WO2020073005A1 - Engineered chimeric nucleic acid guided nucleases, compositions, methods for making, and systems for gene editing - Google Patents

Abstract

Embodiments of the present disclosure relate to methods for creating and using engineered chimeric nucleic acid guided nuclease libraries for improved and commercially viable nuclease constructs for targeted and improved gene editing. In certain embodiments, libraries of chimeric nucleases having modules derived from two, three or more Cas12a-type species can be constructed to form chimeras representing two or more different species. In some embodiments, engineered libraries disclosed herein can be used for rapid production, identification and use for improved targeted genomic editing in a subject. In other embodiments, PAM sequence recognition by a native Cas12a-type nuclease can be selected against in libraries disclosed herein creating novel Cas12a-like chimeric nuclease libraries having increased targeting capabilities across species for improved genomic editing efficiency, reducing off-targeting and/or creating expanded target specificity.

Description

ENGINEERED CHIMERIC NUCLEIC ACID GUIDED NUCLEASES, COMPOSITIONS, METHODS FOR MAKING, AND SYSTEMS FOR GENE

EDITING

PRIORITY

[001] This PCT application claims priority to U.S. Provisional Application No. 62/741,475 filed October 04, 2018 and U.S. Provisional Application No. 62/741,470 filed on the same day, October 04, 2018. These applications are incorporated herein by reference in their entirety for all purposes.

STATEMENT REGARDING GOVERNMENT FUNDING

[002] This invention was made with government support under grant number DE- SC0008812 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

[003] The instant application contains a Sequence Listing which has been submitted via ASCII copy created on October 04, 2019 referred to as‘CU4839B_Final_For_ST25.txt’ and is 380 kilobytes having 136 sequences. Further, the provisional application as filed contained sequence listings in Appendix A, B and C and are hereby incorporated by reference in their entirety for all purposes.

FIELD

[004] Embodiments of the present disclosure relate to methods for creating and using engineered chimeric nucleic acid guided nuclease for improved and commercially viable constructs for targeted and improved gene editing. In certain embodiments, engineered chimeric nucleic acid guided nucleases can include fragments of two, three or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from Prevotella and Francisella 1 (Casl2a) or Casl2a-like start regions constructed to form a single chimera and referenced as Casl2-like chimeric nucleases or engineered chimeric nucleic acid guided nucleases. Casl2a is an RNA-guided endonuclease of a class II CRISPR/Cas system, a putative class 2 CRISPR effector. In certain embodiments, Casl2a chimeras can be created to form a chimeric Casl2a having at least one of improved efficiency and improved genetic editing accuracy. In some embodiments, Casl2a chimeras disclosed herein can include 2 Casl2a fragments to form a single chimera of use for improved genomic editing in a subject. Certain Casl2a chimeras disclosed herein can be created using crossover residue engineering of CAS nuclease chimeras. In accordance with these embodiments, residues, modules, structural motifs, and other physical features are identified and used for generating novel, improved, functional chimeric CAS nuclease enzymes. In certain embodiments, a cross-over region can include a 10 to 50 base pair (bp) region of one or more selected Casl2as.

BACKGROUND

[005] CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats. In a palindromic repeat, the sequence of nucleotides is the same in both directions. Each of these palindromic repetitions is followed by short segments of spacer DNA. Small clusters of Cas (CRISPR-associated system) genes are located next to CRISPR sequences. The CRISPR/Cas system is a prokaryotic immune system that can confer resistance to foreign genetic elements such as those present within plasmids and phages providing the prokaryote a form of acquired immunity. RNA harboring a spacer sequence assists Cas (CRISPR-associated) proteins to recognize and cut exogenous DNA. CRISPR sequences are found in approximately 50% of bacterial genomes and nearly 90% of sequenced archaea has selected for efficient and robust metabolic and regulatory networks that prevent unnecessary metabolite biosynthesis and optimally distribute resources to maximize overall cellular fitness. The complexity of these networks with limited approaches to understand their structure and function and the ability to re-program cellular networks to modify these systems for a diverse range of applications has complicated advances in this space. Certain approaches to re-program cellular networks are directed to modifying single genes of complex pathways but as a consequence of modifying single genes, unwanted modifications to the genes or other genes can result, getting in the way of identifying changes necessary to achieve a particular endpoint as well as complicating the endpoint sought by the modification.

