which can serve as a nucleotide sequence for use in a guide nucleic acid. In some embodiments, a guide nucleic acid may comprise a CRISPR RNA (crRNA) or a single guide RNA (sgRNA). The sgRNA is formed by combining a spacer sequence (which hybridizes to a target sequence in a target nucleic acid) with a handle sequence, where both sequences are covalently linked. The spacer and handle sequences can be linked by a phosphodiester bond or by one or more linked nucleotides. In some cases, a guide nucleic acid may include a spacer sequence, a repeat sequence, a handle sequence, or a combination thereof, with the handle sequence potentially comprising part or all of a repeat sequence. In some embodiments, the composition may include a tracrRNA. The crRNAand tracrRNA may function as separate, unlinked molecules, or they may be covalently linked. The linkage can occur via a phosphodiester bond or through one or more linked nucleotides. In certain embodiments, the composition may not include a tracrRNA. A guide nucleic acid may be a naturally occurring guide nucleic acid or a non-naturally occurring one, which may include chemical or biochemical modifications. The guide RNA may be synthesized chemically or produced recombinantly. The sequence of the guide nucleic acid, or a portion of it, may differ from that of a naturally occurring nucleic acid. In some embodiments, the compositions, systems, and methods of the present disclosure may include one or more additional guide nucleic acids or their uses. For example, these may include two or more additional guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, or more guide nucleic acids) that can target an effector protein to different locations within the target nucleic acid by binding to distinct portions of that nucleic acid. A guide nucleic acid can bind to a segment of the target nucleic acid that is upstream or downstream of a target gene, allowing for modifications at two different locations. This dual-targeting approach, referred to as "dual-cutting," may involve two effector proteins, each corresponding to a guide RNA, or a single effector protein with two different guide RNAs to achieve the dual-cutting effect. In some cases, multiple effector proteins (e.g., 2, 3, 4, 5, 6, 7, 9, 10, or more) may be employed in the dual-guided systems described herein. In some embodiments, the guide nucleic acid may include 10, 11,12,13,14,15,16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, or 30 linked nucleotides. Generally, a guide nucleic acid will consist of at least a certain number of linked nucleotides, with some embodiments specifying at least 25 linked nucleotides. The guide nucleic acid may range from 10 to 50 linked nucleotides in length. In some cases, the guide nucleic acid may consist of or essentially consist of about 12 to about 80, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19, about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleotides. In some embodiments, the guide nucleic acid may have about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides. In some embodiments, the engineered guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. This eukaryotic sequence refers to a nucleotide sequence present in a host eukaryotic cell, distinguishing it from sequences found in prokaryotic cells or viruses. Such sequences can be located within a gene, an exon, an intron, a non-coding region (e.g., a promoter or enhancer), a selectable marker, tag, signal, or similar elements. In some embodiments, the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a nucleotide sequence as described herein (e.g., TABLE 4, TABLE 5, TABLE 6, or TABLE 8). These nucleotide sequences can be characterized as either DNA or RNA; however, it is understood that such sequences can be adapted as needed for describing a sequence within a guide nucleic acid or the sequence that encodes it, such as a nucleotide sequence for a vector. Additionally, the disclosure of these nucleotide sequences also includes their complementary sequences, reverse sequences, and reverse complement sequences, any of which can serve as a nucleotide sequence for use in a guide nucleic acid as described herein. In some embodiments, compositions, systems and methods provided herein comprise a sequence listed in TABLE 1, TABLE 2 or any combination thereof; and further comprises one or more sequence modification or mutation. Sequence mutation(s) or modification(s) can include modification of one or more nucleic acid residues of a nucleotide sequence, such as chemical modification of one or more nucleobases; or chemical modifications to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to guide nucleic acid nucleotide sequence or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that, when transcribed, produces a guide nucleic acid). Methods of modifying a nucleic acid are known. One of ordinary skill in the art will appreciate that the sequence modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vv / ro-transcription. cloning, enzymatic, or chemical cleavage, etc. In some instances, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions within the nucleic acid.

[247] A person of ordinary skill in the field would recognize that the terms nucleotide(s) and / or nucleoside(s), when discussed in relation to a nucleic acid molecule containing multiple residues, are used interchangeably to refer to the sugar and base components of those residues. Likewise, a skilled practitioner would understand that linked nucleotides and / or linked nucleosides, as mentioned in the context of a nucleic acid with multiple linked residues, are also interchangeable terms that describe the linked sugars and bases present in the nucleic acid molecule. When discussing a nucleobase or linked nucleobase within a nucleic acid molecule, it can be interpreted as referring to the base component of the residue in the nucleic acid, such as the base of a nucleotide, nucleoside, or linked nucleotide or nucleoside. A person of ordinary skill in the art would also be aware of the distinctions between RNA and DNA, particularly the substitution of uridine for thymidine or vice versa. The presence of nucleoside analogs, like modified uridines, does not affect the identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have corresponding complements (for example, adenosine complements all forms of thymidine, uridine, or modified uridine; similarly, cytosine and 5-methylcytosine both have guanosine or modified guanosine as their complement). Thus, the sequence 5'-AXG, where X represents any modified uridine (such as pseudouridine, Nl-methyl pseudouridine, or 5-methoxyuridine), is considered 100% identical to AUG, as both sequences are perfectly complementary to the same sequence (5'-CAU). In some embodiments, the guide nucleic acid or a nucleic acid encoding the guide nucleic acid may include a sequence that is at least 70%, 80%, 85%, 90%, 95%, or even 100% identical to any of the nucleotide sequences listed in TABLE 2. Exemplary PAM sequences useful for effectors listed in TABLE 1 and variants thereof are disclosed in WO2022258753. It is understood that the guide nucleic acid, as well as any components therein (e.g., crRNA, spacer region, repeat region, etc.) may comprise may include deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more sequence modifications as described herein), or any combination thereof. In some embodiments, the guide nucleic acid can comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. Such elements can be one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.). Spacer Region In general, a guide nucleic acid comprises a spacer region that hybridizes to a target sequence of a target nucleic acid. The spacer region may comprise complementarity with (e.g., hybridize to) a target sequence of a target nucleic acid. The spacer sequence can function to direct the guide nucleic acid to the target nucleic acid for detection and / or modification. The spacer sequence can be designed (e.g., by genetic engineering) to hybridize to any desired target sequence (e.g., while taking the PAM into account) within the target nucleic acid. In some embodiments, a spacer region may function to direct an RNP complex comprising a guide nucleic acid to a target nucleic acid for detection and / or modification. The spacer region may function to direct the RNP complex to the target nucleic acid for detection and / or modification. A spacer region may be complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein described herein. In certain embodiments, the spacer region consists of linked nucleosides ranging from 15 to 28 in number. In other instances, the spacer region may contain 15 to 26,15 to 24,15 to 22,15 to 20, 15 to 18, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 17 to 26, 17 to 24, 17 to 22, 17 to 20, 17 to 18, 18 to 26, 18 to 24, or 18 to 22 linked nucleosides. Additionally, the spacer region could specifically be 18 to 24 linked nucleosides in length. In some cases, the spacer region is at least 15 linked nucleosides long. It may also be at least 16,18, 20, or 22 linked nucleosides in length. The spacer region can comprise a minimum of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, or 30 nucleotides. In certain embodiments, the spacer region has a minimum length of 17 linked nucleosides. It may also be at least 18 or 20 linked nucleosides long. In some cases, the spacer region exhibits at least 80%, 85%, 90%, 95%, or even 100% complementarity to a target sequence of the target nucleic acid. The term "% complementarity" refers to the percentage of nucleotides in two sequences of equal length that can form stable base pairs at two or more corresponding positions in an antiparallel manner. Consequently, nucleic acid sequences that lack complete complementarity throughout their entire length contain one or more mismatches, which can occur at any position among the opposing nucleotides that are not complementary. The percentage of complementarity is determined by dividing the total number of complementary residues by the total number of nucleotides in one of the equal-length sequences and multiplying the result by 100. A nucleotide sequence exhibiting complete or total complementarity means that 100% of its residues are complementary to those in a reference nucleotide sequence. In contrast, a partially complementary nucleotide sequence has at least 20%, but less than 100%, of its residues complementary to those in a reference sequence. In some instances, at least 50%, but less than 100%, of the residues of a nucleotide sequence are complementary to those in a reference nucleotide sequence. Additionally, there may be cases where at least 70%, 80%, 90%, or 95%, but less than 100%, of the residues of a nucleotide sequence are complementary to a reference sequence. Noncomplementary nucleotide sequences are defined as having less than 20% of their residues being complementary to those in a reference sequence. In some embodiments, the spacer region is completely complementary to the target sequence of the target nucleic acid. Additionally, in certain instances, the spacer region comprises at least 15 contiguous nucleotides that are complementary to the target nucleic acid, or at least 17 contiguous nucleotides that are complementary to the target nucleic acid. It is recognized that the sequence of a spacer region does not have to be 100% complementary to the target sequence of a target nucleic acid in order to hybridize or achieve specific hybridization to that target sequence. The guide nucleic acid may include at least one uracil within nucleic acid residues 5 to 20 of the spacer region, which is not complementary to the corresponding nucleoside in the target sequence. Additionally, the guide nucleic acid may contain at least one uracil located between nucleic acid residues 5 to 9, 10 to 14, or 15 to 20 of the spacer region that does not match the corresponding nucleoside of the target sequence. In some cases, the portion of the target nucleic acid that complements the spacer region may feature an epigenetic or post-transcriptional modification. Such modifications can include, but are not limited to, acetylation, methylation, or thiol modifications. Generally, a spacer sequence consists of a spacer region that hybridizes with a target sequence of a target nucleic acid. Therefore, in certain embodiments, the spacer sequence, or a part of it, is capable of hybridizing to a target sequence of a target nucleic acid. In some instances, the spacer sequence may be an RNA sequence that is at least 65%, 70%, 80%, 90%, 92%, 95%, 97%, 99%, or even 100% identical to any of SEQ. ID NO: 1-61 or 216-261. In general, a guide nucleic acid contains a repeat region that interacts with the effector protein. This repeat region may also be referred to as a "protein-binding segment" and is typically located adjacent to the spacer region. For instance, a guide RNA that engages with an effector protein includes a repeat region positioned 5' to the spacer region. In some instances, the repeat region precedes the spacer region in the 5' to 3' direction. The length of the repeat region may range from 15 to 50 nucleotides, with some embodiments specifying a length between 19 and 37 nucleotides. Additionally, the guide nucleic acid may contain more than one repeat region. In some embodiments, the guide nucleic acid consists of a first repeat sequence, followed by a spacer sequence and a second repeat sequence in the 5' to 3' direction. The first and second repeat sequences may be identical or different. The spacer and repeat sequences can be directly linked, or there may be a short linker of 1, 2, or 3 nucleotides between them. In some cases, the spacer sequence and the repeat sequences of the guide nucleic acid may be present in separate molecules that are linked through base pairing interactions. In certain embodiments, a repeat sequence is adjacent to an intermediary RNA, which may be positioned 3' to the repeat sequence. An intermediary RNA might be followed by a repeat sequence, which in turn is followed by a spacer sequence in the 5' to 3' direction. The repeat sequence can be linked to a spacer sequence and / or an intermediary RNA, either directly or through suitable linkers. In some embodiments, the protein-binding segment may consist of two complementary repeat sequences that hybridize to form a double-stranded RNA duplex (dsRNA duplex). This dsRNA duplex region may contain 5-25 base pairs (bp), and not all nucleotides in the duplex region need to be paired, allowing for the presence of bulges. The repeat region, which may include the dsRNA, can contain one or more bulges. Additionally, the repeat region may form a hairpin structure, particularly in the 3' portion, comprising a double-stranded stem and a single-stranded loop. In such cases, one strand of the stem may contain a sequence that is at least partially complementary to the other strand. Linker for Nucleic Acids In some embodiments, a guide nucleic acid for use in the compositions, systems, and methods described herein may include one or more linkers, or a nucleic acid encoding one or more linkers. The guide nucleic acid may contain at least one, two, three, four, five, six, seven, eight, nine, or ten linkers. It is also possible for the guide nucleic acid to comprise more than one linker, with at least two of these linkers being identical or different. A linker may consist of one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, a linker may have a nucleotide sequence of 5'-GAAA-3'. The guide nucleic acid may include one or more linkers that connect one or more repeat sequences. It may also comprise linkers connecting one or more repeat sequences to one or more spacer sequences, and in some cases, at least two repeat sequences may be connected by a linker. Intermediary RNA Guide nucleic acids described herein may include one or more intermediary RNAs. An intermediary RNA is generally a nucleotide sequence found in a handle sequence that can non-covalently bind to an effector protein, forming a complex (e.g., a ribonucleoprotein (RNP) complex). Typically, the intermediary RNA is not transactivated or transactivating. It may also be referred to as an intermediary sequence, which can contain deoxyribonucleotides in addition to ribonucleotides and / or modified bases. The intermediary RNA primarily binds non-covalently to an effector protein, and in some embodiments, it may form a secondary structure, such as in a cellular context, allowing the effector protein to bind to this structure. The length of the intermediary RNA can vary, with some embodiments indicating a minimum length of at least 30, 50, 70, 90,110,130,150, 170, 190, or 210 linked nucleotides. In other instances, it may be restricted to a maximum of 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides, with specific ranges such as approximately 30 to 210, 60 to 210, 90 to 210, 120 to 210, 150 to 210, 180 to 210, 30 to 180, 60 to 180, 90 to 180, 120 to 180, or 150 to 180 linked nucleotides. An intermediary RNA can also adopt a secondary structure, such as one or more hairpin loops, which may aid in the binding of an effector protein to a guide nucleic acid and / or enhance the modification activity of the effector protein on a target nucleic acid. The intermediary RNA typically consists of a 5' region, a hairpin region, and a 3' region, with the 5' region potentially hybridizing with the 3' region. In some cases, the 5' region may not hybridize to the 3' region. The hairpin region may comprise a first sequence and a second sequence that is reverse complementary to the first, connected by a stem-loop structure. The stem region may include 4 to 8 linked nucleotides, with particular lengths ranging from 5 to 6 or 4 to 5 linked nucleotides. The intermediary RNA may also feature a pseudoknot, which is a secondary structure that involves a stem partially hybridizing with another stem or half-stem structure. The effector protein may interact with an intermediary RNA that contains either a single stem region or multiple stem regions, with the nucleotide sequences of these regions being identical or different. In some embodiments, the terms "intermediary RNA" and "intermediary sequence" denote a nucleotide sequence within a handle sequence that can non-covalently bind to an effector protein to form a complex (e.g., a ribonucleoprotein (RNP) complex). An intermediary sequence is not considered a transactivating nucleic acid within the systems, methods, and compositions described herein. A Single Nucleic Acid System In certain embodiments, the compositions, systems, and methods described herein may incorporate a single nucleic acid system that comprises a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, along with one or more effector proteins or a nucleotide sequence encoding those effector proteins. Within this single nucleic acid system, a first region (FR) of the guide nucleic acid non-covalently interacts with the effector proteins. A second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. Notably, in this arrangement, the effector protein is not transactivated by the guide nucleic acid, indicating that the activity of the effector protein does not depend on binding to a second non-target nucleic acid molecule. Examples of guide nucleic acids suitable for this single nucleic acid system include crRNA or sgRNA. The guide nucleic acids and their components may be derived from a CRISPR array found within the genome of a host organism. A crRNA can be generated through the processing of a longer precursor CRISPR RNA (pre-crRNA) by cleavage within each direct repeat sequence, resulting in shorter, mature crRNAs. There are various mechanisms for generating crRNAs, including the action of specific endonucleases (e.g., Cas6 or Cas5d in Type I and III systems), the coupling of a host endonuclease (e.g., RNase III) with tracrRNA (Type II systems), or the inherent ribonuclease activity of the effector protein itself (e.g., Cpfl in Type V systems). Additionally, a crRNA can be produced independently of pre-crRNA processing and can be directly associated with an effector protein in vivo or in vitro. In some embodiments, a crRNA serves as a guide nucleic acid in a single nucleic acid system for the compositions, methods, and systems described herein. Within this context, a guide nucleic acid comprises a crRNA in which a repeat sequence is capable of linking the crRNA to an effector protein. In some cases, the guide nucleic acid may include a crRNA that is linked to another nucleotide sequence that can non-covalently bind to an effector protein. In these instances, the repeat sequence of the crRNA can be connected to an intermediary RNA. Thus, a single nucleic acid system may comprise a guide nucleic acid that includes a crRNA and an intermediary RNA. In general, a crRNA can comprise a spacer region that hybridizes to a target sequence of a target nucleic acid. In some embodiments, the crRNA of the guide nucleic acid comprises a repeat region and a spacer region, wherein the repeat region binds to the effector protein and the spacer region hybridizes to a target sequence of the target nucleic acid. In some embodiments, the compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g., a single nucleic acid system), wherein the guide RNA is a sgRNA. A sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, deoxyribonucleotides, ribonucleotides, chemically modified nucleotides, or any combination thereof. A sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and / or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). Such a sequence can be contained within a handle sequence as described herein. In some embodiments, a sgRNA comprises one or more of a handle sequence, an intermediary RNA, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof. For example, a sgRNA comprises a handle sequence and a spacer sequence; an intermediary RNA and an crRNA; an intermediary RNA, a repeat sequence and a spacer sequence; and the like. In some embodiments, a sgRNA comprises an intermediary RNA and an crRNA. In some embodiments, an intermediary RNA is 5' to a crRNA in an sgRNA. In some embodiments, a sgRNA comprises a linked intermediary RNA and crRNA. In some embodiments, an intermediary RNA and a crRNA are linked in an sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond) In some embodiments, an intermediary RNA and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein. In certain embodiments, a sgRNA consists of a handle sequence and a spacer sequence. In some cases, the handle sequence is positioned 5' to the spacer sequence within the sgRNA. The sgRNA may contain a linked handle sequence and spacer sequence, where these two components are directly connected (e.g., covalently linked, such as through a phosphodiester bond). Alternatively, the handle sequence and spacer sequence may be linked by any suitable linker, with examples provided herein. In some instances, a sgRNA may also include an intermediary RNA, a repeat sequence, and a spacer sequence. The intermediary RNA may be located 5' to the repeat sequence in the sgRNA. Additionally, the sgRNA may feature a linked intermediary RNA and repeat sequence, where these elements are directly connected (e.g., covalently linked via a phosphodiester bond) or linked through a suitable linker, as mentioned earlier. Furthermore, in some embodiments, a repeat sequence is positioned 5' to the spacer sequence within the sgRNA. The sgRNA may also include a linked repeat sequence and spacer sequence, which can be directly linked (e.g., covalently linked via a phosphodiester bond) or connected by a suitable linker, as described in the examples provided. A Dual Nucleic Acid System In certain embodiments, the compositions, systems, and methods described herein may involve a dual nucleic acid system that includes a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector proteins or a nucleotide sequence encoding those effector proteins. In this dual nucleic acid system, the crRNA and tracrRNA exist as separate, unlinked molecules. A repeat hybridization region of the tracrRNA is capable of hybridizing with an equal-length portion of the crRNA to form a tracrRNA-crRNA duplex. Notably, this equal-length portion of the crRNA does not contain the spacer sequence, which is instead capable of hybridizing to a target sequence within the target nucleic acid. In the dual nucleic acid system, where the complex consists of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA, meaning that the activity of the effector protein is dependent on its binding to the tracrRNA molecule. In some embodiments, the repeat hybridization sequence is located at the 3' end of the tracrRNA. The length of this repeat hybridization sequence may vary, with possible lengths of about 1, 2, 3, 4, 5, 6, 7, 8, 9,10,12,14,16,18, or 20 linked nucleotides. In some cases, the repeat hybridization sequence may range from 1 to 20 linked nucleotides in length. A tracrRNA and / or tracrRNA-crRNA duplex may adopt a secondary structure that enhances the binding of an effector protein to either the tracrRNA or the tracrRNA-crRNA complex. In certain embodiments, this secondary structure can modify the activity of the effector protein on a target nucleic acid. The secondary structure may consist of a stem-loop configuration, which includes both a stem region and a loop region. In some instances, the stem region may range from 4 to 8 linked nucleotides in length, with specific embodiments indicating that the stem region could be 5 to 6 linked nucleotides or 4 to 5 linked nucleotides in length. Additionally, the secondary structure may feature a pseudoknot, which is defined as a configuration where a stem is at least partially hybridized to a second stem or half-stem structure. An effector protein may interact with a secondary structure that contains multiple stem regions. In some cases, the nucleotide sequences of these multiple stem regions may be identical, while in other instances, at least one of the stem regions may have a different sequence. The secondary structure may include at least two, three, four, or five stem regions. Furthermore, it may also comprise one or more loops, with the number of loops potentially being at least one, two, three, four, or five. tracrRNA A tracrRNA and / or a tracrRNA-crRNA duplex may form a secondary structure that aids in the binding of an effector protein to either the tracrRNA or the tracrRNA-crRNA complex. In some instances, this secondary structure can modify the activity of the effector protein on a target nucleic acid. The secondary structure may consist of a stem-loop configuration that includes a stem region and a loop region. The stem region may vary in length, typically ranging from 4 to 8 linked nucleotides, with some embodiments specifying a length of 5 to 6 or 4 to 5 linked nucleotides. Additionally, the tracrRNA may feature a pseudoknot, which is a secondary structure that involves a stem partially hybridizing with another stem or half-stem structure. An effector protein may recognize a tracrRNA that contains multiple stem regions, which may either have identical nucleotide sequences or differ among them. In some embodiments, the tracrRNA may include at least two, three, four, or five stem regions. The length of the tracrRNA can vary, with some embodiments indicating that it should not exceed 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. Other embodiments may specify that the length of the tracrRNA is approximately 30 to 120 linked nucleotides, or more specifically between 50 and 105, 50 and 95, 50 and 73, 50 and 71, 50 and 68, or 50 and 56 linked nucleotides. In certain cases, the length of the tracrRNA may fall within 56 to 105 linked nucleotides, or even 40 to 60 nucleotides. An exemplary tracrRNA may include, from 5' to 3', a 5' region, a hairpin region, a repeat hybridization region, and a 3' region. In some instances, the 5' region may hybridize to the 3' region, while in other cases, it may not. The 3' region may be covalently linked to the crRNA (for instance, via a phosphodiester bond). Additionally, a tracrRNA may contain an un-hybridized region at its 3' end, which may range in length from about 1 to 20 linked nucleotides. In some embodiments, the composition that includes an effector protein and a guide RNA may not have a tracrRNA. In other instances, an effector protein may not require a tracrRNA to locate or cleave a target nucleic acid. The crRNA in the guide nucleic acid may consist of a repeat region and a spacer region, where the repeat region binds to the effector protein and the spacer region hybridizes to a target sequence within the target nucleic acid. The repeat sequence of the crRNA may interact with an effector protein, facilitating the formation of a ribonucleoprotein (RNP) complex. Furthermore, the repeat region may also be referred to as a "protein-binding segment," and it is typically adjacent to the spacer region. For instance, a guide RNA that interacts with an effector protein may include a repeat region positioned 5' to the spacer region.V. Modifications