[006] CRISPR-Cas driven genome editing and engineering has dramatically impacted biology and biotechnology in general. CRISPR-Cas editing systems require a polynucleotide guided nuclease, a guide polynucleotide (e.g. a guide RNA (gRNA)) that directs by homology the nuclease to cut a specific region of the genome, and, optionally, a donor DNA cassette that can be used to repair the cut dsDNA and thereby incorporate programmable edits at the site of interest. The earliest demonstrations and applications of CRISPR-Cas editing used Cas9 nucleases and associated gRNA. These systems have been used for gene editing in a broad range of species encompassing bacteria to higher order mammalian systems such as animals and in certain cases, humans. It is well established, however, that key editing parameters such as protospacer adjacent motif (PAM) specificity, editing efficiency, and off- target rates, among others, are species, loci, and nuclease dependent. There is increasing

1 interest in identifying and rapidly characterizing novel nuclease systems that can be exploited to broaden and improve overall editing capabilities.

[007] One version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to provide useful tools for editing genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut/edited at a predetermined location, allowing existing genes to be removed and/or new ones added. These systems are useful but have some important limitations regarding efficiency and accuracy of targeted editing, imprecise editing complications, as well as, impediments when used for commercially relevant situations such as gene replacement. Therefore, a need exists for improved nucleic acid guided nuclease constructs for directed and accurate editing with improved efficiency.

SUMMARY

[008] Embodiments of the present disclosure relate to methods for creating and using engineered chimeric nucleic acid guided nuclease construct libraries. In certain embodiments, methods for creating engineered chimeric Casl2a-like nucleic acid guided nuclease are directed to improved targeting and efficiency for genomic editing in a wide range of species and applications. Certain embodiments disclosed herein concern creating designer chimeric Casl2a-like constructs of nucleic acid guided nucleases for commercial use. In accordance with these embodiments, engineered chimeric nucleic acid guided nucleases can include combining fragments of two, three or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from Prevotella and Francisella 1 (referred to as Cpfl or Casl2a) or Casl2a-type start regions to form a single chimera construct. In certain embodiments, Casl2a-like chimeras can be created to form chimeric Casl2a-like nucleases having at least one of improved efficiency, altered protospacer adjacent motif (PAM) sequence recognition and/or improved genetic editing accuracy. PAM is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas nuclease in the CRISPR. In some embodiments, Casl2a-like chimeras disclosed herein are made up of two or more Cas 12a fragments combined to form a single chimera of use for improved genomic editing in a subject in need thereof.

[009] In other embodiments, a PAM sequence recognized by a native Cas 12a can be modified in these Cas 12a- like chimeric constructs of the instant application, where the Cas 12a- like chimeric constructs recognize novel PAM sequences creating Cas 12a- like chimeras having increased genomic editing efficiency or improved target recognition. In certain embodiments, Casl2a-like chimeric constructs of the instant invention have improved

2 editing efficiency over naturally-occurring Casl2as that recognize standard PAM sequences such as the TTTN recognition site.

[0010] In other embodiments, certain Casl2a-like chimeras disclosed herein can be created using crossover residue engineering of CAS nuclease chimeras to select for chimeras having the same or altered PAM recognition capabilities or recognize different sites PAM- like sites. In accordance with these embodiments, residues, modules, structural motifs, and other physical features are identified and used for generating novel, improved, functional chimeric CAS nuclease enzymes with improved characteristics over a wild-type Casl2a. In certain embodiments, chimeric constructs disclosed herein do not recognize TTTN PAM sequences or recognize these TTTN regions but do not excise/cut at this site. In other embodiments, chimeric constructs disclosed herein recognize different PAM sequences other than TTTN sequences of wild-type Casl2a. In yet other embodiments, novel Casl2a-like chimeric constructs disclosed herein recognize the same PAM sites as a wild-type control Casl2a nuclease (e.g., TTTN and CTTN PAM sites) but have reduced off- targeting rates to increase accuracy of editing. In yet other embodiments, Casl2a-like chimeric constructs disclosed herein create designer nucleic acid guided Casl2a-like nucleases where the crossover points of each of the respective wild-type Casl2a (e.g., derived from two or more different wild type Casl2a nucleases) create nucleases having altered PAM specificity for genome editing. In certain embodiments, an altered PAM recognition sequence can be GAAA.