[325] Effector proteins and nucleic acids (e.g., engineered guide nucleic acids) described herein can be further modified as described throughout and as further described herein. Examples are modifications of interest that do not alter primary sequence, including chemical derivatization of effector proteins, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of an effector protein during its synthesis and processing or in further processing steps; e.g. by exposing the effector protein to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. Modifications described herein may also encompass alterations to the effector proteins and / or engineered guide nucleic acids via any suitable methods, including molecular biological techniques and synthetic chemistry. These modifications aim to enhance resistance to proteolytic degradation, adjust target sequence specificity, optimize solubility, alter protein activity (such as transcriptional modulation or enzymatic activity), or make them more suitable for their intended applications. Analogs of these effector proteins may include those that incorporate residues other than the naturally occurring L-amino acids, such as D-amino acids or synthetic non-naturally occurring amino acids. D-amino acids can replace some or all of the amino acid residues. Additionally, modifications may involve the incorporation of non-naturally occurring unnatural amino acids. The specific sequence and method of preparation will depend on factors such as convenience, cost, required purity, and other considerations. Further modifications may involve the introduction of various functional groups to the effector proteins and / or engineered guide nucleic acids described herein. For instance, these groups can be added during the synthesis or expression of the effector protein, enabling linkage to other molecules or surfaces. For example, cysteine residues can be utilized to form thioethers, histidine residues can be used for binding to metal ion complexes, and carboxyl groups can facilitate the formation of amides or esters, while amino groups can also be employed to create amides. Moreover, modifications may include the alteration of nucleic acids described herein (e.g., engineered guide nucleic acids) to impart new or enhanced features, such as improved stability. Such modifications can involve base modifications, backbone modifications, sugar modifications, or combinations thereof, affecting one or more nucleotides, nucleosides, or nucleobases within the nucleic acid. In some embodiments, nucleic acids (e.g., engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2'0-methyl modified nucleotides, 2' Fluoro modified nucleotides; locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5' cap (e.g., a 7-methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage; phosphorothioate and / or heteroatom intemucleoside linkages, such as -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -C H2-O-N(C H ,)-CH2-. -CHj-NfCHs)-N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester intemucleotide linkage is represented as -0-P(=0)(0H)-0-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH2 component parts; and combinations thereof. Vectors and Multiplexed Expression Vectors The compositions, systems, and methods described herein may include a vector or its application. A vector can contain a nucleic acid of interest. In certain embodiments, the nucleic acid of interest encompasses one or more components of a composition or system outlined herein. This nucleic acid may consist of a nucleotide sequence that encodes one or more components of the described composition or system. Components may include effector proteins, guide nucleic acids, target nucleic acids, and donor nucleic acids. In some instances, the component may be a nucleic acid encoding an effector protein, a donor nucleic acid, and a guide nucleic acid or a nucleic acid that encodes the guide nucleic acid. The vector may be part of a vector system, which consists of a library of vectors, each designed to encode one or more components of the compositions or systems described herein. In certain embodiments, the components (including an effector protein, a guide nucleic acid, and / or a target nucleic acid) may all be encoded by a single vector. Alternatively, these components may be encoded by different vectors within the system. In some cases, a vector may include a nucleotide sequence that encodes one or more effector proteins as described herein. In specific embodiments, the one or more effector proteins may consist of at least two effector proteins. These effector proteins may be identical in some instances, or they may differ from one another in others. The nucleotide sequence within the vector is typically operably linked to a promoter that functions in a target cell, such as a eukaryotic cell. In some embodiments, the vector may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more effector proteins. In some embodiments, a fusion effector protein, as described herein, can be inserted into a vector. The vector may also contain one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and / or transcriptional terminator sequences.

[333] In some embodiments, a vector may encode one or more of any system components, including but not limited to effector proteins, guide nucleic acids, donor nucleic acids, and target nucleic acids as described herein. In some embodiments, a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector may encode 1, 2, 3, 4 or more of any system components. For example, a vector may encode two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence. A vector may encode an effector protein and a guide nucleic acid. A vector may encode an effector protein, a guide nucleic acid, and a donor nucleic acid. In some embodiments, a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein. In some embodiments, the one or more guide nucleic acids comprise at least two guide nucleic acids. In some embodiments, the at least two guide nucleic acids are the same. In some embodiments, the at least two guide nucleic acids are different from each other. In some embodiments, the guide nucleic acid or the nucleotide sequence encoding the guide nucleic acid is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids. In some embodiments, the vector comprises a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids. In some embodiments, a vector comprises one or more donor nucleic acids as described herein. In some embodiments, the one or more donor nucleic acids comprise at least two donor nucleic acids. In some embodiments, the at least two donor nucleic acids are the same. In some embodiments, the at least two donor nucleic acids are different from each other. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donor nucleic acids. In certain embodiments, a vector may include or encode one or more regulatory elements. These regulatory elements refer to sequences that control transcription and translation, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals. These elements facilitate and / or regulate the transcription of a non-coding sequence (e.g., a guide nucleic acid) or a coding sequence (e.g., effector proteins, fusion proteins, etc.), and they may also regulate the translation of an encoded effector protein. Additionally, a vector may comprise or encode one or more supplementary elements, including replication origins, antibiotic resistance genes (or nucleic acids encoding them), tags (or nucleic acids encoding them), selectable markers, and similar components. In some instances, a vector may also include elements such as ribosome binding sites and RNA splice sites. Vectors described herein can encode a promoter, which is a regulatory region on a nucleic acid, such as a DNA sequence, that is capable of initiating transcription of a downstream (3' direction) coding or non-coding sequence. A promoter can be linked at its 3' end to a nucleic acid whose expression or transcription is desired, extending upstream (5' direction) to encompass the bases or elements necessary for initiating transcription or inducing expression, which can be measured at a detectable level. The promoter comprises a nucleotide sequence, referred to as a "promoter sequence," which may include a transcription initiation site and one or more protein binding domains essential for the binding of transcription machinery, such as RNA polymerase. Eukaryotic promoters may contain elements like "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, can be employed to drive the expression, or transcriptional activation, of the nucleic acid of interest. Consequently, in some embodiments, the nucleic acid of interest may be operably linked to a promoter. Promotors may be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline -regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and / or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human HI promoter (HI). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 2 fold, 5 fold, 10 fold, 50 fold, by 100 fold, 500 fold, or by 1000 fold, or more. In addition, vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and / or a nucleic acid that encodes an effector protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and / or the effector protein. In general, the plasmids and vectors described herein contain at least one promoter. In some embodiments, these promoters are constitutive promoters, while in other instances, they are inducible promoters. Additionally, some embodiments may feature prokaryotic promoters, which drive the expression of a gene in a prokaryotic cell, whereas other embodiments may include eukaryotic promoters that facilitate gene expression in eukaryotic cells. Exemplary promoters include, but are not limited to, ApoE, TBG, CMV, EFla, SV40, PGK1, Ube, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIla, GALI-10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, CaMV35S, SV40, CMV, and HSV TK promoter. In some cases, the promoter may be CMV, EFla, ApoE, TBG, or ubiquitin. Furthermore, the vectors may be bicistronic or polycistronic, meaning they contain two or more loci that are responsible for generating proteins, and may include an internal ribosome entry site (IRES) for cap-independent translation initiation. In general, the vectors provided herein comprise at least one promoter or a combination of promoters that drive the expression or transcription of one or more genome editing tools described herein. In some embodiments, the vector includes a nucleotide sequence for a promoter, and in other instances, the vector may contain two or three promoters. The length of the promoter can vary, being less than about 500, 400, 300, or 200 linked nucleotides in some cases. Conversely, in other embodiments, the length of the promoter may be at least 100, 200, 300, 400, or 500 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ube, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIla, GALI-10, Hl, TEF1, GDS, ADH1, CaMV35S, HSVTK, Ubi, U6, MNDU3, MSCV, MND, and CAG.

[341] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter only drives expression of its corresponding coding sequence (e.g., effector protein or guide nucleic acid) when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline -inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence. In some embodiments, a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA. In certain embodiments, a vector described herein functions as a delivery vector. This delivery vector may be a eukaryotic vector, a prokaryotic vector (such as a bacterial vector), a viral vector, or any combination of these. In some cases, the delivery vehicle may be a non-viral vector. Additionally, it may take the form of a plasmid, which can comprise either DNA or RNA. Examples of plasmids include circular double-stranded DNA or linear forms. The plasmid may contain one or more genes of interest along with various regulatory elements. Typically, it includes a bacterial backbone that has an origin of replication and an antibiotic resistance gene or another selectable marker to facilitate plasmid amplification in bacteria. In some scenarios, the plasmid may be a minicircle plasmid. Additionally, the plasmid can contain genes that provide a selective marker to encourage target cells to retain the plasmid. Formulations for delivery may include preparations for injection using a syringe or for electroporation. Plasmids can also be engineered using synthetic methods or other suitable techniques known in the field. For instance, genetic elements may be assembled through restriction digestion of the desired genetic sequence from a donor plasmid or organism, creating ends that can be easily ligated to another genetic sequence. Generally, a guide nucleic acid includes a repeat region that interacts with the effector protein. This repeat region, sometimes referred to as a "protein-binding segment," is usually located adjacent to the spacer region. For instance, a guide RNA that interacts with an effector protein features a repeat region positioned 5' to the spacer region. In some cases, the repeat region is situated before the spacer region in the 5' to 3' direction. The length of the repeat region can vary, typically ranging from 15 to 50 nucleotides, with some embodiments specifying a length between 19 and 37 nucleotides. Moreover, the guide nucleic acid may contain multiple repeat regions. In specific embodiments, the guide nucleic acid may consist of a first repeat sequence, followed by a spacer sequence and a second repeat sequence in the 5' to 3' direction. The first and second repeat sequences may be identical or different from each other. The spacer and repeat sequences can be directly linked, or a short linker of 1, 2, or 3 nucleotides may be present between them. In some instances, the spacer sequence and the repeat sequences may exist in separate molecules that are connected through base pairing interactions. Additionally, a repeat sequence may be adjacent to an intermediary RNA, which could be positioned 3' to the repeat sequence. An intermediary RNA may be followed by a repeat sequence, which in turn is followed by a spacer sequence in the 5' to 3' direction. The repeat sequence can be linked to a spacer sequence and / or an intermediary RNA, either directly or through suitable linkers. In some instances, the protein-binding segment may consist of two complementary repeat sequences that hybridize to form a double-stranded RNA duplex (dsRNA duplex). This dsRNA duplex region may contain 5-25 base pairs (bp), and it is not necessary for all nucleotides in this region to be paired, allowing for the formation of bulges. The repeat region, which may include the dsRNA, can feature one or more bulges. Additionally, the repeat region may form a hairpin structure, particularly in the 3' portion, consisting of a double-stranded stem and a single-stranded loop. In such cases, one strand of the stem may have a sequence that is at least partially complementary to the other strand. Linker for Nucleic Acids In certain embodiments, a guide nucleic acid utilized in the compositions, systems, and methods described herein may incorporate one or more linkers, or a nucleic acid that encodes one or more linkers. The guide nucleic acid may contain at least one, two, three, four, five, six, seven, eight, nine, or ten linkers. It is also feasible for the guide nucleic acid to include multiple linkers, with at least two of these linkers being either identical or different. A linker may consist of one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some instances, a linker may have a nucleotide sequence of 5'-GAAA-3'. The guide nucleic acid may feature one or more linkers that connect various repeat sequences. It may also include linkers that connect one or more repeat sequences to one or more spacer sequences, and in some cases, at least two repeat sequences may be linked by a linker. Intermediary RNA Guide nucleic acids described herein may include one or more intermediary RNAs. An intermediary RNA is generally a nucleotide sequence found in a handle sequence that can non-covalently bind to an effector protein, forming a complex (e.g., a ribonucleoprotein (RNP) complex). Typically, the intermediary RNA is not transactivated or transactivating. It may also be referred to as an intermediary sequence, which can contain deoxyribonucleotides in addition to ribonucleotides and / or modified bases. The intermediary RNA primarily binds non-covalently to an effector protein, and in some embodiments, it may form a secondary structure, such as in a cellular context, allowing the effector protein to bind to this structure. The length of the intermediary RNA may vary, with some embodiments specifying a length of at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In other cases, it may be limited to no more than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides, with specific length ranges such as about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides. An intermediary RNA may also form a secondary structure, such as one or more hairpin loops, which can facilitate the binding of an effector protein to a guide nucleic acid and / or enhance the modification activity of the effector protein on a target nucleic acid. The intermediary RNA may consist of a 5' region, a hairpin region, and a 3' region, with the 5' region potentially hybridizing to the 3' region. In some embodiments, the 5' region does not hybridize to the 3' region. The hairpin region may include a first sequence and a second sequence that is reverse complementary to the first, connected by a stem-loop structure. The stem region may consist of 4 to 8 linked nucleotides, with specific lengths ranging from 5 to 6 or 4 to 5 linked nucleotides. The intermediary RNA may also include a pseudoknot, which is a secondary structure involving a stem that partially hybridizes with another stem or half-stem structure. The effector protein may interact with an intermediary RNA that has a single stem region or multiple stem regions, with the nucleotide sequences of these regions being identical or different. In some embodiments, the terms "intermediary RNA" and "intermediary sequence" refer to a nucleotide sequence in a handle sequence that can non-covalently bind to an effector protein to form a complex (e.g., an RNP complex). An intermediary sequence is not a transactivating nucleic acid in the systems, methods, and compositions described herein. A Single Nucleic Acid System In certain embodiments, the compositions, systems, and methods described herein may incorporate a single nucleic acid system that consists of a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, along with one or more effector proteins or a nucleotide sequence encoding those effector proteins. Within this single nucleic acid system, a first region (FR) of the guide nucleic acid interacts non-covalently with the effector proteins. A second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In this configuration, the effector protein is not transactivated by the guide nucleic acid, indicating that the activity of the effector protein does not depend on binding to a second non-target nucleic acid molecule. Examples of guide nucleic acids suitable for this single nucleic acid system include crRNA or sgRNA. The guide nucleic acids and their components may be derived from a CRISPR array found within the genome of a host organism. A crRNA can be generated through the processing of a longer precursor CRISPR RNA (pre-crRNA) by cleavage at each direct repeat sequence, resulting in shorter, mature crRNAs. Various mechanisms can produce crRNAs, including those involving dedicated endonucleases (such as Cas6 or Cas5d in Type I and III systems), the coupling of a host endonuclease (for example, RNase III) with tracrRNA (in Type II systems), or the inherent ribonuclease activity of the effector protein itself (e.g., Cpfl in Type V systems). Furthermore, a crRNA can also be generated independently of pre-crRNA processing and can be directly associated with an effector protein in vivo or in vitro. In some instances, a crRNA functions as a guide nucleic acid within the single nucleic acid system for the compositions, methods, and systems described herein. In this context, the guide nucleic acid includes a crRNA in which a repeat sequence facilitates the linkage between the crRNA and an effector protein. In certain cases, the guide nucleic acid may consist of a crRNA that is connected to another nucleotide sequence capable of non-covalently binding to an effector protein. In these scenarios, the repeat sequence of the crRNA may be linked to an intermediary RNA. Therefore, a single nucleic acid system may encompass a guide nucleic acid that consists of a crRNA and an intermediary RNA. The methods, systems, and compositions described herein are capable of editing or modifying a target nucleic acid, with such editing or modification being quantifiable through indel activity. Indel activity refers to the extent of change in a target nucleic acid (e.g., nucleotide deletions and / or insertions) when compared to a target nucleic acid that has not been exposed to a polypeptide as described in the compositions, systems, and methods herein. For instance, indel activity can be assessed using next-generation sequencing of one or more target loci within the target nucleic acid, where the indel percentage is calculated as the ratio of sequencing reads that contain insertions or deletions in relation to an unedited reference sequence. In certain embodiments, methods, systems, and compositions that include an effector protein and a guide nucleic acid can demonstrate indel activity ranging from approximately 0.0001% to about 65% or higher when in contact with a target nucleic acid, compared to a target nucleic acid that has not been treated with the described compositions, systems, or methods. For example, such methods, systems, and compositions may exhibit indel activity levels of about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or even higher. Compositions The compositions disclosed herein include one or more effector proteins or nucleic acids that encode these effector proteins, as well as one or more guide nucleic acids or nucleic acids encoding the guide nucleic acids, or combinations of these components. In certain embodiments, the effector protein may be any protein exhibiting nuclease activity that can recognize any of the PAM sequences outlined herein. Exemplary effector proteins are detailed throughout the specification. In some cases, one or more repeat sequences within the guide nucleic acids can interact with the effector proteins. Additionally, the spacer sequences of the guide nucleic acids may hybridize with a target sequence of the target nucleic acid. The compositions may also include one or more donor nucleic acids as described herein. These compositions are capable of editing a target nucleic acid within a cell or subject. They may facilitate the editing of a target nucleic acid or its expression in a cell, tissue, organ, or in vitro, in vivo, or ex vivo. Furthermore, the compositions can edit a target nucleic acid found in a sample containing the target nucleic acid. In some embodiments, the compositions may consist of plasmids, viral vectors, non-viral vectors, or combinations thereof. Some embodiments specifically include viral vectors, and in particular, may comprise adeno-associated viruses (AAVs). Additionally, the compositions may include liposomes (such as cationic or neutral lipids), dendrimers, lipid nanoparticles (LNPs), or cell-penetrating peptides. In certain cases, the compositions may specifically include an LNP. In some embodiments, the compositions are utilized for editing the human albumin gene. The editing may result in the expression of at least a partially functional acid alpha-glucosidase. In certain instances, this editing may lead to a partial or complete cure of acid alpha-glucosidase deficiency. Pharmaceutical Compositions and Modes of Administration In some embodiments, compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein and a pharmaceutically acceptable carrier or diluent. Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.

[389] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, fusion effector proteins, guide nucleic acids, or donor nucleic acid as described herein and any combination thereof. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo, or ex vivo. In certain embodiments, pharmaceutical compositions may include one or more nucleic acids that encode an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination of these components, along with a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or their combinations may be any of those described herein. The nucleic acids may take the form of a plasmid, a nucleic acid expression vector, or a viral vector. In some instances, the compositions—particularly pharmaceutical compositions—may consist of a viral vector that encodes a fusion effector protein and a guide nucleic acid, where at least a portion of the guide nucleic acid binds to the effector protein within the fusion effector protein. The vector may be formulated for delivery via injection using a syringe. Other formulations may include delivery by electroporation or through chemical methods. The pharmaceutical compositions may comprise either a viral vector or a non-viral vector. In some cases, the pharmaceutical compositions may contain a virus comprising a viral vector that encodes a fusion effector protein, an effector protein, a fusion partner, a guide nucleic acid, or a combination of these elements, along with a pharmaceutically acceptable carrier or diluent. Pharmaceutical compositions described herein may also include a salt. In some embodiments, the salt may be a sodium salt, while in other embodiments, it may be a potassium salt or a magnesium salt. Specific examples of salts include NaCI, KNO3, and Mg2+ SO4. In some embodiments, pharmaceutical compositions are in the form of a solution (e.g., a liquid). In some embodiments, the solution may be formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7. Systems Disclosed herein, in certain aspects, are systems designed for modifying or editing a target nucleic acid, which include the effector proteins or nucleic acids encoding these effector proteins, or a multimeric complex thereof. These systems can be utilized to alter or edit a target nucleic acid, as well as to insert a donor nucleic acid into a target nucleic acid. In some embodiments, the systems consist of an effector protein or a nucleic acid encoding the effector protein described herein, along with a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, a reagent as described herein, a donor nucleic acid, a support medium, or any combination of these elements. In certain instances, the effector protein may be an effector protein or a fusion protein as described herein. In some embodiments, systems comprise an effector protein described herein, a guide nucleic acid described herein, a reagent, support medium, or a combination thereof. In some embodiments, the effector protein comprises an effector protein, or a fusion protein thereof, described herein. In some embodiments, effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences recited in TABLE 1. In some embodiments, the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences recited in TABLE 1 In certain embodiments, the systems described herein may consist of separate compositions, solutions, containers, kits, vectors, or similar items, each containing an effector protein, a nucleic acid encoding the effector protein, a guide nucleic acid, a nucleic acid encoding the guide nucleic acid, a donor nucleic acid, or a combination of these elements. These systems facilitate the individual delivery of the effector protein, the nucleic acid encoding the effector protein, the guide nucleic acid, the nucleic acid encoding the guide nucleic acid, or the donor nucleic acid as described herein. Additionally, in some embodiments, the systems may comprise a composition, solution, container, kit, vector, or similar entity that includes two or more of the following: an effector protein, a nucleic acid encoding the effector protein, a guide nucleic acid, a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid. Such systems enable the delivery of multiple components, including the effector protein, the nucleic acid encoding the effector protein, the guide nucleic acid, the nucleic acid encoding the guide nucleic acid, and the donor nucleic acid. Additional System Components In some embodiments, systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass, plastic, or polymers. The system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use. A system may include labels that detail the contents and / or provide instructions for use, along with package inserts containing usage guidelines. Typically, a set of instructions will be included as well. In one embodiment, a label is affixed to or associated with the container. A label is considered to be on a container when letters, numbers, or other characters are attached, molded, or etched directly into the container itself; it is deemed associated with a container when it is included within a receptacle or carrier that also holds the container, such as a package insert. In one embodiment, a label may indicate that the contents are intended for a specific therapeutic application. The label may also provide directions for using the contents, as outlined in the methods described herein. After the product is packaged and wrapped or boxed to ensure a sterile barrier, it may undergo terminal sterilization through methods such as heat sterilization, gas sterilization, gamma irradiation, or electron beam sterilization. Alternatively, the product might be prepared and packaged using aseptic processing. Amplification Reagents / Components In some embodiments, systems described herein comprise a reagent or component for amplifying a nucleic acid. Non-limiting examples of reagents for amplifying a nucleic acid include polymerases, primers, and nucleotides. In some embodiments, systems comprise reagents for nucleic acid amplification of a target nucleic acid in a sample. Nucleic acid amplification of the target nucleic acid may improve at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. In some embodiments, nucleic acid amplification is isothermal nucleic acid amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification. In some embodiments, amplification of the target nucleic acid increases the concentration of the target nucleic acid in the sample relative to the concentration of nucleic acids that do not correspond to the target nucleic acid. The reagents for nucleic acid amplification may comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, a polymerase, or a combination thereof that is suitable for an amplification reaction. Non-limiting examples of amplification reactions are transcription mediated amplification (TMA), helicase dependent amplification (HDA), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA). Certain System Conditions Certain conditions that may enhance the activity of an effector protein include a certain salt presence or salt concentration of the solution in which the activity occurs. For example, cis-cleavage activity of an effector protein may be inhibited or halted by a high salt concentration. The salt may be a sodium salt, a potassium salt, or a magnesium salt. In some embodiments, the salt is NaCI. In some embodiments, the salt is KNOB. In some embodiments, the salt concentration is less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM.

[407] Certain conditions that may enhance the activity of an effector protein include the pH of a solution in which the activity. For example, increasing pH may enhance trans cleavage activity. For example, the rate of trans cleavage activity may increase with increase in pH up to pH 9. In some embodiments, the pH is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH is less than 7. In some embodiments, the pH is greater than 7. Certain conditions that may enhance the activity of an effector protein include the temperature at which the activity is conducted. In some embodiments, this temperature ranges from about 25°C to about 50°C. Other embodiments may specify temperatures of about 20°C to about 40°C, about 30°C to about 50°C, or about 40°C to about 60°C. Additionally, the temperature may be set at about 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C. Methods and Formulations for Introducing Systems and Compositions into a Target Cell A guide nucleic acid (or a nucleic acid containing a nucleotide sequence encoding it) and / or an effector protein as described herein can be introduced into a host cell using various established methods. For example, a guide nucleic acid and / or effector protein may be combined with a lipid. Alternatively, these components may be formulated into or combined with a particle. The methods for introducing various components to a host are described herein. A host can refer to any suitable entity, such as a host cell. When mentioned, a host cell may be an in vivo or in vitro eukaryotic cell, a prokaryotic cell (such as bacterial or archaeal), or a cell from a multicellular organism (for instance, a cell line) cultured as a unicellular entity. These eukaryotic or prokaryotic cells can serve as recipients for the introduction methods described herein and may include progeny of the original cell that has been transformed by these methods. It is understood that the progeny of a single cell may not be completely identical in morphology or genomic content to the original parent due to natural, accidental, or intentional mutations. A host cell can be classified as a recombinant host cell or a genetically modified host cell if a heterologous nucleic acid, such as an expression vector, has been introduced into the cell. [4H] Methods of introducing a nucleic acid and / or protein into a host cell are known in the art, and any convenient method may be used to introduce a subject nucleic acid (e.g., an expression construct / vector) into a target cell (e.g., a human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle -mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023), and the like. In some embodiments, the nucleic acid and / or protein are introduced into a disease cell comprised in a pharmaceutical composition comprising the guide nucleic acid and / or effector protein and a pharmaceutically acceptable excipient. In certain embodiments, molecules of interest, such as nucleic acids, are introduced into a host. This may include the introduction of effector proteins to the host as well. Additionally, vectors, such as lipid particles and / or viral vectors, can also be introduced to the host. The introduction may be intended for contact with the host or for assimilation into the host, such as introduction into a host cell. In some embodiments, methods are described for introducing one or more nucleic acids, including a nucleic acid that encodes an effector protein, a nucleic acid that, upon transcription, produces an engineered guide nucleic acid, and / or a donor nucleic acid, or combinations of these, into a host cell. Various suitable methods can be employed to introduce a nucleic acid into a cell. Examples of these methods include viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and others. Additional methods are detailed throughout the text.

[414] Introducing one or more nucleic acids into a host cell may occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing one or more nucleic acids into a host cell may be carried out in vivo or ex vivo. Introducing one or more nucleic acids into a host cell may be carried out in vitro. In certain embodiments, an effector protein may be delivered in the form of RNA. This RNA can be produced through direct chemical synthesis or transcribed in vitro from a DNA sequence that encodes the effector protein. After synthesis, the RNA can be introduced into a cell using various techniques suitable for nucleic acid delivery (e.g., microinjection, electroporation, transfection, etc.). In some instances, the introduction of one or more nucleic acids may involve the use of a vector and / or a vector system. Therefore, in some embodiments, the compositions and systems described herein may include a vector and / or a vector system. Vectors can be introduced directly into a host. In some cases, host cells may be treated with one or more of the described vectors, and in certain instances, these vectors may be taken up by the cells. Methods for delivering vectors to cells include, but are not limited to, electroporation, calcium chloride transfection, microinjection, lipofection, and direct contact with the cells or particles that contain the molecules of interest. Components described herein can also be introduced directly into a host. For example, an engineered guide nucleic acid can be delivered to a host, specifically into a host cell. Methods for introducing nucleic acids, such as RNA, into cells include, but are not limited to, direct injection, transfection, or any other method suitable for nucleic acid delivery. Effector proteins described herein may also be introduced directly to a host. In some embodiments, these effector proteins may be modified to facilitate their introduction into a host. For instance, modifications may be made to enhance the solubility of the effector proteins. Such modifications may involve fusing the effector protein to a polypeptide domain that increases solubility. This domain can be linked to the effector protein via a defined protease cleavage site, such as a TEV sequence that is cleaved by TEV protease. The linker may also contain one or more flexible sequences, such as 1 to 10 glycine residues. In some cases, cleavage of the polypeptide is performed in a buffer that maintains the solubility of the product, for example, in the presence of 0.5 to 2 M urea, or in the presence of polypeptides and / or polynucleotides that enhance solubility. Domains of interest may include endosomolytic domains, such as the influenza HA domain, as well as other polypeptides that assist in production, like the IF2 domain, GST domain, GRPE domain, and others. Additionally, effector proteins may be modified to improve their stability. For example, PEGylation of effector proteins can enhance their lifespan in the bloodstream due to the presence of polyethylene glycol groups. Effector proteins may also be modified to facilitate uptake by a host, such as a host cell. For instance, an effector protein may be fused to a cell-penetrating peptide to promote cellular uptake. Various suitable permeant domains can be utilized in the non-integrating polypeptides described herein, including peptides, peptidomimetics, and non-peptide carriers. Examples include penetratin, derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapaedia; the HIV-1 tat basic region amino acid sequence (e.g., amino acids 49-57 of the naturally occurring tat protein); and poly-arginine motifs, such as the region of amino acids 34-56 of the HIV-1 rev protein, nonaarginine, octa-arginine, and similar sequences. The site of fusion may be chosen to optimize the biological activity, secretion, or binding characteristics of the effector protein, with the optimal site determined through appropriate methods. The formulations described herein are designed for introducing the systems and compositions to a host. In certain embodiments, these formulations, systems, and compositions may include an effector protein along with a carrier (such as an excipient, diluent, vehicle, or filling agent). In some aspects of the present invention, the effector protein is incorporated into a pharmaceutical composition that consists of the effector protein and any pharmaceutically acceptable excipient, carrier, or diluent. A pharmaceutically acceptable excipient, carrier, or diluent refers to any substance that is formulated alongside the active ingredient in a pharmaceutical composition, allowing the active ingredient to maintain its biological activity while being non-reactive with the subject's immune system. These substances can serve various purposes, including long-term stabilization, bulking solid formulations that contain potent active ingredients in minimal amounts, or enhancing the therapeutic properties of the active ingredient in the final dosage form. This enhancement may involve improving absorption, reducing viscosity, or increasing solubility. The choice of suitable substances may depend on factors such as the route of administration, dosage form, active ingredient, and other considerations. Compositions containing these substances can be formulated using established methods. Gene of Interest (GOI) / Donor nucleic acid / Donor template The systems or compositions according to the invention are useful for the treatment of a wide variety of diseases by incorporating a specific GOI into the Albumin locus. The following provides an illustrative and non-exhaustive list of GOIs and the respective diseases that can be potentially treated with the systems according to the invention. a) Factor IX (F9) - Useful in the treatment of Hemophilia B b) Factor VIII (F8) - Useful in the treatment of Hemophilia A. c) Alpha-1 Antitrypsin (SERPINA1) - Useful in the treatment of Alpha-1 Antitrypsin Deficiency. d) Glucocerebrosidase (GBA) - Useful in the treatment of Gaucher disease. e) Phenylalanine hydroxylase (PAH) - Useful in the treatment of Phenylketonuria (PKU). f) Urokinase-type plasminogen activator (PLAU) - Potentially useful in thrombolytic therapy g) Lipoprotein lipase (LPL) - Useful in the treatment of hyperlipoproteinemia. h) Cl Esterase Inhibitor (SERPING1) - Useful in the treatment of Hereditary Angioedema. i) Sphingomyelinase (SMPD1) - Useful in the treatment of Niemann-Pick disease. j) N-acetylglucosamine-l-phosphate transferase (GNPTAB) - Useful in the treatment of Mucolipidosis II (l-Cell disease). k) Cystic fibrosis transmembrane conductance regulator (CFTR) - Useful in the treatment of Cystic Fibrosis. I) Dipeptidyl peptidase IV (DPP4) - Potentially useful in diabetes treatment. m) Adenosine deaminase (ADA) - Useful in the treatment of Severe Combined Immunodeficiency (SCID). m) Ornithine transcarbamylase (OTC) - Useful in the treatment of Ornithine Transcarbamylase Deficiency. n) Beta-glucuronidase (GUSB) - Useful in the treatment of Mucopolysaccharidosis VII (MPS VII). o) Cystathionine beta-synthase (CBS) - Useful in the treatment of Homocystinuria. p) Sodium-dependent glucose transporter 1 (SGLT1) - Useful in the treatment of GlucoseGalactose Malabsorption. q) Fumarase (FH) - Useful in the treatment of Fumarase Deficiency. r) Arginase (ARG1) - Useful in the treatment of Arginase Deficiency. s) Glutamate decarboxylase (GADI) - Useful in the treatment of Autoimmune Encephalitis. t) Acid alpha-glucosidase (GAA) - Useful in the treatment of Pompe disease. u) Hexosaminidase A (HEXA) - Useful in the treatment of Tay-Sachs disease. v) UDP-glucuronosyltransferase 1A1 (UGT1A1) - Useful in the treatment of Crigler-Najjar syndrome. w) Sulfatase (ARSB) - Useful in the treatment of Maroteaux-Lamy syndrome. x) Epidermal growth factor receptor (EGFR) - Useful in targeted therapy for various cancers. y) BRCA1 (BRCA1) - Useful in the treatment of hereditary breast and ovarian cancer syndromes, z) TP53 (TP53) - Useful in targeted therapies for cancers associated with p53 mutations. aa) Cystic fibrosis transmembrane conductance regulator (CFTR) - Useful in the treatment of Cystic Fibrosis. bb) Nerve growth factor (NGF) - Potentially useful in neurodegenerative diseases. cc) Insulin (INS) - Useful in the treatment of Diabetes Mellitus. dd) Cystathionine gamma-lyase (CTH) - Useful in the treatment of Cystathionine Beta-Synthase Deficiency. In one aspect the compositions and methods described herein comprise a donor nucleic acid encoding acid alphaglucosidase enzyme, and methods described herein comprise inserting a donor nucleic acid encoding acid alpha-glucosidase enzyme into intron 1 of a human albumin gene. In some embodiments, the donor nucleic acid is a cDNA encoding acid alpha-glucosidase enzyme. The term "GAA". as used herein, refers to a gene encoding acid alpha glucosidase enzyme. The human GAA gene is located at chromosome 17q25.2-q25.3. An exemplary amino acid sequence of human acid alpha glucosidase enzyme encoded by the human GAA gene, UniProtKB protein P10253 (LYAG_HUMAN) as accessed on 2024-12-17. In some embodiments, methods comprise inserting a donor nucleic acid encoding acid alphaglucosidase enzyme into intron 1 of a human albumin gene in a human cell. In some instances, the human cell comprises a GAA gene. In some embodiments, GAA gene comprise a mutation. In some embodiments, a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof. A point mutation optionally comprises a substitution, insertion, or deletion. In some embodiments, a mutation comprises a chromosomal mutation. A chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation. In some embodiments, a mutation comprises a copy number variation. A copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. The mutation may be located in a non-coding region or a coding region of a gene. In some embodiments, GAA gene comprise a mutation, wherein the mutation is a SNP. The single nucleotide mutation or SNP may be associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. The SNP, in some embodiments, is associated with altered phenotype from wild type phenotype. The SNP may be a synonymous substitution or a nonsynonymous substitution. The nonsynonymous substitution may be a missense substitution, or a nonsense point mutation. The synonymous substitution may be a silent substitution. The mutation may be a deletion of one or more nucleotides. Often, the single nucleotide mutation, SNP, or deletion is associated with a disease such as a genetic disorder. The mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell. In some embodiments, a diseased cell is a cell comprising pathway conditions or pathway systems that are not conducive to cell survival, tissue survival, systemic survival, or organism survival. In some embodiments, GAA gene comprise a mutation, wherein the mutation is a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. The mutation may be a deletion of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. The mutation may be a deletion of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides. In some embodiments, the GAA gene comprises a mutation associated with disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, or pathological state. In some examples, a mutation associated with a disease, disorder or pathological state refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder or pathological state. A mutation associated with a disease may also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. The mutation may cause a disease. The disease may comprise, at least in part, an inherited disorder. In some embodiments, the disease, disorder or pathological state comprises a hereditary condition. The disease may comprise, at least in part, an inherited disorder. The disease may comprise, at least in part, a glycogen storage disorder. In some embodiments, the glycogen storage disorder is a Pompe disease. Pompe disease is a genetic metabolic disorder that is related to acid alpha glucosidase deficiencies. Pompe disease is an autosomal recessive condition. Acid alpha glucosidase enzyme helps digesting glycogen in lysosomal. However, an individual, who is suffering from Pompe disease, cannot digest glycogen resulting in excessive accumulation of glycogen. The condition impairs the ability of Pompe patient to function normally by causing irreversible damage to muscle. Accordingly, in some embodiments, one or more mutations or aberrant expression of an acid alpha glucosidase protein is associated with Pompe disease. The mutations associated with Pompe disease can be a point mutation, a single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof. Mutations in GAA gene can be associated with acid alpha glucosidase enzyme expression, acid alpha glucosidase enzymic activity, and acid alpha glucosidase structural stability. As a result, the mutation may cause reduced expression of acid alpha glucosidase, reduced or no acid alpha glucosidase enzymic activity, reduced half-life of acid alpha glucosidase enzyme, increased lysosomal glycogen concentration, or combinations thereof. Alternatively, mutations in a region responsible for GAA gene expression may result in abnormal or low expression of acid alpha glucosidase enzyme. Disclosed herein are compositions, systems, methods for expressing acid alpha glucosidase, and method of treating acid alpha glucosidase deficiencies. Accordingly, in some embodiments, compositions, systems, and methods discloses herein relates to inserting a donor nucleic acid into a cleaved target nucleic acid, wherein the donor nucleic acid comprises a nucleotide sequence that encodes the acid alpha glucosidase enzyme. The donor nucleic acid may be inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid. In some embodiments, the donor nucleic acid may be inserted within, directly adjacent to, directly adjacent to, about one to twenty nucleotides adjacent to, about one to ten nucleotides adjacent to, about one to five nucleotides adjacent to, about five to twenty nucleotides adjacent to, about five to ten nucleotides adjacent to, or about ten to twenty nucleotides adjacent to the target sequence. In some embodiments, the donor nucleic acid encodes an amino acid sequence of a functional acid alpha glucosidase. In some embodiments, the functional acid alpha glucosidase has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 180%, at least 200%, at least 300%, at least 400% enzymatic activity compared to wildtype acid alpha glucosidase. In some embodiments, the functional acid alpha glucosidase comprises wildtype acid alpha glucosidase. In some embodiments, the wildtype acid alpha glucosidase comprises human wildtype acid alpha glucosidase amino acid sequence. In some embodiments, the donor nucleic acid encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to the wildtype sequence. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein comprising an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1, thereby introducing a single -stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein, optionally comprising an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences recited in TABLE 1, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally via HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site or in between two cleavage sites). XI11. Methods of Nucleic Acid Editing Provided herein are methods of editing target nucleic acids. In general, editing refers to modifying the nucleotide sequence of a target nucleic acid. However, compositions and systems disclosed herein may also be capable of making epigenetic modifications of target nucleic acids. Effector proteins, multimeric complexes thereof and systems described herein may be used for editing or modifying a target nucleic acid. Editing a target nucleic acid may comprise one or more of cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or modifying (e.g, methylating, demethylating, deaminating, or oxidizing) of one or more nucleotides of the target nucleic acid. Methods of editing may comprise contacting a target nucleic acid with an effector protein described herein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1. Editing may introduce a mutation (e.g., point mutations, insertions, deletions) in a target nucleic acid relative to a corresponding wildtype nucleotide sequence. Editing may remove or insert a nucleic acid sequence to produce a corresponding wildtype protein. Editing may remove / insert tissue specific nucleic acid sequence in a target nucleic acid. Editing may be used to generate gene knock-in, gene editing, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a cell. Editing may comprise single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof. In some embodiments, cleavage (single -stranded or double -stranded) is site specific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer region. In some cases, the cleavage occurs directly adjacent to, or about one to ten nucleotides adjacent to the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer region. In some cases, the effector proteins introduce a single -stranded break in a target nucleic acid to produce a cleaved nucleic acid. In some cases, the effector protein is capable of introducing a break in a single stranded RNA (ssRNA). The effector protein may be coupled to a guide nucleic acid that targets a particular region of interest in the ssRNA. In some embodiments, the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g., homology directed repair (HDR)) or non-homologous end joining (NHEJ). In some cases, a double-stranded break in the target nucleic acid may be repaired (e g., by NHEJ or HDR) without insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the double-stranded break.