[0011] In other embodiments, an altered PAM recognition sequence of chimeric nuclease constructs disclosed herein can include a recognition sequence of TTTN where at least one thymidine nucleotide recognized by a designer nuclease is a C, A or a G. In other examples, a recognition sequence of designer constructs disclosed herein can be CCCN, AAAN, GGGN, GAGG, GAAT, GAAA, etc. or other combination wherein the recognition sequence does not include TTTN and is from 2 to 6 nucleotides in length.

[0012] Embodiments of the present disclosure relate to engineering designer chimeric nucleic acid guided nucleases for improved targeted gene editing. In certain embodiments, the engineered designer chimeric nucleic acid guided nucleases can be used for genome editing in an organism. In certain embodiments, organisms contemplated herein can be bacteria, yeast, plants, mammals such as humans, pets and livestock and further can include birds, fish or other aquatic animals. In accordance with these embodiments, a library can be generated for a targeted genome application(s) in order to be edited by one or more engineered designer Casl2a-like chimeric nucleic acid guided nucleases in the library to

3 remove and/or insert one or more genes or gene fragments into the targeted genome or edit a targeted gene providing methods for producing a targeted result (e.g. removing or replacing a defective gene, increasing or decreasing expression of a gene, etc.)·

[0013] In some embodiments, designer constructs disclosed here can further include one or more mutations, one or more manipulations or modifications that increase gene editing efficiency or accuracy. In some embodiments, the one or more mutations can include one or more point mutations, single nucleotide polymorphism (SNP), an insertion or a deletion of two or more nucleotides or other mutation to alter PAM recognition of the designer chimeric constructs or reduce off-targeting rates of constructs disclosed herein.

[0014] In certain embodiments, designer engineered chimeric nucleic acid guided nuclease construct libraries described herein can be created from Casl2a as known in the art including, but not limited to, Succinivibrio dextrinosolvens (SD_Casl2a), Candidatus Methanoplasma termitum (CT_Casl2a), Porphyromonas crevioricanis (PC_Casl2a), Thiomicrospira sp.

XS5, (TX_Casl2a), Candidatus Methanomethylophilus alvus (CA_Casl2a), Candidatus Methanomethylophilus alvus (CA_Casl2a), Eubacterium rectale, and Flavobacterium branchiophilum (FB_Casl2a) or other Casl2a-type. In certain embodiments, chimeric construct libraries disclosed herein are obtained starting with two Casl2a-type nucleases in order to generate chimeras having one or more cross-over recombination events. In other embodiments, chimeric construct libraries disclosed herein can be obtained using three different Casl2a-type nucleases to generate chimera libraries by use of cross-over recombination technologies. In certain embodiments, chimeric Casl2a-like nuclease constructs can include constructs with reduced off-targeting rates and/or improved editing functions compared to a control or wild-type Casl2a nuclease.

[0015] In certain embodiments, two or more Casl2a-type sequences can be used to recombine and create non-naturally occurring chimera at one or more of crossover positions occurring between REC1 and REC2; REC2 and WEDII; one or more of 2 positions occurring between PI and WEDIII; WEDIII and RuvC-l; and/or between RuvC-II and Nuc. In certain embodiments, recombination can result in chimera having rearranged domains that lead to recombinations at 1) REC1; 2) REC1/REC2; 3) REC1/REC2/WED-II/PI; 4) WED-III/RuvC- PBH/RuvC-II/Nuc/RuvC-III; 5) RuvC-PBH/RuvC-II/Nuc/RuvC-III; 6) Nuc/RuvC-III or other combination where each of these domains or modules can be derived from one or more recombinations. In some embodiments, where two Casl2a-types are recombined for example, SD_Casl2a with FB_Casl2a, recombinations can include all recombinations at the contemplated 6 recombination sites (See Fig. 7C) leading to for example, SDREC1 on an FB-

4 backbone; FBREC1 on an SD backbone; SD-REC1/FB-REC2/SD-WED-II/FB-PI or FB- REC1/SD-REC2/FB-WED-IFSD-PI or other recombination; SD-WED-III/FB-RuvC- FBH/RuvC-IFSD-Nuc/RuvC-III or FB-WED-III/SD-RuvC-I/BH/RuvC-II/FB-Nuc/RuvC-III where some or all possible recombinations at the 6 recombination sites are represented in a targeted library.