[425] In some embodiments, an indel, sometimes referred to as an insertion-deletion or indel mutation, is a type of genetic mutation that results from the insertion and / or deletion of nucleotides in a target nucleic acid. An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including sequencing. If the number of nucleotides in the insertion / deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation. In some embodiments, an indel percentage is based on a percentage of sequencing reads that show at least one nucleotide has been edited from the insertion and / or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides edited. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given effector protein.

[427] In some embodiments, wherein the compositions, systems, and methods of the present disclosure comprise an additional guide nucleic acid or a use thereof, the dual-guided compositions, systems, and methods described herein can modify the target nucleic acid in two locations. In some cases, dual-guided editing can comprise cleavage of the target nucleic acid in the two locations targeted by the guide RNAs. In certain embodiments, upon removal of the sequence between the guide nucleic acids, a new nucleotide sequence can be inserted. Accordingly, in some embodiments, compositions, systems, and methods described herein can edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid. In certain embodiments, 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides can be edited by the compositions, systems, and methods described herein. In some embodiments, 10, 20, 30, 40, 50, 60, 70, 80 90, 100 or more nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein. In some embodiments, 100, 200, 300,400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between, can be edited by the compositions, systems, and methods described herein. Methods may comprise use of two or more effector proteins. An illustrative method for introducing a break in a target nucleic acid comprises contacting the target nucleic acid with: (a) a first engineered guide nucleic acid comprising a region that binds to a first effector protein, wherein the effector protein comprises a sequence that is at least 75% identical to any one of the amino acid sequences recited in TABLE 1; and (b) a second engineered guide nucleic acid comprising a region that binds to a second effector protein, wherein the effector protein comprises a sequence that is at least 75% identical to any one of the amino acid sequences recited in TABLE 1, wherein the first engineered guide nucleic acid comprises an additional region that binds to the target nucleic acid and wherein the second engineered guide nucleic acid comprises an additional region that binds to the target nucleic acid.

[430] In some embodiments, editing a target nucleic acid comprises genome editing. Genome editing may comprise modifying a genome, chromosome, plasmid, or other genetic material of a cell or organism. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vivo. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in a cell. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vitro. For example, a plasmid may be modified in vitro using a composition described herein and introduced into a cell or organism. In some embodiments, modifying a target nucleic acid may comprise deleting a sequence from a target nucleic acid. In some embodiments, modifying a target nucleic acid may comprise replacing a sequence in a target nucleic acid with a second sequence. In some embodiments, modifying a target nucleic acid may comprise introducing a sequence into a target nucleic acid. In some embodiments, methods comprise editing a target nucleic acid with two or more effector proteins. Editing a target nucleic acid may comprise introducing a two or more single stranded breaks in a target nucleic acid. In some embodiments, a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid. The guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting the effector protein to the region of the target nucleic acid. Binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid. In some embodiments, modifying a target nucleic acid may comprise introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid. For example, modifying a target nucleic acid may comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second programmable nickase and hybridizes to a second region of the target nucleic acid. The first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid, and the second effector protein may introduce a second break in a second strand at the second region of the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be removed, thereby modifying the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be replaced (e.g., with donor nucleic acid), thereby modifying the target nucleic acid. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences of TABLE 1. In some embodiments, methods comprise inserting a donor nucleic acid into a cleaved target nucleic acid. The donor nucleic acid may be inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1, thereby introducing a single -stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein, optionally comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences recited in TABLE 1, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally via HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site or in between two cleavage sites). In some embodiments, methods comprise editing a target nucleic acid with two or more effector proteins. Editing a target nucleic acid may comprise introducing a two or more single stranded breaks in a target nucleic acid. In some embodiments, a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid. The guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting the effector protein to the region of the target nucleic acid. Binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid. In some embodiments, modifying a target nucleic acid may comprise introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid. For example, modifying a target nucleic acid may comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second programmable nickase and hybridizes to a second region of the target nucleic acid. The first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid, and the second effector protein may introduce a second break in a second strand at the second region of the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be removed, thereby modifying the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be replaced (e.g, with donor nucleic acid), thereby modifying the target nucleic acid. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1

[433] In some embodiments, editing is achieved by fusing an effector protein to a heterologous sequence. The heterologous sequence may be a suitable fusion partner, e.g., a protein that provides recombinase activity by acting on the target nucleic acid. In some embodiments, the fusion protein comprises an effector protein fused to a heterologous sequence by a linker. The heterologous sequence or fusion partner may be a base editing domain. The base editing domain may be an ADAR1 / 2 or any functional variant thereof. The heterologous sequence or fusion partner may be fused to the C-terminus, N-terminus, or an internal portion (e.g., a portion other than the N- or C-terminus) of the effector protein. The heterologous sequence or fusion partner may be fused to the effector protein by a linker. A linker may be a peptide linker or a non-peptide linker. In some embodiments, the linker is an XTEN linker. In some embodiments, the linker comprises one or more repeats a tri -peptide GGS. In some embodiments, the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 10 to 27 amino acids in length. A non-peptide linker may be a polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene / propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker. In certain embodiments, editing or modification of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit or modify a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising sequence deletion, sequence knock-in, or any combination thereof. For example, editing or modification of a specific locus can effect sequence knock-in. In certain embodiments, sequence knock-in is a modification where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in. In certain embodiments, editing or modification of a specific locus can effect sequence knockin and sequence deletion. In certain embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both. In certain embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise an effector protein described herein and a guide nucleic acid described herein. In certain embodiments, edition or modification of a target nucleic acid is specific to intron 1 of mammalian albumin gene. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vitro. For example, a plasmid may be modified in vitro using a compositions, systems and methods described herein and introduced into a cell or organism. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed ex vivo. For example, methods may comprise obtaining a cell from a subject, modifying a target nucleic acid in the cell with methods described herein, and returning the cell to the subject. Transfection Donor Nucleic Acids In reference to a viral vector, the term transfection donor nucleic acid refers to a sequence of nucleotides that will be or has been introduced into a cell following transfection of the viral vector. The transfection donor nucleic acid may be introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to, integration into the genome of the cell or introduction of an episomal plasmid or viral genome. As another example, when used in reference to the activity of an effector protein, the term donor nucleic acid refers to a sequence of nucleotides that will be or has been inserted at the site of cleavage by the effector protein (cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break -nuclease activity). As yet another example, when used in reference to homologous recombination, the term donor nucleic acid refers to a sequence of DNA that serves as a template in the process of homologous recombination, which may carry the modification that is to be or has been introduced into the target nucleic acid. By using this donor nucleic acid as a template, the genetic information, including the modification, is copied into the target nucleic acid by way of homologous recombination. Donor nucleic acids of any suitable size may be integrated into a target nucleic acid or genome. In some embodiments, the donor polynucleotide integrated into a genome is less than 3, about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 kilobases in length. In some embodiments, donor nucleic acids are more than 500 kilobases (kb) in length. The donor nucleic acid may comprise a sequence that is derived from an animal. The animal may be human. The animal may be a non-human animal, such as, by way of non-limiting example, a mouse, rat, hamster, rabbit, pig, bovine, deer, sheep, goat, chicken, cat, dog, ferret, a bird, non-human primate (e.g., marmoset, rhesus monkey). The non-human animal may be a domesticated mammal or an agricultural mammal. Genetically Modified Cells and Organisms Methods of editing described herein may be employed to generate a genetically modified cell. The cell may be a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., an archaeal cell). The cell may be derived from a multicellular organism and cultured as a unicellular entity. The cell may comprise a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation. The cell may be progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell. A genetically modified cell may comprise a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived. In some embodiments, upon modification of a target nucleic acid by compositions, systems, and methods described herein, the target nucleic acid can comprise an intron deletion, intron knock-in, or a combination thereof. Methods may comprise contacting a cell with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences recited in TABLE 1. Methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding a guide nucleic acid, a tracrRNA, an intermediary RNA, a crRNA, or any combination thereof. Contacting may comprise electroporation, acoustic poration, optoporation, viral vector-based delivery, iTOP, nanoparticle delivery (e.g, lipid or gold nanoparticle delivery), cellpenetrating peptide (CPP) delivery, DNA nanostructure delivery, or any combination thereof. Methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding a guide nucleic acid, a tracrRNA, an intermediary RNA, a sgRNA, or any combination thereof. Methods may comprise contacting a cell with an effector protein or a multimeric complex thereof, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences recited in TABLE 1. In certain embodiments, the compositions, systems, and methods described herein may include an effector protein or a nucleic acid encoding the effector protein, where the effector protein features one or more substitutions compared to any of the amino acid sequences outlined in TABLE 1. These substitutions can range from at least one to twenty or more, and may be classified into various ranges such as one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty substitutions relative to the sequences found in TABLE 1. The substitutions may include one or more conservative substitutions, one or more non-conservative substitutions, or a combination of both. In some instances, the effector protein or the nucleic acid encoding it may contain one or more conservative substitutions in relation to any of the amino acid sequences in TABLE 1, again falling within the previously mentioned ranges. Similarly, the effector protein may also contain one or more non-conservative substitutions relative to any of the sequences in TABLE 1, with the substitutions potentially varying from one to twenty or more. In some cases, the amino acid modifications may lead to changes in the activity of the effector protein compared to its naturally occurring counterpart. For example, these modifications might enhance or diminish the catalytic activity or binding affinity of the effector protein. In certain embodiments, the alterations could result in a catalytically inactive variant of the effector protein. The effector proteins described herein can perform enzymatic reactions similar to those of the wildtype (WT) effector protein. Variants of the WT effector protein may include modifications that provide beneficial characteristics, such as increased activity (e.g., enhanced indel activity, catalytic activity, specificity, selectivity, or affinity for substrates like target nucleic acids or guide nucleic acids). In some cases, the activity of the effector proteins may be equal to or greater than that of the WT effector protein, indicating that they exhibit one or more activities that are the same as or higher than those of the effector protein without any variant at the same amino acid positions. For instance, variants may show increased activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or even 200% compared to the WT effector protein. The activity of the effector proteins or their variants can be evaluated in relation to the WT effector protein through a cleavage assay. In some embodiments, the effector proteins may feature one or more amino acid substitutions compared to any of the sequences listed in TABLE 1, with the remaining amino acid sequence being at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the reference amino acid sequence from TABLE 1. Additionally, the remaining amino acid sequence of the variant may also demonstrate at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the corresponding reference sequence. In some instances, the substitutions may involve one or more positively charged amino acid residues, such as Lys (K), Arg (R), His (H), or combinations thereof. In some embodiments, the term "in vitro" refers to processes that occur outside of a living organism, typically in a controlled environment like a test tube or culture dish. Effector proteins described herein may include both coded and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids, and proteins with altered peptide backbones. These effector proteins may contain one or more mutations, engineered modifications, or both. It is understood that the coding sequences for these effector proteins do not necessarily need to include a codon for an N-terminal Methionine (M) or Valine (V). A skilled individual in the field would recognize that a start codon can be substituted with one that encodes an amino acid residue sufficient for initiating translation in a host cell. Additionally, the compositions, systems, and methods may include heterologous peptides or polypeptides, which can be fusion proteins comprising an effector protein along with one or more fusion partner proteins. The fusion partner proteins can be located at the N-terminus of the effector protein. In such cases, the start codon for the heterologous peptide or polypeptide may also serve as the start codon for the effector protein, allowing for the removal or absence of the natural start codon that typically encodes an amino acid residue necessary for translation initiation. Heterologous polypeptides may consist of at least two different polypeptide sequences that are not found together in nature. A heterologous system may include at least one component that does not naturally occur in conjunction with the other components. In certain embodiments, a heterologous peptide or polypeptide may contain a subcellular localization signal, such as a nuclear localization signal (NLS) that facilitates the targeting of a nucleic acid, protein, or small molecule to the nucleus within a cell. Other types of localization signals may include nuclear export signals (NES), signals for retaining an effector protein in the cytoplasm, mitochondrial localization signals, chloroplast localization signals, endoplasmic reticulum retention signals, and others. In specific cases, an effector protein may not include a subcellular localization signal to prevent targeting to the nucleus, which can be beneficial when the target nucleic acid is an RNA present in the cytosol. In some embodiments, the heterologous polypeptide may be an endosomal escape peptide (EEP), designed to disrupt the endosome quickly to minimize the time a delivered molecule, such as an effector protein, remains in the endosomal environment, thus avoiding entrapment in endosomal vesicles and subsequent degradation in the lysosomal compartment. Additionally, a heterologous polypeptide may be a cell-penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD), which facilitates the traversal of lipid bilayers, cell membranes, organelle membranes, or vesicle membranes. In certain instances, a heterologous peptide or polypeptide may include a protein tag, which can serve as a purification tag or a fluorescent protein. Such tags may be detectable for the purpose of identifying or purifying the effector protein. Various protein tags can be utilized depending on the intended application, including but not limited to fluorescent proteins, histidine tags (e.g., 6XHis), hemagglutinin (HA) tags, FLAG tags, Myc tags, and maltose binding proteins (MBP). Examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato. A heterologous polypeptide may be positioned at or near the amino terminus (N-terminus) or the carboxy terminus (C-terminus) of the effector protein. In some cases, the heterologous polypeptide may be located internally within the effector protein at a suitable insertion site. Guide Nucleic Acids The compositions, systems, and methods of the present disclosure may include a guide nucleic acid or its use. These may also encompass compositions, systems, and methods that feature one or more guide nucleic acids and DNA molecules encoding these guide nucleic acids. A person skilled in the art understands that a DNA molecule that "encodes" a nucleic acid, such as a guide nucleic acid, refers to a DNA molecule with a nucleotide sequence that produces an RNA molecule (e.g., a guide nucleic acid) upon transcription. It is understood that mention of a guide nucleic acid also includes a DNA molecule encoding that guide nucleic acid. A guide nucleic acid, along with its components (e.g., spacer sequence, repeat sequence, linker nucleotide sequence, handle sequence, intermediary RNA, etc.), may consist of one or more deoxyribonucleotides (DNA), ribonucleotides (RNA), or a combination of both (e.g., RNA containing a thymine base), as well as biochemically or chemically modified nucleotides. Such nucleotide sequences can be described as either DNA or RNA; however, regardless of the form, it is understood that these sequences can be adapted to be RNA or DNA as required for describing a sequence within a guide nucleic acid or the sequence that encodes it, such as a nucleotide sequence for a vector. The disclosure of the nucleotide sequences also includes their complementary sequences, reverse sequences, and reverse complement sequences, any of which can serve as a nucleotide sequence for use in a guide nucleic acid. In some embodiments, a guide nucleic acid may comprise a CRISPR RNA (crRNA) or a single guide RNA (sgRNA). The sgRNA is formed by combining a spacer sequence (which hybridizes to a target sequence in a target nucleic acid) with a handle sequence, where both sequences are covalently linked. The spacer and handle sequences can be linked by a phosphodiester bond or by one or more linked nucleotides. In some cases, a guide nucleic acid may include a spacer sequence, a repeat sequence, a handle sequence, or a combination thereof, with the handle sequence potentially comprising part or all of a repeat sequence. In some embodiments, the composition may include a tracrRNA. The crRNA and tracrRNA may function as separate, unlinked molecules, or they may be covalently linked. The linkage can occur via a phosphodiester bond or through one or more linked nucleotides. In certain embodiments, the composition may not include a tracrRNA. A guide nucleic acid may be a naturally occurring guide nucleic acid or a non-naturally occurring one, which may include chemical or biochemical modifications. The guide RNA may be synthesized chemically or produced recombinantly. The sequence of the guide nucleic acid, or a portion of it, may differ from that of a naturally occurring nucleic acid. In some embodiments, the compositions, systems, and methods of the present disclosure may include one or more additional guide nucleic acids or their uses. For example, these may include two or more additional guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more guide nucleic acids) that can target an effector protein to different locations within the target nucleic acid by binding to distinct portions of that nucleic acid. A guide nucleic acid can bind to a segment of the target nucleic acid that is upstream or downstream of a target gene, allowing for modifications at two different locations. This dual-targeting approach, referred to as "dual-cutting," may involve two effector proteins, each corresponding to a guide RNA, or a single effector protein with two different guide RNAs to achieve the dual-cutting effect. In some cases, multiple effector proteins (e.g., 2, 3, 4, 5, 6, 7, 9, 10, or more) may be employed in the dual-guided systems described herein. In some embodiments, the guide nucleic acid may include 10, 11,12,13,14,15,16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, or 30 linked nucleotides. Generally, a guide nucleic acid will consist of at least a certain number of linked nucleotides, with some embodiments specifying at least 25 linked nucleotides. The guide nucleic acid may range from 10 to 50 linked nucleotides in length. In some cases, the guide nucleic acid may consist of or essentially consist of about 12 to about 80, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19, about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleotides. In some embodiments, the guide nucleic acid may have about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides. In some embodiments, the engineered guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. This eukaryotic sequence refers to a nucleotide sequence present in a host eukaryotic cell, distinguishing it from sequences found in prokaryotic cells or viruses. Such sequences can be located within a gene, an exon, an intron, a non-coding region (e.g., a promoter or enhancer), a selectable marker, tag, signal, or similar elements. In some embodiments, the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a nucleotide sequence as described herein (e.g., TABLE 4, TABLE 5, TABLE 6, or TABLE 8). These nucleotide sequences can be characterized as either DNA or RNA; however, it is understood that such sequences can be adapted as needed for describing a sequence within a guide nucleic acid or the sequence that encodes it, such as a nucleotide sequence for a vector. Additionally, the disclosure of these nucleotide sequences also includes their complementary sequences, reverse sequences, and reverse complement sequences, any of which can serve as a nucleotide sequence for use in a guide nucleic acid. TABLE 1 Exemplary CRISPR Cas nucleases / effectors used herein: SEQID NO: Name 185 B-GEn.l 186 B-GEn.1.1 TABLE 2. TABLE of Sequences SEQ ID NO: Identifier Sequence 1 insert of pBLR2542 TTCTATTGTTCAACI111ATTCT 2 insert of pBLR2543 TTCAACI111 AlIGIAl 11ICCC 3 insert of pBLR2545 TAI 1 1 1CCCAGTAAAATAAAGTT 4 insert of pBLR2546 TCCCAGTAAAATAAAG11 1 1 AG 1 5 insert of pBLR2544 TACTGGGAAAATAGAATAAAAGT 6 insert of pBLR2547 TTTAAAGATGCAGAGTTTACTAA 7 insert of pBLR2549 Illi GGCATTTA HILI AAAATG 8 insert of pBLR2550 TGGCATTTA HILI AAAATGGCA 9 insert of pBLR2551 A1 1 1C1AAAATGGC AT AGTATTT 10 insert of pBLR2552 CTAAAATGGCATAGTA1 1 1 1G1A 11 insert of pBLR2548 TAGAAATAAATGCCAAAATAATT 12 insert of pBLR2553 TGTATTTGTGAAGTCTTACAAGG 13 insert of pBLR2554 GTGAAGTCTTACAAGGTTATCTT 14 insert of pBLR2557 ATAAAATTCAAACATCCTAGGTA 15 insert of pBLR2555 ATAAGATAACCTTGTAAGACTTC 16 insert of pBLR2559 TGACCI 1 1 1 1 1 1 1 1 1 11IACCTA 17 insert of pBLR2561 TTTAGTGACTGTAA1 1 1 1C1 1 1 1 18 insert of pBLR2560 C AGTC ACT AAACAATTCTG ACCT 19 insert of pBLR2562 TCI 1 1 1GCGCACTAAGGAAAGTG 20 insert of pBLR2563 TGTGAAGTTTCAGTCACTCTAAG 21 insert of pBLR2565 AATTCATAACTATCCCAAAGACC 22 insert of pBLR2564 AATCTTCAACCCTATTCTGTGAA 23 insert of pBLR2537 ATAACTATCCCAAAGACCTATCC 24 insert of pBLR2538 CACTATGCTTTATTTAAAAACCA 25 insert of pBLR2539 AAAAACCACAAAACCTGTGCTGT 26 insert of pBLR2540 ATGAGATCAACAGCACAGG Illi T1 insert of pBLR2541 ATAI HAI 1 1 ICAI 1 1 IAGTCTG 28 insert of pBLR2566 Al 11ICAI11 IAGTCTGTCTTCT 29 insert of pBLR2567 TCAI 1 1 1AGTCTGTCTTCTTGGT 30 insert of pBLR2568 TAGTCTGTCTTCTTGGTTGCTGT 31 insert of pBLR2570 GATATTATCTAAGTTTGAATATA 32 insert of pBLR2571 TCTAAGTTTGAATATAAGGCTAT 33 insert of pBLR2572 ATAGCCTTATATTCAAACTTAGA 34 insert of pBLR2576 AATAAI 1 1 1 1AAAATAGTATTCT 35 insert of pBLR2573 AATATTTATAGCCTTATATTCAA 36 insert of pBLR2574 TTAAATATTTATAGCCTTATATT 37 insert of pBLR2577 1 IAAAAIAGIAI ICI IGGIAAI 1 38 insert of pBLR2575 TAAAAATTATTAAATATTTATAG 39 insert of pBLR2527 TTGGTAATTGAATTATTCTTCTG 40 insert of pBLR2526 CCAAGAATACTAI 1 1 IAAAAATT 41 insert of pBLR2579 AATTATTCTTCTGTTTAAAGGCA 42 insert of pBLR2578 AATTACCAAGAATACTAI 1 1 1AA 43 insert of pBLR2522 TTCTTCTGTTTAAAGGCAGAAGA 44 insert of pBLR2529 TTCTGTTTAAAGGCAGAAGAAAT 45 insert of pBLR2536 CTTCTGCCTTTAAACAGAAGAAT 46 insert of pBLR2523 TTTCTTCTGCCTTTAAACAGAAG 47 insert of pBLR2580 AACATCATCCTGAG1 1 111L1G1 48 insert of pBLR2530 CTACAGAAAAACTCAGGATGATG 49 insert of pBLR2581 GGCTCTGATTCCTACAGAAAAAC 50 insert of pBLR2533 TGAAACAAATGCATAATCTAAGT 51 insert of pBLR2532 GTTTCAAAATATTGGGCTCTGAT 52 insert of pBLR2524 TGCATTTGTTTCAAAATATTGGG 53 insert of pBLR2535 CTTTCCATTTGACTTAGATTATG 54 insert of pBLR2521 TTACTTCTTG1111L11CAGTAT 55 insert of pBLR2525 CTTCTTG1 1 1 1L1 1CAGTATTTA 56 insert of pBLR2531 AACAATCC1 1 1 1 1 1 1 1L11CCCT 57 insert of pBLR2582 TTAAATACTGAAGAAAACAAGAA 58 insert of pBLR2558 AAACATCCTAGGTAAAAAAAAAA 59 insert of pBLR2556 TATTAATAAGATAACCTTGTAAG 60 insert of pBLR2569 AAACTTAGATAATATCTAATACT 61 insert of pBLR2534 GACTTAGATTATGCATTTGTTTC 62 gRNA sequence for B-GEn.l human albumin target 1 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCU AUUGUUCAACUUUUAUUCU 63 gRNA sequence for B-GEn.l human albumin target 2 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCA ACUUUUAUUCUAUUUUCCC 64 gRNA sequence for B-GEn.l human albumin target 3 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAUU UUCCCAGUAAAAUAAAGUU 65 gRNA sequence for B-GEn.l human albumin target 4 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCCC AG U AAAAU AAAG U U U U AG U 66 gRNA sequence for B-GEn.l human albumin target 5 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUACU GGGAAAAUAGAAUAAAAGU 67 gRNA sequence for B-GEn.l human albumin target 6 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUA AAGAUGCAGAGUUUACUAA 68 gRNA sequence for B-GEn.l human albumin target 7 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUU GGCAUUUAUUUCUAAAAUG 69 gRNA sequence for B-GEn.l human albumin target 8 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGGC AUUUAUUUCUAAAAUGGCA 70 gRNA sequence for B-GEn.l human albumin target 9 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUUU CUAAAAUGGCAUAGUAUUU 71 gRNA sequence for B-GEn.l human albumin target 10 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUAA AAUGGCAUAGUAUUUUGUA 72 gRNA sequence for B-GEn.l human albumin target 11 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAGA AAUAAAUGCCAAAAUAAUU 73 gRNA sequence for B-GEn.l human albumin target 12 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGUA UUUGUGAAGUCUUACAAGG 74 gRNA sequence for B-GEn.l human albumin target 13 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGUGA AGUCUUACAAGGUUAUCUU 75 gRNA sequence for B-GEn.l human albumin target 14 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAA AAUUCAAACAUCCUAGGUA 76 gRNA sequence for B-GEn.l human albumin target 15 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAA GAUAACCUUGUAAGACUUC 77 gRNA sequence for B-GEn.l human albumin target 16 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGAC CUUUUUUUUUUUUUACCUA 78 gRNA sequence for B-GEn.l human albumin target 17 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUA GUGACUGUAAUUUUCUUUU 79 gRNA sequence for B-GEn.l human albumin target 18 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCAGU CACUAAACAAUUCUGACCU 80 gRNA sequence for B-GEn.l human albumin target 19 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCUU UUGCGCACUAAGGAAAGUG 81 gRNA sequence for B-GEn.l human albumin target 20 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGUG AAGUUUCAGUCACUCUAAG 82 gRNA sequence for B-GEn.l human albumin target 21 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUU CAUAACUAUCCCAAAGACC 83 gRNA sequence for B-GEn.l human albumin target 22 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUC UUCAACCCUAUUCUGUGAA 84 gRNA sequence for B-GEn.l human albumin target 23 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAA CUAUCCCAAAGACCUAUCC 85 gRNA sequence for B-GEn.l human albumin target 24 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCACU AUGCUUUAUUUAAAAACCA 86 gRNA sequence for B-GEn.l human albumin target 25 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAA ACCACAAAACCUGUGCUGU 87 gRNA sequence for B-GEn.l human albumin target 26 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUGA GAUCAACAGCACAGGUUUU 88 gRNA sequence for B-GEn.l human albumin target 27 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAU UUAUUUUCAUUUUAGUCUG 89 gRNA sequence for B-GEn.l human albumin target 28 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUUU UCAUUUUAGUCUGUCUUCU 90 gRNA sequence for B-GEn.l human albumin target 29 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCAU UUUAGUCUGUCUUCUUGGU 91 gRNA sequence for B-GEn.l human albumin target 30 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAGU CUGUCUUCUUGGUUGCUGU 92 gRNA sequence for B-GEn.l human albumin target 31 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGAUA UUAUCUAAGUUUGAAUAUA 93 gRNA sequence for B-GEn.l human albumin target 32 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCUA AGUUUGAAUAUAAGGCUAU 94 gRNA sequence for B-GEn.l human albumin target 33 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAG CCU UAUAU UCAAACU UAGA 95 gRNA sequence for B-GEn.l human albumin target 34 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUA AUUUUUAAAAUAGUAUUCU 96 gRNA sequence for B-GEn.l human albumin target 35 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUA UUUAUAGCCUUAUAUUCAA 97 gRNA sequence for B-GEn.l human albumin target 36 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAA AUAUUUAUAGCCUUAUAUU 98 gRNA sequence for B-GEn.l human albumin target 37 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAA AAUAGUAUUCUUGGUAAUU 99 gRNA sequence for B-GEn.l human albumin target 38 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAAA AAUUAUUAAAUAUUUAUAG 100 gRNA sequence for B-GEn.l human albumin target 39 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUGG UAAUUGAAUUAUUCUUCUG 101 gRNA sequence for B-GEn.l human albumin target 40 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCCAA GAAUACUAUUUUAAAAAUU 102 gRNA sequence for B-GEn.l human albumin target 41 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUU AUUCUUCUGUUUAAAGGCA 103 gRNA sequence for B-GEn.l human albumin target 42 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAUU ACCAAGAAUACUAUUUUAA 104 gRNA sequence for B-GEn.l human albumin target 43 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCU UCUGUUUAAAGGCAGAAGA 105 gRNA sequence for B-GEn.l human albumin target 44 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCU GUUUAAAGGCAGAAGAAAU 106 gRNA sequence for B-GEn.l human albumin target 45 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUUC UGCCUUUAAACAGAAGAAU 107 gRNA sequence for B-GEn.l human albumin target 46 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUC UUCUGCCUUUAAACAGAAG 108 gRNA sequence for B-GEn.l human albumin target 47 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAACA UCAUCCUGAGUUUUUCUGU 109 gRNA sequence for B-GEn.l human albumin target 48 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUAC AGAAAAACUCAGGAUGAUG 110 gRNA sequence for B-GEn.l human albumin target 49 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGGCU CUGAUUCCUACAGAAAAAC 111 gRNA sequence for B-GEn.l human albumin target 50 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGAA ACAAAUGCAUAAUCUAAGU 112 gRNA sequence for B-GEn.l human albumin target 51 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGUUU CAAAAUAUUGGGCUCUGAU 113 gRNA sequence for B-GEn.l human albumin target 52 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGCA UUUGUUUCAAAAUAUUGGG 114 gRNA sequence for B-GEn.l human albumin target 53 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUUU CCAUUUGACUUAGAUUAUG 115 gRNA sequence for B-GEn.l human albumin target 54 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAC UUCUUGUUUUCUUCAGUAU 116 gRNA sequence for B-GEn.l human albumin target 55 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUUC UUGUUUUCUUCAGUAUUUA 117 gRNA sequence for B-GEn.l human albumin target 56 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAACA AUCCUUUUUUUUCUUCCCU 118 gRNA sequence for B-GEn.l human albumin target 57 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAA AUACUGAAGAAAACAAGAA 119 gRNA sequence for B-GEn.l human albumin target 58 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAC AUCCUAGGUAAAAAAAAAA 120 gRNA sequence for B-GEn.l human albumin target 59 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAUU AAUAAGAUAACCUUGUAAG 121 gRNA sequence for B-GEn.l human albumin target 60 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAC UUAGAUAAUAUCUAAUACU 122 gRNA sequence for B-GEn.l human albumin target 61 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGACU UAGAUUAUGCAUUUGUUUC 123 pBLR2542 SEQ. ID NO: 123-184 see sequence listing 124 pBLR2543 125 pBLR2545 126 pBLR2546 127 pBLR2544 128 pBLR2547 129 pBLR2549 130 pBLR2550 131 pBLR2551 132 pBLR2552 133 pBLR2548 134 pBLR2553 135 pBLR2554 136 pBLR2557 137 pBLR2555 138 pBLR2559 139 pBLR2561 140 pBLR2560 141 pBLR2562 142 pBLR2563 143 pBLR2565 144 pBLR2564 145 pBLR2537 146 pBLR2538 147 pBLR2539 148 pBLR2540 149 pBLR2541 150 pBLR2566 151 pBLR2567 152 pBLR2568 153 pBLR2570 154 pBLR2571 155 pBLR2572 156 pBLR2576 157 pBLR2573 158 pBLR2574 159 pBLR2577 160 pBLR2575 161 pBLR2527 162 pBLR2526 163 pBLR2579 164 pBLR2578 165 pBLR2522 166 pBLR2529 167 pBLR2536 168 pBLR2523 169 pBLR2580 170 pBLR2530 171 pBLR2581 172 pBLR2533 173 pBLR2532 174 pBLR2524 175 pBLR2535 176 pBLR2521 177 pBLR2525 178 pBLR2531 179 pBLR2582 180 pBLR2558 181 pBLR2556 182 pBLR2569 183 pBLR2534 184 pBLR709 185 SEQID NO:2from WO2022 / 258753 (here also named B- GEn.l) MPIRSFKLKLVTHNGDSTYMDKLRRGLWKTHVIINRGIAYYMNTLALMRQ EPYGSKSREEVRLDLLSTLREQQRRNNWSEQTGTDDELLSLSRRVYELLVPS AIGEKGDAQMLSRKFLSPLVDPNSEGGRGTAKSGRKPRWKKMMEEGHPD WEKEKEKDAAKKAEDPTASILADLEAVGLLPLFPLFSDEQKEIRWLPKKKRQ FVRTWDRDMFQQALERMLSWESWNRRVAEEYLKLQAQRDEVYAKYLED AGSWLNDLQTFEKQREEELAEVSFEPNSEYLITRRQIRGWKEVYEKWSKTSE NASQEQLWRMVADVQTAMAGAFGDPKVYQFLSQPKHHHIWREHPNRL FYYSKYNEVREKLNRAKKQAAFTLPDPVEHPLWTRFDARGGNIHDYEISKV GKQYHVTFSSLILPEAQSWVEIENVTVGIGNSLQLKRQIRLDGYADKKQKVK YYDYSSRFELTGVLGGAKIQFDRKHLKKAAHRLAEGETGPIFLNVVVDVEPFL EVKNGRLRTPLGQVLQVNTRDWPKVVDYKAKELSVLMENTQIGNENGVS TIEAGMRIMSIDLGQRTAAAVSIFEVISKKPDEKETKLFYPIADTDLYAVHRRS LLLRLPGEEISSKKMIEKRKERARIRSLVRYQIRLLSEVLRLHTQGTAEQRRFKL DELLVSIQKKLELDQSEWISELEKLFDYIDESAEKWKEALVVAHRTLEPIVVEA VRNWKKSLSKENKDRRRIAGISIWSIEELEETRKLLIAWSKHSREPGIPKRLEK EETFAPEHLQHIQNVKDDRLKQMANLFVMTALGYKYDEGNKRWVEAYPA CQVILFEDLSRYRFALDRPRRENNRLMKWAHRSIPRLTYMQAELFGIQVGD VYSAYTSRFHAKTGAPGIRCHALTEADLQSNSYWNQLIKDKFIQDNQTEILK AGQIVPWQGGELFVTFADRSGASLAVIHADINAAQNLQKRFWQHNSEVF RVPCKVVKGGLVPVYEKMRKLFGKGLFVNIDDPESKEVYRWEHSTKMKSK TTPVDLESEDIDHEELSDEWEDMQEGYKTLLRDPSGFFWSSDSWIPQKDF WIRVKSRIGKSLREQIR 186 SEQID NO:39 from WO2022 / 258753 MPIRSFKLKLVTHNGDSTYMDKLRRGLWKTHVIINRGIAYYMNTLALMRQ EPYGSKSREEVRLDLLSTLREQQRRNNWSEQTGTDDELLSLSRRVYELLVPS AIGEKGDAQMLSRKFLSPLVDPNSEGGRGTAKSGRKPRWKKMMEEGHPD WEKEKEKDAAKKAEDPTASILADLEAVGLLPLFPLFSDEQKEIRWLPKKKRQ FVRTWDRDMFQQALERMLSWESWNRRVAEEYQKLQAQRDEVYAKYLED AGSWLNDLQTFEKQREEELAEVSFEPNSEYLITRRQIRGWKEVYEKWSKTSE NASQEQLWRMVADVQTAMAGAFGDPKVYQFLSQPKHHHIWREHPNRL FYYSKYNEVREKLNRAKKQAAFTLPDPVEHPLWTRFDARGGNIHDYEISKV GKQYHVTFSSLILPEAQSWVEIENVTVGIGNSLQLKRQIRLDGYADKKQKVK YYDYSSRFELTGVLGGAKIQFDRKHLKKAAHRLAEGETGPIFLNVVVDVEPFL EVKNGRLRTPLGQVLQVNTRDWPKVVDYKAKELSVLMENTQIGNENGVS TIEAGMRIMSIDLGQRTAAAVSIFEVISKKPDEKETKLFYPIADTDLYAVHRRS LLLRLPGEEISSKKMIEKRKERARIRSLVRYQIRLLSEVLRLHTQGTAEQRRFKL DELLVSIQRKLELDQSEWISELEKLFDYIDESAEKWKEALVVAHRTLEPIVVEA VRNWKKSLSKENKDRRRIAGISIWSIEELEETRKLLIAWSKHSREPGIPKRLEK EETFAPEHLQHIQNVKDDRLKQMANLFVMTALGYKYDEGNKRWVEAYPA CQVILFEDLSRYRFALDRPRRENNRLMKWAHRSIPRLTYMQAELFGIQVGD VYSAYTSRFHAKTGAPGIRCHALTEADLQSNSYWNQLIKDKFIQDNQTEILK AGQIVPWQGGELFVTFADRSGASLAVIHADINAAQNLQKRFWQHNSEVF RVPCKVVKGGLVPVYEKMRKLFGKGLFVNIDDPESKEVYRWEHSTKMKSK TTPVDLESEDIEHEELSDEWEDMQEGYKTLLRDPSGFFWSSDSWIPQKDF WIRVKSRIGKSLREQIR 187 hm alb intron 1 GTAAGAAATCCA1 1 1 1 1C1A1 1G FTCAAC1 1 1 1A1 TCTA Illi CCCAGTAA AATAAAG1 1 1 1 AG 1AAAC1C1GCAIC1 1 1AAAGAA1 1A1 1 1 1GGCATTTAT TTCTAAAATGGCATAGTAI 1 1 1GTATTTGTGAAGTCTTACAAGGTTATCTT ATTAATAAAATTCAAACATCCTAGGTAAAAAAAAAAAAAGGTCAGAATT GTTTAGTGACTGTAAI INCH 1 1GCGCACTAAGGAAAGTGCAAAGTAAC TTAGAGTGACTGAAACTTCACAGAATAGGGTTGAAGATTGAATTCATAA CTATCCCAAAGACCTATCCATTGCACTATGCTTTATTTAAAAACCACAAA ACCTGTGCTGTTGATCTCATAAATAGAACTTGTATTTATATTTAI 1 1 ICAT TTTAGTCTGTCTTCTTGGTTGCTGTTGATAGACACTAAAAGAGTATTAGA TATTATCTAAGTTTGAATATAAGGCTATAAATATTTAATAAI 1 1 1 IAAAAT AGTAI 1 Cl 1GGTAATTGAATTATTCTTCTGTTTAAAGGCAGAAGAAATAA TTGAACATCATCCTGAG1 1 1 1 1C1G1AGGAA1CAGAGCCCAA1A1 1 1 1 GA AACAAATGCATAATCTAAGTCAAATGGAAAGAAATATAAAAAGTAACAT TATTACTTCTTG1 1 1 1 Cl ICAGIAI 1 IAACAAICCI 1 1 1 1 1 1 1 Cl ICCCTTGC CCAG 188 amplicon_T51 ACTTTCCTTAGTGCGCAAAAGAAAATTACAGTCACTAAACAATTCTGACC 1 1 1 1 1 1 1 1 1 1 1 1 IACCIAGGAIGI 1 IGAAI 1 1 1ATTAATAAGATAACCTTGT AAGACTTCACAAATACAAAATACTATGCCAI 1 1 1AGAAATAAATGCCAAA ATAA1 1C1 1 1AAAGA1GCAGAG1 1 1 AC 1AAAAC1 1 1A1 1 1 1ACTGGGAAA ATAGAATAAAAGTTGAACAATAGAAAAATGGATTTCTTACGTGCATCTC GACGAAACACACCCCTGGAATAAGCCGAGCTAAAG 189 amplicon_T18 GCATCTTTAAAGAATTA Illi GGCA1 1 1A1 1 1C1AAAATGGCATAGTATTT TGTATTTGTGAAGTCTTACAAGGTTATCTTATTAATAAAATTCAAACATC CTAGGTAAAAAAAAAAAAAGGTCAGAATTGTTTAGTGACTGTAAI 1 1 IC Illi GCGCACTAAGGAAAGTGCAAAGTAACTTAGAGTGACTGAAACTTC ACAGAATAGGGTTGAAGATTGAATTCATAACTATCCCAAAGACCTATCC ATTGCACTATG 190 amplicon_T42 TCI 1 1 1GCGCACTAAGGAAAGTGCAAAGTAACTTAGAGTGACTGAAACT TCACAGAATAGGGTTGAAGATTGAATTCATAACTATCCCAAAGACCTAT CCATTGCACTATGCTTTATTTAAAAACCACAAAACCTGTGCTGTTGATCT CATAAATAG AACTTGTATTTATA1 1 1A1 1 1 1C A1 1 1 1AGTCTGTC1 1C1 1G GTTGCTGTTGA 191 amplicon_T27 CTGTCTTCTTGGTTGCTGTTGATAGACACTAAAAGAGTATTAGATATTAT CTAAGTTTGAATATAAGGCTATAAATATTTAATAAI 1 1 1 1AAAATAGTAT TCTTGGTAATTGAATTATTCTTCTGTTTAAAGGCAGAAGAAATAATTGAA CATCATCCTGAG1 1 1 1 1C1G1AGGAA1CAGAGCCCAA1A1 1 1 1GAAACAA ATGCATAATCTAAGTCAAATGGA 192 amplicon_T50 1 1 1C1G1AGGAATCAGAGCCCAATA Illi GAAACAAATGCATAATCTAAG TCAAATGGAAAGAAATATAAAAAGTAACATTATTACTTCTTG Illi CTTC AGTATTTAACAATCCI 1 1 1 1 1 1 1 Cl 1CCCTTGCCCAGACAAGAGTGAGGTT GCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGTAAG TTAAAATATTGATGAATCAAATTTAATG1 1 1C1AATAGTGTTGTTTATTAT TCTAAAGTGCTTATATTTCCTTGTCATCAGGGTTCAG 193 amplicon_T72 GAACATCATCCTGAG1 1 1 1 1C1 GT AGGAATCAGAGCCCAATA Illi GAAA CAAATGCATAATCTAAGTCAAATGGAAAGAAATATAAAAAGTAACATTA TTACTTCTTGI 1 1 ICIICAGIAI 1 IAACAAICCI 1 Illi 1 1C1 1CCCTTGCCC AGACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAA TTTCAAAGCCTTGTAAGTTAAAATATTGATGAATCAAATTTAATG1 1 1 Cl AATAGTGTTGTTTATTATTCTAAAGTGCTTATATTTCCTTGTCATCAGGGT TCAGATTCTAAAACAGTGCTGCCTCGTAGA 194 amplicon_T55 GAG Illi ATG1 1 1 1 1 1CATCTCTGCTTGTA1 1 1 1 1C1AGTAATGGAAGCCT GGTAI 1 1 1AAAAI AG 1 1 AAAI 1 1 ICCI 1 IAGIGCIGAI 1 ICIAGATTATTA TTACTGTTGTTGTTGTTATTATTGTCATTATTTGCATCTGAGAACCCTTAG GTGGTTATATTATTGATATAI 1 1 1 1GGTATCTTTGATGACAATAATGGGG GA 1 1 1 1GAAAGC1 1AGC1 1 1 AAA 1 1 1C1 1 1 1AATTAAAAAAAAATGCTAG GCAGAATGACTCAAATTAC 195 B-h-181 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrCrCrCrArGrUrArArArArUrArArArGrUrUrUrU*mA*mG* mU 196 B-h-234 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrArUrUrUrCrUrArArArArUrGrGrCrArUrArGrUrA*mU*mU* mU 197 B-h-235 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrArUrArArGrArUrArArCrCrUrUrGrUrArArGrArC*mU*mU* mC 198 B-h-236 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrGrUrGrArArGrUrUrUrCrArGrUrCrArCrUrCrU*mA*mA* mG 199 B-h-237 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrArArUrUrArCrCrArArGrArArUrArCrUrArUrUrU*mU*mA* mA 200 B-h-213 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrU rCrCrCrArGrU rArArArArUrArArArG *mU*mU*mU 201 Sp-h-154 mU*mA*mA*rArGrCrArUrArGrUrGrCrArArUrGrGrArUrGrUrUrUrUr ArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGr CrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCr ArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU 202 Sp-h-155 mU*mA*mA*rUrArArArArUrUrCrArArArCrArUrCrCrUrGrUrUrUrUrA rGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrC rUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrllrGrGrCrA rCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU 203 gRNA sequence for B-GEn.l mouse albumin target 1 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCU AGUAAUGGAAGCCUGGUAU 204 gRNA sequence for B-GEn.l mouse albumin target 4 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAAA AUACCAGGCU UCCAU UACU 205 gRNA sequence for B-GEn.l mouse albumin target 7 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUAG AUUAUUAUUACUGUUGUUG 206 gRNA sequence for B-GEn.l mouse albumin target 8 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAU UACUGUUGUUGUUGUUAUU 207 gRNA sequence for B-GEn.l mouse albumin target 18 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCAU CAAAGAUACCAAAAAUAUA 208 B-ms-1 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrUrCrUrArGrUrArArUrGrGrArArGrCrCrUrGrG*mU*mA* mU 209 B-ms-4 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrArArArArUrArCrCrArGrGrCrUrUrCrCrArUrU*mA*mC*m U 210 B-ms-7 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrCrUrArGrArUrUrArUrUrArUrUrArCrUrGrUrUrG*mU*mU* mG 211 B-ms-8 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrUrArUrUrArCrUrGrUrUrGrUrUrGrUrUrGrUrU*mA*mU* mU 212 B-ms-18 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrCrArUrCrArArArGrArUrArCrCrArArArArArU*mA*mU*m A 213 Control2 AAV Sequence SEQID No. 263-270 from US20200270617A1 TCTAGAGCTAGCATATGGATCCATCGATTTAGGGATAACAGGGTAATTA TCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCT GATATCTGTACACTTAAGGGCTAGATCTTAGCTTACGTCACTAGAGGGT CCACGTTTAGI 1 1 1 1AAGATCCATTGATCTCCTAAACGCTGCAAGATTCG CAACCTGGTATACTTAGCCTAGGCGCTAGGTCCTAGTGCAGCGGGACTT TTTTTCTAAAGTCGTTGAGAGGAGGAGTCGTCAGACCAGATAGCTTTGA TGTCCTGATCGGAAGGATCGTTGGCCCCCCTGCAGGCAGCTGTTAATTA AACGCTAGCCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTTG TTAATGATTAACCCGCCATGCTACTTATCTACGGCGCGCCACACGCGTTC TTGACACI 1 ICI 1AAGAGA1G1AAAA1 1 1 ICAIGAIGI 1 1ICI1111 IGCT AAAACTAAAGAATTATTC Illi ALA 1 1 1 LAG 1 1 1 1 1C1 1GATCATGAAAAC GCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGG AAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGT GTAG Illi GAAGAAGCAGTATTCACTTTGGAGGACTTTGTCGGTGACTG GAGGCAAACCGCTGGTTATAATCTCGACCAAGTACTGGAACAGGGCGG GGTAAGTTCCCTCTTTCAGAATTTGGGTGTAAGCGTCACACCAATCCAGC GGATTGTGTTGTCTGGAGAGAACGGACTCAAAATTGACATCCATGTTAT CATTCCATATGAAGGTCTCAGTGGAGACCAAATGGGGCAGATCGAGAA GAI 1 1 1CAAGGTAGTTTACCCAGTCGACGATCACCACTTCAAAGTCATTC TCCACTATGGCACACTTGTTATCGACGGAGTAACTCCTAATATGATTGAT TACTTTGGTCGCCCGTATGAGGGCATCGCAGTGTTTGATGGCAAAAAGA TCACCGTAACAGGAACGTTGTGGAATGGGAACAAGATAATCGACGAGA GATTGATAAATCCAGACGGGTCACTCCTGTTCAGGGTTACAATTAACGG CGTCACAGGATGGAGACTCTGTGAACGAATACTGGCCACAAAI 1 1 1 1 CA CTCCTGAAGCAGGCCGGAGACGTGGAGGAAAACCCAGGGCCCGTGAG CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCG AGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTAC GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT CTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGA GGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGT GAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG GGATCACTCTCGGCATGGACGAGCTGTACAAGGGAGGAGGAAGCCCG AAGAAGAAGAGAAAGGTCTAACCTCGACTGTGCCTTCTAGTTGCCAGCC ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG CTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTA TCCCCAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAA TGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATA AAGCAATAGCATCACAAATTTCACAAATAAAGCATTTmTCACTGCATT CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTTACA 214 Control2 AAV Sequence SEQID No. 263-270 from US20200270617A1 CCTTCCTCTTCTTCTTGGGGCTGCCGCCGCCCTTGTACAGCTCGTCCATG CCCAGGGTGATGCCGGCGGCGGTCACGAACTCCAGCAGCACCATGTGG TCCCTCTTCTCGTTGGGGTCCTTGCTCAGGGCGCTCTGGGTGCTCAGGTA GTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTG CTGGTAGTGGTCGGCCAGCTGCACGCTGCCGTCCTCGATGTTGTGCCTG ATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATGTA CACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGGCCCAGGATGTTG CCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATCCTGTTCACCAGGGT GTCGCCCTCGAACTTCACCTCGGCCCTGGTCTTGTAGTTGCCGTCGTCCT TGAAGAAGATGGTCCTCTCCTGCACGTAGCCCTCGGGCATGGCGCTCTT GAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTACCTGCTGAAGCACTGC ACGCCGTAGGTCAGGGTGGTCACCAGGGTGGGCCAGGGCACGGGCAG CTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGC GTCGCCCTCGCCCTCGCCGCTCACGCTGAACTTGTGGCCGTTCACGTCGC CGTCCAGCTCCACCAGGATGGGCACCACGCCGGTGAACAGCTCCTCGCC CTTGCTCACGGGGCCGGGGTTCTCCTCCACGTCGCCGGCCTGCTTCAGC AGGCTGAAGTTGGTGGCCAGGATCCTCTCGCACAGCCTCCAGCCGGTCA CGCCGTTGATGGTCACCCTGAACAGCAGGCTGCCGTCGGGGTTGATCA GCCTCTCGTCGATGATCTTGTTGCCGTTCCACAGGGTGCCGGTCACGGT GATCTTCTTGCCGTCGAACACGGCGATGCCCTCGTAGGGCCTGCCGAAG TAGTCGATCATGTTGGGGGTCACGCCGTCGATCACCAGGGTGCCGTAGT GCAGGATCACCTTGAAGTGGTGGTCGTCCACGGGGTACACCACCTTGAA AATCTTCTCGATCTGGCCCATCTGGTCGCCGCTCAGGCCCTCGTAGGGG ATGATCACGTGGATGTCGATCTTCAGGCCGTTCTCGCCGCTCAGCACGA TCCTCTGGATGGGGGTCACGCTCACGCCCAGGTTCTGGAACAGGCTGCT CACGCCGCCCTGCTCCAGCACCTGGTCCAGGTTGTAGCCGGCGGTCTGC CTCCAGTCGCCCACGAAGTCCTCCAGGGTGAACACGGCCTCCTCGAAGC TGCACTTCTCCTCCATGCACTCCCTCTCCAGGTTGCCCTGCACGAACTCCT CCAGCTTGCCGCTGTTGTACCTCTTGGGCCTGTTCAGGATCTTGTTGGCG TTCTCGTGGTCCAGGAAAACTGAAATGTAAAAGAATAATTCTTTAG Illi AGCAAAAAAGAAAACATCATGAAAAI 1 1 1ACATCTCTTAAGAAAGTGTT TAAACACGCGGCCGCGTAGATAAGTAGCATGGCGGGTTAATCATTAACT ACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTT TGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGTTAATTAAGGCG CCCTAGGCCGACCCTTAGACTCTGTACTCAGTTCTATAAACGAGCCATTG GATACGAGATCCGTAGATTGATAAGGGACACGGAATATCCCCGGACGC AATAGACACCGGTGGACAGCTTGGTATCCTGAGCACAGTCGCGCGTCC GAATCTAGCTCTACTTTAGAGGCCCCGGATTCTGATGGTCGTAGACCGC AGAACCGATTGGGGGGATGAGATCTACTAGTTATCAGCACACAATTGCC CATTATACGCGCGTATAATGGACTATTGTGTGCTGATATAGGGATAACA GGGTAATTCTAGAGCTAGCATATGGATCCATCGATTTGATGCGGTAI 1 1 1 CTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCA TAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTT TCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAG CTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCAT CGCCCTGATAGACGG1 1 1 1 1CGCCC1 1 1GACG1 1GGAG1CCACG1 1L1 1 1 AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGG CTATTCI 1 1 IGAI 1 IAIAAGGGAI 1 1 1GCCGATTTCGGTCTATTGGTTAAA AAATGAGCTGATTTAACAAAAATTTAACGCGAAI 1 1 1AACAAAATATTAA CGI 1 IACAAI 1 1 1 Al GGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA TAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC CGGGAGCTGCATGTGTCAGAGG Illi CACCGTCATCACCGAAACGCGCG AGACGAAAGGGCCTCGTGATACGCCTAI 1 1 1 1ATAGGTTAATGTCATGA TAATAATGGTTTCTTAGACGTCAGGTGGCACI 1 1 1CGGGGAAATGTGCG CGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCA ACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGG GCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCA GAGTTGI 1 ICIGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAG ATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGAC CATCAAGCAI 1 1 1ATCCGTACTCCTGATGATGCATGGTTACTCACCACTG CGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTC AGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCAT TCGATTCCTGTTTGTAATTGTCCI 1 1 1AACAGCGATCGCGTATTTCGTCTC GCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATT TTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAA TGCATAAAC Illi GCCATTCTCACCGGATTCAGTCGTCACTCATGGTGAT TTCTCACTTGATAACCTTAI 1 1 1 1GACGAGGGGAAATTAATAGGTTGTAT TGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATC CTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTT TCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATT TGATGCTCGATGAGTTTTTCTAAGCGTATAATGGTCTAGAGCTAGCATAT GGATCCATCGATTCCATTATACGCCTGTCAGACCAAGTTTACTCATATAT ACTTTAGATTGATTTAAAACTTCAI 1 1 1 1AATTTAAAAGGATCTAGGTGA AGATCCI 1 1 1 1G A1AA1C1CA1G ACCAAAA1CCC1 1AACG1G AG 1 1 1 ICGT TCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGA TCC1 1 1 1 1 1 1C1GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAG TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGAI 1 1 1 1GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA ACGCCAGCAACGCGGCC1 1 1 1 1ACGG1 1CC1GGCC Illi GCTGGCCTTTT GCTCACATGT 215 mRNA_2xNLS-B-GEn_l-Hys-Tdep_Trilink AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC CAUGGGCAAGAGGCCAGCCGCCACAAAGAAGGCCGGCCAGGCCAAGA AGAAGAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCAGCAUGCCCAUCCGGAGCUUCAAGCUGAAGCUGGUG ACCCACAACGGCGACAGCACCUACAUGGACAAGCUGCGGCGGGGCCU GUGGAAGACCCACGUGAUCAUCAACCGGGGCAUCGCCUACUACAUG AACACCCUGGCCCUCAUGCGGCAGGAGCCCUACGGCUCCAAGAGCCG GGAGGAGGUGCGGCUGGACCUGCUGAGCACCCUGCGGGAGCAGCAG CGGCGGAACAACUGGAGCGAGCAGACCGGCACCGACGACGAGCUGCU GAGCCUGAGCCGGCGGGUGUACGAGCUGCUGGUGCCCAGCGCCAUC GGCGAGAAGGGCGACGCCCAGAUGCUGAGCCGGAAGUUCCUGAGCC CCCUGGUGGACCCCAACAGCGAGGGCGGGCGGGGCACCGCCAAGAGC GGCCGGAAGCCCCGGUGGAAGAAGAUGAUGGAGGAGGGCCACCCCG ACUGGGAGAAGGAGAAGGAGAAGGACGCCGCCAAGAAGGCCGAGGA CCCCACCGCCAGCAUCCUGGCCGACCUGGAGGCCGUGGGCCUGCUGC CCCUGUUCCCCCUGUUCAGCGACGAGCAGAAGGAGAUCCGGUGGCU GCCCAAGAAGAAGCGGCAGUUCGUGAGGACCUGGGACCGGGACAUG UUCCAGCAGGCCCUGGAGCGAAUGCUGAGCUGGGAAAGCUGGAACC GGCGGGUGGCCGAGGAGUACCUGAAGCUGCAGGCCCAGCGGGACGA GGUGUACGCCAAGUACCUGGAGGACGCCGGCAGCUGGCUGAACGAC CUGCAGACCUUCGAGAAGCAGCGGGAAGAGGAGCUGGCCGAGGUGA GCUUCGAGCCCAACAGCGAGUACCUGAUCACCCGGCGGCAGAUCCGC GGCUGGAAGGAGGUGUACGAGAAGUGGAGCAAGACCAGCGAGAACG CCAGCCAGGAGCAGCUGUGGCGGAUGGUGGCCGACGUGCAGACCGC CAUGGCCGGCGCCUUCGGCGACCCCAAGGUGUACCAGUUCCUGAGCC AGCCCAAGCACCACCACAUCUGGCGGGAGCACCCCAACAGACUGUUC UACUACAGCAAGUACAACGAGGUGCGGGAGAAGCUGAACAGAGCCA AGAAGCAGGCCGCCUUCACCCUGCCCGACCCCGUGGAGCACCCCCUG UGGACCCGGUUCGACGCCCGGGGCGGCAACAUCCACGACUACGAGAU CAGCAAGGUGGGCAAGCAGUACCACGUGACCUUCAGCAGCCUGAUCC UGCCCGAGGCCCAGAGCUGGGUGGAGAUCGAGAACGUGACCGUGGG CAUCGGCAACAGCCUGCAGCUGAAGCGGCAGAUCCGCCUGGACGGCU ACGCCGACAAGAAGCAGAAGGUGAAGUACUACGACUACAGCAGCAGA UUCGAGCUGACAGGCGUACUGGGCGGCGCCAAGAUCCAGUUCGAUA GAAAGCACCUGAAGAAGGCCGCCCACAGACUGGCCGAGGGCGAGACC GGCCCCAUCUUCCUGAAUGUGGUGGUGGACGUGGAGCCCUUCCUGG AGGUGAAGAACGGCCGGCUGCGGACCCCCCUGGGCCAGGUGCUGCA GGUGAACACCAGAGACUGGCCCAAGGUGGUGGACUACAAGGCCAAG GAGCUGAGCGUGCUGAUGGAGAACACCCAAAUCGGCAACGAGAACG GCGUGAGCACCAUCGAGGCCGGAAUGAGAAUCAUGAGCAUCGACCU GGGCCAGAGAACCGCCGCCGCCGUGAGCAUCUUCGAGGUCAUCAGCA AGAAGCCCGACGAGAAGGAGACCAAGCUGUUCUACCCCAUCGCCGAC ACCGACCUGUACGCCGUGCACAGACGGAGCCUGCUGCUGAGACUGCC CGGCGAGGAGAUCAGCAGCAAGAAGAUGAUCGAGAAGCGGAAGGAG CGCGCCCGGAUCCGGAGCCUGGUGAGAUACCAGAUCAGACUGCUGA GCGAGGUGCUGCGGCUGCACACCCAGGGCACCGCCGAGCAGAGAAGA UUCAAGCUGGACGAGCUGCUGGUGAGCAUCCAGAAGAAGCUGGAGC UGGACCAGAGCGAGUGGAUCAGCGAGCUGGAGAAGCUGUUCGACUA CAUCGACGAGAGCGCCGAGAAGUGGAAGGAGGCCCUGGUCGUGGCA CACAGAACCCUGGAGCCCAUCGUGGUGGAGGCCGUCAGAAACUGGA AGAAGAGCCUGAGCAAGGAGAACAAGGACAGACGGAGAAUCGCCGG AAUCUCCAUCUGGAGCAUCGAGGAGCUGGAGGAGACCCGGAAGCUG CUGAUCGCCUGGAGCAAGCACAGCAGAGAGCCCGGCAUCCCCAAGAG ACUGGAGAAGGAGGAGACCUUCGCCCCCGAGCACCUGCAGCACAUCC AGAACGUGAAGGACGACCGGCUGAAGCAGAUGGCCAACCUGUUCGU GAUGACCGCCCUGGGCUAUAAGUAUGACGAGGGCAACAAGCGGUGG GUGGAGGCCUACCCCGCCUGCCAGGUGAUCCUGUUCGAGGACCUGA GCAGAUACCGGUUCGCCCUGGACAGACCCAGGCGGGAGAACAAUCG GCUGAUGAAGUGGGCCCACCGCAGCAUCCCCAGACUGACCUACAUGC AGGCCGAGCUGUUCGGCAUCCAGGUGGGCGACGUCUACAGCGCCUA CACCAGCAGAUUCCACGCCAAGACCGGCGCCCCCGGGAUCCGGUGCC ACGCCCUGACCGAGGCCGACCUGCAGAGCAACAGCUACGUGGUGAAC CAGCUGAUCAAGGACAAGUUCAUCCAGGACAACCAGACCGAGAUCCU GAAGGCCGGCCAGAUCGUGCCCUGGCAGGGCGGCGAGCUGUUCGUC ACCUUCGCCGACAGAAGCGGCGCCAGCCUGGCCGUGAUCCACGCCGA UAUCAACGCCGCCCAGAACCUGCAGAAGAGAUUCUGGCAGCACAACA GCGAGGUGUUCCGGGUGCCCUGCAAGGUGGUGAAGGGCGGCCUGG UGCCCGUGUACGAGAAGAUGCGGAAGCUGUUCGGCAAAGGCCUGUU CGUGAACAUCGACGACCCCGAGAGCAAGGAGGUGUACCGGUGGGAG CACUCCACCAAGAUGAAGAGCAAGACCACCCCAGUGGACCUGGAGAG CGAGGACAUCGACCACGAGGAGCUGAGCGACGAGUGGGAGGACAUG CAGGAGGGCUACAAGACCCUGCUGCGGGACCCCUCAGGCUUCUUCU GGAGCAGCGACAGCUGGAUCCCCCAGAAGGACUUCUGGAUCAGAGU GAAGAGCAGAAUCGGCAAGAGCCUGCGGGAGCAGAUCCGGGGUGGA AGCGGAGGCAGCGGGGGCAGCGGUCCCCCUAAGAAAAAGCGGAAGG UGGGCGGUGGCGGCGGCAGCCAUCAUCAUCACCAUCAUUGAGCUGC CUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUG CACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAA 216 insert of pBLR3106b TTCTAGTAATGGAAGCCTGGTAT 217 insert of pBLR3107 CTAGAAAAATACAAGCAGAGATG 218 insert of pBLR3108 TAAAATAGTTAAA1 1 1 1CC1 1 1A 219 insert of pBLR3109 TAAAATACCAGGCTTCCATTACT 220 insert of pBLR3110 AACTAI 1 1 IAAAATACCAGGCTT 221 insert of pBLR3111 TCC1 1 1AGTGCTGA HILI AGAT 222 insert of pBLR3112 CTAGATTATTATTACTGTTGTTG 223 insert of pBLR3113 TTATTACTGTTGTTGTTGTTATT 224 insert of pBLR3114 TTACTGTTGTTGTTGTTATTATT 225 insert of pBLR3115 CTGTTGTTGTTGTTATTATTGTC 226 insert of pBLR3116 TTGTCATTATTTGCATCTGAGAA 227 insert of pBLR3117 TCATTATTTGCATCTGAGAACCC 228 insert of pBLR3118 TTTGCATCTGAGAACCCTTAGGT 229 insert of pBLR3119 GCATCTGAGAACCCTTAGGTGGT 230 insert of pBLR3120 TTGATATA1 1 1 1 1GG fATCTTTG 231 insert of pBLR3121 ATATA1 1 1 1 1 GG 1ATCTTTGATG 232 insert of pBLR3122 TTGGTATCTTTGATGACAATAAT 233 insert of pBLR3123 TCATCAAAGATACCAAAAATATA 234 insert of pBLR3124 TTGTCATCAAAGATACCAAAAAT 235 insert of pBLR3125 TGAAAGCTTAGC1 1 1 AAA HILI 236 insert of pBLR3126 AAAGCTAAGCTTTCAAAATCCCC 237 insert of pBLR3127 Cl 1 1 1AATTAAAAAAAAATGCTA 238 insert of pBLR3128 AAAGAAATTTAAAGCTAAGCTTT 239 insert of pBLR3129 AAAAAAAATGCTAGGCAGAATGA 240 insert of pBLR3130 1 1 1 1 1 1AATTAAAAGAAATTTAA 241 insert of pBLR3131 TGCCTAGCAI1 1 1 1 1 1 1IAATTA 242 insert of pBLR3132 GAGTCATTCTGCCTAGCAI Illi 243 insert of pBLR3133 CGTTGGATACAGTTGAATTTATT 244 insert of pBLR3134 AACTGTATCCAACGTAATTTGAG 245 insert of pBLR3135 ATTACGGTCTCATAGGGCCTGCC 246 insert of pBLR3136 CGGTCTCATAGGGCCTGCCTGCT 247 insert of pBLR3137 TTAGTATAGCATGGTCGAGCAGG 248 insert of pBLR3138 AAAGTGTGTGTTACTAAI 1 1 IAI 249 insert of pBLR3139 GTAACACACAC1 1 1 IAAI 1 1 1 IA 250 insert of pBLR3140 TATAAATGGAGTTTCCATTTATA 251 insert of pBLR3141 ATAAAATTAGTAACACACACTTT 252 insert of pBLR3142 ATATTTACCTTTATTTCTTATTT 253 insert of pBLR3143 ACCTTTATTTCTTATTTACCATT 254 insert of pBLR3144 CTTATTTACCATTGTCTTAGTAG 255 insert of pBLR3145 ACCATTGTCTTAGTAGATATTTA 256 insert of pBLR3146 TCTTAGTAGATATTTACAAACAT 257 insert of pBLR3147 ACAAACATGACAGAAACACTAAA 258 insert of pBLR3148 AGTGI 1 ICIGICAIGI 1 IGTAAA 259 insert of pBLR3149 AAACTCAAGATTTAGTG1 1 1L1G 260 insert of pBLR3150 TTAAAACCCGTTAAGTGTTTATA 261 insert of pBLR3151 1 1 1 11AAAACCCGTTAAGTGTTT 262 insert of pBLR3152 TTAI 1 1 1 IAAAACCCGTTAAGTG 263 insert of pBLR3153 TTTCACCAACATTATTAI 1 1 1 IA 264 insert of pBLR3154 CCACCTTCAGAI 1 1 ICCTGTAAC 265 insert of pBLR3155 TCCTGTAACGATCGGGAACTGGC 266 insert of pBLR3156 CAGTTAGTTGTCTTCATCAATCT 267 insert of pBLR3157 ATGAAGACAACTAACTGTAATAT 268 insert of pBLR3158 AAAGATTGATGAAGACAACTAAC 269 gRNA sequence for B-GEn.l mouse albumin target 1 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUCU AGUAAUGGAAGCCUGGUAU 270 gRNA sequence for B-GEn.l mouse albumin target 2 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUAG AAAAAUACAAGCAGAGAUG 271 gRNA sequence for B-GEn.l mouse albumin target 3 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAAA AUAGUUAAAUUUUCCUUUA 272 gRNA sequence for B-GEn.l mouse albumin target 4 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAAA AUACCAGGCU UCCAU UACU 273 gRNA sequence for B-GEn.l mouse albumin target 5 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAACU AUUUUAAAAUACCAGGCUU 274 gRNA sequence for B-GEn.l mouse albumin target 6 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCCU UUAGUGCUGAUUUCUAGAU 275 gRNA sequence for B-GEn.l mouse albumin target 7 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUAG AUUAUUAUUACUGUUGUUG 276 gRNA sequence for B-GEn.l mouse albumin target 8 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAU UACUGUUGUUGUUGUUAUU Til gRNA sequence for B-GEn.l mouse albumin target 9 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAC UGUUGUUGUUGUUAUUAUU 278 gRNA sequence for B-GEn.l mouse albumin target 10 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUGU UGUUGUUGUUAUUAUUGUC 279 gRNA sequence for B-GEn.l mouse albumin target 11 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUGU CAUUAUUUGCAUCUGAGAA 280 gRNA sequence for B-GEn.l mouse albumin target 12 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCAU UAUUUGCAUCUGAGAACCC 281 gRNA sequence for B-GEn.l mouse albumin target 13 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUG CAUCUGAGAACCCUUAGGU 282 gRNA sequence for B-GEn.l mouse albumin target 14 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGCAU CUGAGAACCCUUAGGUGGU 283 gRNA sequence for B-GEn.l mouse albumin target 15 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUGA UAUAUUUUUGGUAUCUUUG 284 gRNA sequence for B-GEn.l mouse albumin target 16 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAU AUUUUUGGUAUCUUUGAUG 285 gRNA sequence for B-GEn.l mouse albumin target 17 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUGG UAUCUUUGAUGACAAUAAU 286 gRNA sequence for B-GEn.l mouse albumin target 18 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCAU CAAAGAUACCAAAAAUAUA 287 gRNA sequence for B-GEn.l mouse albumin target 19 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUGU CAUCAAAGAUACCAAAAAU 288 gRNA sequence for B-GEn.l mouse albumin target 20 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGAA AGCUUAGCUUUAAAUUUCU 289 gRNA sequence for B-GEn.l mouse albumin target 21 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAG C UAAGC U U U CAAAAU CCCC 290 gRNA sequence for B-GEn.l mouse albumin target 22 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUUU UAAUUAAAAAAAAAUGCUA 291 gRNA sequence for B-GEn.l mouse albumin target 23 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAG AAAUUUAAAGCUAAGCUUU 292 gRNA sequence for B-GEn.l mouse albumin target 24 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAA AAAAUGCUAGGCAGAAUGA 293 gRNA sequence for B-GEn.l mouse albumin target 25 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUU UUAAUUAAAAGAAAUUUAA 294 gRNA sequence for B-GEn.l mouse albumin target 26 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUGCC UAGCAUUUUUUUUUAAUUA 295 gRNA sequence for B-GEn.l mouse albumin target 27 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGAGU CAUUCUGCCUAGCAUUUUU 296 gRNA sequence for B-GEn.l mouse albumin target 28 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCGUU GGAUACAGUUGAAUUUAUU 297 gRNA sequence for B-GEn.l mouse albumin target 29 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAACU GUAUCCAACGUAAUUUGAG 298 gRNA sequence for B-GEn.l mouse albumin target 30 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUUA CGGUCUCAUAGGGCCUGCC 299 gRNA sequence for B-GEn.l mouse albumin target 31 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCGGU CUCAUAGGGCCUGCCUGCU 300 gRNA sequence for B-GEn.l mouse albumin target 32 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAG UAUAGCAUGGUCGAGCAGG 301 gRNA sequence for B-GEn.l mouse albumin target 33 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAG UGUGUGUUACUAAUUUUAU 302 gRNA sequence for B-GEn.l mouse albumin target 34 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACGUAA C AC AC AC U U U UAAU U U U UA 303 gRNA sequence for B-GEn.l mouse albumin target 35 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUAUA AAUGGAGUUUCCAUUUAUA 304 gRNA sequence for B-GEn.l mouse albumin target 36 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAA AAU U AGU AACACACACU U U 305 gRNA sequence for B-GEn.l mouse albumin target 37 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUAU UUACCUUUAUUUCUUAUUU 306 gRNA sequence for B-GEn.l mouse albumin target 38 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACACCU UUAUUUCUUAUUUACCAUU 307 gRNA sequence for B-GEn.l mouse albumin target 39 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCUUA UUUACCAUUGUCUUAGUAG 308 gRNA sequence for B-GEn.l mouse albumin target 40 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACACCA UUGUCUUAGUAGAUAUUUA 309 gRNA sequence for B-GEn.l mouse albumin target 41 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCUU AG U AGAU AU U U ACAAACAU 310 gRNA sequence for B-GEn.l mouse albumin target 42 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACACAA ACAUGACAGAAACACUAAA 311 gRNA sequence for B-GEn.l mouse albumin target 43 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAGUG UUUCUGUCAUGUUUGUAAA 312 gRNA sequence for B-GEn.l mouse albumin target 44 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAC UCAAGAUUUAGUGUUUCUG 313 gRNA sequence for B-GEn.l mouse albumin target 45 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAA AACCCGUUAAGUGUUUAUA 314 gRNA sequence for B-GEn.l mouse albumin target 46 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUU UAAAACCCGUUAAGUGUUU 315 gRNA sequence for B-GEn.l mouse albumin target 47 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUAU UUUUAAAACCCGUUAAGUG 316 gRNA sequence for B-GEn.l mouse albumin target 48 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUUUC ACCAACAUUAUUAUUUUUA 317 gRNA sequence for B-GEn.l mouse albumin target 49 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCCACC UUCAGAUUUUCCUGUAAC 318 gRNA sequence for B-GEn.l mouse albumin target 50 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACUCCU GUAACGAUCGGGAACUGGC 319 gRNA sequence for B-GEn.l mouse albumin target 51 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACCAGU UAGUUGUCUUCAUCAAUCU 320 gRNA sequence for B-GEn.l mouse albumin target 52 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAUGA AGACAACUAACUGUAAUAU 321 gRNA sequence for B-GEn.l mouse albumin target 53 GUUAGCUAUAGGCUAAUAAGAUAGUUGUGUCAAGUGCUUCGGAGA CCUAACACGUCAUCCAGUCACAACGGCUAAAAAUAGCCAGCACAAAG AUUGAUGAAGACAACUAAC 322 pBLR3106b SEQ. ID NO: 322-374 see sequence listing 323 pBLR3107 324 pBLR3108 325 pBLR3109 326 pBLR3110 327 pBLR3111 328 pBLR3112 329 pBLR3113 330 pBLR3114 331 pBLR3115 332 pBLR3116 333 pBLR3117 334 pBLR3118 335 pBLR3119 336 pBLR3120 337 pBLR3121 338 pBLR3122 339 pBLR3123 340 pBLR3124 341 pBLR3125 342 pBLR3126 343 pBLR3127 344 pBLR3128 345 pBLR3129 346 pBLR3130 347 pBLR3131 348 pBLR3132 349 pBLR3133 350 pBLR3134 351 pBLR3135 352 pBLR3136 353 pBLR3137 354 pBLR3138 355 pBLR3139 356 pBLR3140 357 pBLR3141 358 pBLR3142 359 pBLR3143 360 pBLR3144 361 pBLR3145 362 pBLR3146 363 pBLR3147 364 pBLR3148 365 pBLR3149 366 pBLR3150 367 pBLR3151 368 pBLR3152 369 pBLR3153 370 pBLR3154 371 pBLR3155 372 pBLR3156 373 pBLR3157 374 pBLR3158 375 Cas9_552 mA*mU*mC*rGrGrGrArArCrUrGrGrCrArUrCrUrUrCrArGrUrUrUrUrA rGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrC rUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrA rCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU 376 amplicon_T56 TTTGCATCTGAGAACCCTTAGGTGGTTATATTATTGATATAI 1 1 1 IGGTAT CTTTGATGACAATAATGGGGGA Illi GAAAGC1 1AGC1 1 1 AAA 1 1 1L1 1 1 TAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGATACA GTTGAATTTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATAC T 377 amplicon_T60 TTTGCATCTGAGAACCCTTAGGTGGTTATATTATTGATATAI 1 1 1 IGGTAT CTTTGATGACAATAATGGGGGA Illi GAAAGC1 1AGC1 1 1 AAA 1 1 1C1 1 1 TAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGATACA GTTGAATTTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATAC T 378 amplicon_T61 CTGAAGGTGGCAATGGTTCCTCTCTGCTACACTCAAAGTTATAI 1 1 1 1 IC ACCAACATTATTAI 1 1 1 1AAAACCCGTTAAGTGTTTATATCTGTGCATTCA AACTCAAGATTTAGTG1 1 1C1GTCATGTTTGTAAATATCTACTAAGACAA TGGTAAATAAGAAATAAAGGTAAATATAAATGGAAACTCCATTTATAAA ATTAGTAACACACAC1 1 1 1AAI 1 1 1 1AGTATAGCATGGTCGAGCAGGCAG GCCCTATGAGACCGTAAT 379 Amplicon_T62 ATACCGATGGGCGATCTCACTCTTGTCTGTGGAAACAGGGAGAGAAAA ACCACACAACATATTTAAAGATTGATGAAGACAACTAACTGTAATATGCT GC1 1 1 1 1GTTCTTCTCTTCACTGACCTAAGCTACTCCCTGAAGATGCCAGT TCCCGATCGTTACAGGAAAATCTGAAGGTGGCAATGGTTCC 380 B-ms-26 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrUrGrCrCrUrArGrCrArUrUrUrUrUrUrUrUrUrArA*mU*mU* mA 381 B-ms-39 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrCrUrUrArUrUrUrArCrCrArUrUrGrUrCrUrUrArG*mU*mA* mG 382 B-ms-40 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrArCrCrArUrUrGrUrCrUrUrArGrUrArGrArUrArU*mU*mU* mA 383 B-ms-49 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrCrCrArCrCrUrUrCrArGrArUrUrUrUrCrCrUrGrU*mA*mA*m C 384 B-ms-52 mG*mU*mU*rArGrCrUrArUrArGrGrCrUrArArUrArArGrArUrArGrUr UrGrUrGrUrCrArArGrUrGrCrUrUrCrGrGrArGrArCrCrUrArArCrArCrGr UrCrArUrCrCrArGrUrCrArCrArArCrGrGrCrUrArArArArArUrArGrCrCrA rGrCrArCrArUrGrArArGrArCrArArCrUrArArCrUrGrUrArA*mU*mA*m U "*" represents a phophorothioate linkage, and "m" stands for 20' methyl The inserts of the pBLR plasmids are also referred to as spacers. Examples Example 1 Cloning and plasmid preparation The cloning of the B-Gen.l expression vector pBLR709, the transfection in HEK293T cells, AMP-Seq, DNA library preparation, and NGS analysis is described in detail in WO2022258753 (with the nuclease of SEQ. ID NO: 2, this is also disclosed in this application under SEQ ID NO: 185). The cloning of the human albumin guide RNA expression vectors was done as follows. Acceptor vector pBLR1936 was digested with Bbsl-HF (New England Biolabs) according to manufacturer's guidelines. Sense and antisense strand matching oligonucleotides, which encompass the respective spacer sequence of SEQID NOs: 1-61 were annealed to generate small double stranded DNA fragments with fitting overhangs. Annealed oligonucleotidea were incubated with pre-cut pBLR1936 in a Golden Gate reaction according to manufacturer's guidelines to generate pBLR2521-2582. Single Golden Gate reactions were transformed and propagated in ToplO electrocompetent E.Coli. Plasmids were Sanger sequenced, and positive clones were propagated as medium (midi) cultures and isolated with the NuceloSnap Plasmid Midi Kit for plasmid DNA (Macherey Nagel). Cell culture of HEK293T cells HEK293T (ATCC® CRL-3216™) cells were cultivated in DMEM medium with 10% FCS and 1% Pen / Strep in T75 flasks. Cells were passaged every third day. Transfection in HEK293T cells One day prior transfection cells were split as described above and counted with the Luna-FL™ Dual Fluorescence Cell Counter Counter (Biocat) according to manufacturer's guidelines. Cells were plated in a poly-D-lysine coated 96 well plate at a density of 18,000 per well in lOOpI Medium. On the day of transfection medium was changed. LipoD293™ (SignaGen) reagent was adjusted to room temperature shortly before transfection (~10min). At the day of transfection plasmid 140ng of pBLR709 DNA was mixed with the 60ng of guide RNA plasmid or no guide RNA plasmid (nuclease only control) DNA and medium without antibiotics and FCS was added to lOpI total. In another tube lOpI DMEM without additives was mixed with 0.3pl of LipoD293. The Lipo mixture was added to the DNA mixture. Plate was sealed, shaked, spun down at 300g for 10 seconds and put at room temperature for 15 minutes. After that transfection mix was added to the cells. After three days post transfection cells were harvested. Medium was aspirated and 50pl of PBS was added per well. After that 25pl of TrypLE was added on top. Cells were trypsinized for 15 minutes at 37C. 60 pl of PBS was added for resuspension. 60 pl of the cell suspension was added to PCR tubes in a 96 well plate format. Cells were spun down at 300g for 3 minutes. Supernatant was discarded and cells were lysed in 60pl Lysis buffer (10 mM Tris pH 7.0, 0.05% SDS) + 1:800 Proteinase K in a heat cycler. AAV preparation AAVs of reporter 2 with a bidirectional promoter-less nanoluc-eGFP-polyA cargo (SEQID NO: 210) were generated. guide RNA (gRNA) Synthetic guides (SEQID NOs: 195-199, 205-209) were ordered as research-use-only (RUO) at IDT or Axolabs. gRNA was reconstituted with IDTE pH 7.5 and stored at -70C in 5pl aliquots, which were used for one time usage. mRNA sourcing The SpCas9 mRNA was sourced from Trilink. The ORF sequence can be found here: trilinkbiotech.com / media / maravai / productattachments / product insert / cas9 catno 1-8106 1-7206 I-7606 .txt as accessed on 2024-12-17. The B-GEn.l mRNA below comprise B-GEn.l ORF SEQID NO: 210. mRNA of B-GEn.l was T depleted manually by changing T-rich codons into T-less codons using a codon table with preferred usage for humans. Codons were chosen to have the minimum possible uridine content while maximizing the expression of the corresponding tRNA in the liver. Reducing uridine content was intended to decrease the innate immune response to the mRNA and provide other benefits. The SpCas9 mRNA and SEQID NO: 210 used mRNA in this study was sourced from Trilink as a research-use-only (RUO) product, created using PCR amplification of a plasmid template to add the poly A sequence. All mRNA had the following characteristics: Nl-methyl pseudo-U modification, a 120 nt polyA tail, CleanCap AG capping reagent, purification with silica membrane, and resuspended in 1 mM Sodium Citrate at pH 6.4. LNP preparation protocol For the experiments described in other examples, the LNPs were prepared using a cross - flow technique utilizing impinging jet mixing of the lipid in ethanol with three volumes of RNA solutions and one volume of water. The lipid in ethanol was mixed through a mixing cross with the three volumes of RNA solution. A fourth stream of water was mixed with the outlet stream of the cross through an inline tee. The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1: 1 v / v ) . Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, lOOkD MWCO ) and then buffer exchanged by diafiltration into 50 mM Tris , 45 mM NaCI, 5 % ( w / v ) sucrose , pH 7.5 ( TSS ) . Alternatively, the final buffer exchange into TSS was completed with PD - 10 desalting columns. If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 um sterile filter. The final LNP was stored at -80 ° C. until further use. The LNPs were formulated at a molar ratio of ionizable lipid: cholesterol: DSPC: PEG2k DMG of 50 : 38 : 9 : 3 , with a lipid amine to RNA phosphate ( N : P ) molar ratio of about 6.0 , and a ratio of gRNA to mRNA of 1: 1 by weight. Cell culture PHH Maintenance Medium -Williams' Medium E (Cat. W1878-500ML, Sigma Aldrich) with 5 % FCS (Cat. S0615, Sigma Aldrich), 1 % P / S (Cat. P4333, Sigma Aldrich), 15 mM HEPES (Cat. 15630-056, Gibco), lx ITS (Cat. I3146-5ML, Sigma Aldrich), final 6.25 pg / mL, Insulin, 6.25 pg / mL Transferrin, 1.25 ng / mL Selenious Acid), lx GlutaMAX (Cat. 35050-038, Gibco), 50 pg / mL Gentamycin (Cat. G1272, Sigma Aldrich), 100 nM Dexamethason (A13449, Gibco) Primary human hepatocytes (Lonza, various Lots.) Seeding and treatment Hepatocytes (PHH) were thawed and resuspended in 35 mL Maintenance Medium. A solution of 90 % Percoll (diluted in lOx PBS) was added. The resuspended cells were mixed carefully and centrifuged for 5 min at 150 x g. The supernatant was discarded, and the pelleted cells were resuspended in 50 mL Maintenance Medium and centrifuged for 5 min at 150 x g. The resulting cell pellet was resuspended carefully. Cells were counted and plated on Tissue Culture treated 96-well plates that have prior been coated for 30 minutes with 0.0006 % Collagen R (Cat. 47254.02, SERVA) at a density of 50,000 cells / well. Plated cells were allowed to settle and adhere for 1 hour at room temperature and 3 hours in a tissue culture incubator at 37 °C and 5 % CO2. After incubation cells were checked for monolayer formation, were washed twice with Maintenance Medium. Transfection of PHH cells Prior to the treatment the Lipid Nano Particles (LNP) containing Cas9 or B-Gen.l mRNA were diluted in phosphate buffered saline (PBS) using a serial dilution of 1:2 starting at 40 pg RNA / mL to 0.625 pg / mL The resulting dilutions were further diluted 1:4 in Maintenance Medium (e.g. 40 pL LNP dilution + 120 pL Maintenance Medium). The AAV containing NanoLuciferase DNA were diluted to an MOI of 2e5 (6.25E10 vg / mL) in Maintenance Medium. Media was aspirated from the cells prior to transfection and 40 pL of each LNP were added to the cells followed by the addition of 120 pL diluted AAV. The cells were incubated in a tissue culture incubator at 37 °C and 5 % CO2. 24 hours after the treatment the supernatant was aspirated, and cells were washed twice with serum free medium. A solution of 20 % Collagen I (diluted in serum free medium) was added to each well. The plates were incubated at 37 °C. After two hours the plates were checked to see whether the gel had solidified, and 200 pL of Maintenance Medium were added. The cells were imaged and the supernatants were collected on day 3, day 6 and day7 post transfection including the replacement of the culture medium. On day 7 the cells were frozen to -20 °C for further analysis. RLU measurement: For experiments involving NanoLuc detection in cell media, one volume of Nano-Gio® Luciferase Assay Substrate was combined with 50 volumes of Nano-Gio® Luciferase Assay Buffer. The assay was run on a Biotek Synergy Neo2 Hybrid Multi-Mode Microplate Reader by Agilent at an integration time of 0.2 sec and a gain of 200. The undiluted samples were combined with assay reagent at a ratio of 1 + 1 (e.g. 10 pl + 10 pl) in black 384 well plates with a closed bottom. After combining samples and reagent the plate was incubated on a plate shaker with moderate speed for 1 minute and measured thereafter. Crude Lysis from cell lines and primary human hepatocytes Frozen cells were lysed in 30 pl (PHH) or 60 pl (HEK293T and HEPA1-6) crude lysis buffer (lOmM Tris pH7.0, 0.005% SDS) supplemented with Proteinase K (1:800 dilution). The solution was transferred in 96 well plates, sealed. Cycler conditions were following: Stepl 60 minutes 37°C, 20 minutes 65°C. Genomic DNA extraction from mice Tissue from liver was cut to approx. 10 mg pieces using a scalpel. Tissue homogenization was done with a the Qiagen TissueLyser II according to the protocol guidelines. DNA extraction was performed with the KingFisher Apex according to manufacturer guidelines. Amplicon generation To generate the amplicon, endpoint PCR with barcoded primer was performed. The PCR reaction was prepared as follows: 12,5 pL 2x Q5 Mastermix, 8 pL H2O, 3pL Barcoded primer, 1.5 pL crude lysis genomic DNA Following cycler conditions were used: Step 1: 98C for 30sec, Step 2: 98C for lOsec, Step 3: 66C for 20sec, Step 4: 72C for 20sec Step 5: 32 cycles of steps 2-4, Step 6: 72C for 2 min, Step 7: 12C hold To allow gene editing assessment amplicons from human albumin intron 1 (SEQID NO: 188-193) and mouse albumin intron 1 (SEQID NOs: 194,376-379) were established. DNA library preparation and NGS analysis All PCR reactions were pooled. For that 5 pl of single reactions were pooled. Total volume of pooled PCR was mixed with equal amount of AMPure XP Bead cleanup. After incubation on a rotor for 5 minutes mixture was placed on a magnet and DNA was extracted according to manufacturer's guidelines. To ligate Illumina adapters to the sheared fragments ends were repaired. For that the NEBNext® Ultra™ II End Repair / dA-Tailing Module was used. In total 2pg of the amplicon (up to 80 pL) was incubated with 6pl of NEBNext Ultra II End Prep Enzyme Mix and 14 pl NEBNext Ultra II End Prep Reaction Buffer. After that it was filled with H2O ad lOOpl. The reaction in Thermocycler used the following protocol: 30 minutes @ 20°C, 30 minutes @ 65°C, Hold at 4°C. Samples were cleaned up with AMPure XP Bead Cleanup and eluted in 32 pl EB buffer. For the ligation we use the Blunt / TA Ligase Maser Mix from NEB and Illumina adapters from the TruSeq DNA PCR-Free LT Sample Prep Kit. 30 pL of End-prepped DNA was mixed with 2pL Illumina and 32 pL of Blunt / TA Ligase Master Mix. This was incubated at lh at 21°C in a thermocycler. Then 34 pl H2O was added to a total of lOOpI and AMPure XP beads cleanup was performed with a final elution step in 50pl EB buffer. Double stranded DNA library was measured with the Qubit 4 HS system according to manufacturer's guidelines. DNA library was diluted to 1 nm with RSB buffer and then dilute to 450 pM. DNA library was loaded onto a NextSeq 1000 Sequencer (Illumina) using the manufacturers protocol. Raw fastq files were quality trimmed using cutadapt version 1.18 (https: / / cutadapt.readthedocs.io / en / vl.18 / index.html, as accessed on 2024-12-17) with a minimum quality score of Q30. Filtered reads were joined using fastq-join version 1.3.1 and demultiplexed using a custom demultiplexing as described in WO2022258753 (storage.googleapis.com). the demultiplexed files were subsequently analyszed using the CRISPresso v. 1.0.13 (doi: 10.1038 / nbt.3583). Final values were plotted with GraphPad Prism 9.50 (GraphPad Software). Example 2 Screening Methods to detect nuclease activity on the human albumin intron 1 locus in mammalian cells (HEK293T). In this Example, HEK293T cells were cultured and transfected with B-GEn.l nuclease plasmid (pBLR709) and gRNA expressing plasmids (see TABLE 3) to assess insertion deletion (indel) formation by nuclease mediated DNA double strand as described in Example 1. As shown in TABLE 3 and FIG. 1 most gRNA tested showed substantial editing. TABLE 3. indel generation in HEK293T cells human albumin intron 1 SEQID NO: pBLR amplicon Activity in % SD 1 pBLR2542 51 2,84 0,79 2 pBLR2543 51 0,94 0,18 3 pBLR2545 51 13,6 3,54 4 pBLR2546 51 74,47 4,67 5 pBLR2544 51 64,69 7,19 6 pBLR2547 51 56,38 0,46 7 pBLR2549 51 -0,21 0,36 8 pBLR2550 51 1,9 1,47 9 pBLR2551 51 55,48 1,85 10 pBLR2552 51 22,23 0 11 pBLR2548 51 18,42 10,61 12 pBLR2553 51 26,38 4,78 13 pBLR2554 51 42,06 4,73 14 pBLR2557 51 47,03 3,96 15 pBLR2555 51 50,77 6,21 16 pBLR2559 51 16,66 4,39 17 pBLR2561 51 5,49 1,05 18 pBLR2560 51 1,58 1,76 19 pBLR2562 42 2,64 0,55 20 pBLR2563 42 56,61 9,68 21 pBLR2565 42 0,17 0,77 22 pBLR2564 42 19,13 21,3 23 pBLR2537 42 0,25 0,16 24 pBLR2538 42 0,32 0,13 25 pBLR2539 42 18,11 7,11 26 pBLR2540 42 45,69 6,64 T1 pBLR2541 42 -0,04 0,43 28 pBLR2566 42 -0,39 0,31 29 pBLR2567 42 -0,28 0,26 30 pBLR2568 42 -0,02 0,03 31 pBLR2570 T1 4,72 2,27 32 pBLR2571 T1 26,07 2,22 33 pBLR2572 T1 20,46 16,38 34 pBLR2576 T1 7,98 2,24 35 pBLR2573 T1 -0,32 2,05 36 pBLR2574 T1 0 0 37 pBLR2577 T1 0 0 38 pBLR2575 T1 0 0 39 pBLR2527 T1 43,86 5,25 40 pBLR2526 T1 0,93 0,33 41 pBLR2579 T1 3,78 1,01 42 pBLR2578 T1 71,34 4,74 43 pBLR2522 T1 6,17 1,85 44 pBLR2529 T1 16,45 3,66 45 pBLR2536 T1 6,44 1,89 46 pBLR2523 T1 14,59 2,74 47 pBLR2580 T1 1,72 0,73 48 pBLR2530 T1 0,35 0,25 49 pBLR2581 T1 9,92 2,96 50 pBLR2533 T1 36,88 0 51 pBLR2532 T1 15,57 13,34 52 pBLR2524 T1 54,35 0 53 pBLR2535 72 35,99 0 54 pBLR2521 72 47,83 5,96 55 pBLR2525 72 48,3 30,17 56 pBLR2531 72 12,62 7,65 57 pBLR2582 72 na 0 58 pBLR2558 51 46,13 3,79 59 pBLR2556 51 66,67 2,13 60 pBLR2569 T1 18,42 2,04 61 pBLR2534 T1 na 0 Example 3 Screening Methods to detect B-GEn.l nuclease and integration activity on the albumin intron 1 locus in primary human hepatocytes (PHH) In this Example, PHH cells were cultured as described, AAV8 infected and transfected with B-GEn.l mRNA and synthetic gRNAs in a LNP format (see TABLE 4 to assess AAV assisted insertion as well as indel formation by nuclease mediated DNA double strand as described in Example 1. As shown in table 4 and FIG. 2a and FIG.2b SEQID 199 showed substantial editing and integration signals. TABLE 4: editing and integration of B-GEn.l in PHH SEQID No. indel activity in % (400ng) SD (indel) RLU activity (400ng) SD (RLU) 195 8,38 1,31 7944,33 3005,78 196 6,44 1,33 5305,67 932,96 197 2,67 0,11 4624 2566,14 198 2,16 0,24 1948 827,96 199 4,56 0,16 2163,67 691,03 200 12,43 5,2 14658,33 6829,86 201 2,65 0,55 24396,33 14786,24 202 4,26 0,94 24778 10655,06 Example 4 Screening Methods to detect nuclease activity on the mouse albumin intron 1 locus in mammalian cells (HEPA1-6). In this Example, HEPA1-6 cells were cultured and transfected with B-GEn.l nuclease plasmid (pBLR709) and gRNA expressing plasmids targeting the mouse albumin intron 1 (see TABLE 5) to assess insertion deletion (indel) formation by nuclease mediated DNA double strand as described in Example 1. As shown in TABLE 5 and FIG. 3 some gRNA showed substantial editing. TABLE 5: indel generation in HEPA1-6 cells targeting mouse albumin intron 1 SEQID NO: pBLR amplicon Activity indel in % SD 322 pBLR3106b 55 11,16 1,4 323 pBLR3107 55 -0,01 0,3 324 pBLR3108 55 2,68 0,1 325 pBLR3109 55 9,71 0,6 326 pBLR3110 55 3,03 1,8 327 pBLR3111 55 2,68 1,4 328 pBLR3112 55 11,48 1,7 329 pBLR3113 55 11,14 1,0 330 pBLR3114 55 7,06 1,6 331 pBLR3115 55 3,41 1,8 332 pBLR3116 55 -0,5 1,3 333 pBLR3117 55 -0,13 1,7 334 pBLR3118 55 8,75 1,5 335 pBLR3119 55 3,67 2,5 336 pBLR3120 55 -0,26 1,3 337 pBLR3121 55 0,18 1,5 338 pBLR3122 55 4,17 0,4 339 pBLR3123 55 10,29 1,1 340 pBLR3124 55 0,79 1,5 341 pBLR3125 55 -0,23 0,5 342 pBLR3126 55 -0,21 0,4 343 pBLR3127 55 0,45 0,2 344 pBLR3128 55 -0,2 0,4 345 pBLR3129 55 -0,42 0,7 346 pBLR3130 55 -0,06 0,2 347 pBLR3131 55 10,58 1,5 348 pBLR3132 55 0,7 0,6 349 pBLR3133 60 0,84 0,9 350 pBLR3134 60 -0,23 0,3 351 pBLR3135 60 0,43 0,5 352 pBLR3136 60 0,08 0,4 353 pBLR3137 61 -0,09 0,2 354 pBLR3138 61 0,23 0,2 355 pBLR3139 61 0,01 0,3 356 pBLR3140 61 0,16 0,2 357 pBLR3141 61 0,26 0,3 358 pBLR3142 61 0,78 0,6 359 pBLR3143 61 4,31 1,0 360 pBLR3144...

Claims

1. A system comprising:a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; andb) a guide RNA (gRNA) comprising a spacer sequence from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 58, 59, 60, or a nucleic acid encoding the gRNA; andc) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof.

2. A system comprising:a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; andb) a guide RNA (gRNA) from any one of SEQ ID NOs: 62 to 122, or nucleic acid encoding the gRNA, or any or any sequence thereof which is 95%, preferably, 98%, more preferably 99% identical to these sequences; andc) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof.

3. A system according to claim 1, wherein the guide RNA (gRNA) comprises a spacer sequence from any one of SEQ ID NOs: 3, 4, 5, 6, 9, 11, 12, 13, 14, 15, 16, 20, 21, 22, 25, 26, 32, 39, 42, 44, 46, 51, 52, 54, 55, 56, 58, 59, 60, or a nucleic acid encoding the gRNA.

4. A system according to claim 1, wherein the guide RNA (gRNA) comprises a spacer sequence from any one of SEQ ID NOs: 4, 5, 6, 9, 12, 13, 14, 15, 20, 26, 32, 39, 42, 52, 54, 55, 58, 59, or a nucleic acid encoding the gRNA.

5. A system according to claim 1, wherein the guide RNA (gRNA) comprises a spacer sequence from any one of SEQ ID NOs: 4, 5, 6, 9, 15, 20, 42, 52, 59, or a nucleic acid encoding the gRNA.

6. A system according to claim 1, wherein the guide RNA (gRNA) comprises a spacer sequence from any one of SEQ ID NOs: 4 or 42, or a nucleic acid encoding the gRNA.

7. The system of any of claims 1-6, wherein the GOI is encoding for Acid alpha-glucosidase(GAA).

8. A method of editing a genome in a cell, the method comprising:providing the following to the cell:a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding said DNA endonuclease, selected from SEQ ID NO: 185 or SEQ ID NO: 186, or any sequence thereof which is 95%, preferably, 98% identical, more preferably 99% identical to these sequences; andb) a guide RNA (gRNA) comprising a spacer sequence from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, T1, 28, 29, 30, 31, 32, 33, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 58, 59, 60, or a nucleic acid encoding the gRNA; andc) a donor nucleic acid comprising a nucleic acid sequence encoding a gene-of-interest (GOI) or functional derivative thereof.

9. A method according to any of claims 1-8, wherein the cell is a hepatocyte.

10. The system of any of claims 1 to 7 for use in the treatment of a disorder or healthcondition in a subject, wherein (a), (b) and (c) are to be provided to a cell in the subject.

11. The system of any one of claims 1 to 7, or the method of claim 8, wherein the components (a) to (c) are encoded by one or separate viral vectors, optionally, such viral vector is an AAV.

12. The system of any one of claims 1 to 7, or the method of claim 8, wherein the components (a) to (c) are formulated in one or separate liposome or lipid nanoparticle(s).

13. A genetically modified cell in which the genome of the cell is edited by the method of any of claims 8 or 9.

14. The system of any of claims 1-7 for use in the treatment of a disorder or health condition5                wherein a), b), and c) are to be provided to a cell in the subject.

15. The system of claim 7 for use in the treatment of Pombe disease, wherein a), b), and c)are to be provided to a cell in the subject.10        16. A kit comprising the elements of the systems of any of claims 1-7, and 11, 12, and furthercomprising instructions for use.

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