Brief Description of the Drawings

[0016] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] FIGS. 1A-1C illustrates a schematic diagram for creating and testing certain designer chimeric constructs (1A), chimeric recombinations (1B) designed by methods and testing by editing efficiency (1C) compared to a positive control of some embodiments disclosed herein.

[0018] FIG. 2 illustrates a schematic of components of use in genetic editing of some embodiments disclosed herein.

[0019] FIG. 3 illustrates exemplary screening of binding and cleavage of an altered PAM recognition sequences for a Casl2a-like chimeric nuclease construct compared to a control of some embodiments disclosed herein.

[0020] FIGS. 4A and 4B represent gene editing efficiencies (4B) of a Casl2a-like chimeric nuclease construct disclosed herein compared with a control when gRNA is conserved (WT) or mutated (4 A) to test off-targeting rates.

[0021] FIGS. 5A-5D represents plots of various Casl2a-like chimeric nuclease constructs off-targeting rates compared to a control using wild-type and altered gRNA sequences in certain embodiments disclosed herein.

[0022] FIGS. 6A-6C represents a schematic illustration of genetic editing (6A) and histogram plots (6B and 6C) of various Casl2a chimeras compared to a control demonstrating editing efficiency relative to induction time of each variant and the control in certain embodiments disclosed herein.

[0023] FIGS. 7A-7D represent Casl2a-type chimera library construction: (7 A) the Casl2a-type protein structure analysis based on AsCasl2a (PDB:5B43), (7B) editing and transformation efficiencies of the Casl2a like nucleases used in certain embodiments disclosed herein. (7C) Crossover points indicated for domain recombination: 6 crossover points are contemplate of use herein; WED-I&REC1, REC2, WED-II&PI, WED-III, RuvC-

5 l&BH&RUVC-II, and Nuc&RuvC-III, respectively, and (7D) represents a schematic distribution of some constructs of a Casl2a-like chimera library containing Casl2a variants. Numbers to the left of variants were the crossover points as illustrated in 1C. Swapped regions are illustrated using different colors; the colors are the same as illustrated in 7B in certain embodiments disclosed herein.

[0024] FIGS. 8A-8I 8A represents an illustration of a schematic for testing editing efficiency of certain constructs created by methods disclosed herein. 8B represents a histogram plot of cutting efficiency of chimeric Casl2a-like proteins using 6 different gRNA plasmids. 8C represents a plot of percent editing efficiency of chimera library variants with different gRNAs of galKl, galK2, lacZl, and lacZ2. 8D is a schematic representation of a Casl2a-type with reduced activity (dCasl2a) in a protein binding assay. 8E-8F represents plasmid systems where one plasmid expresses dCasl2a (Casl2a with reduced activity) using an inducible promoter; a second plasmid expresses a single crRNA a test gene; and a third plasmid expresses the a resistance protein using a constitutive promoter containing a fully complementary (on-target) crRNA binding site as well as an encoded enzyme making the cells sensitive to an agent. 8E and 8F represent cutting efficiency of some chimeric Casl2a like nucleases with different induction times using different gRNAs. (8E) galK_l and (8F) galK_2. 8G is an illustration of an inducible system for testing chimeric nucleases disclosed herein. Three additional plasmid systems were constructed for genome editing: where one plasmid expresses a Casl2a like protein using an inducible promoter; a second plasmid bacterial test proteins using a temperature-inducible promoter; and a third plasmid expresses a single crRNA (with a promoter) targeting a tester gene with homology arm (HM) containing a test protein-inactivating mutation as a template for recombineering. (8H and 81) The editing efficiency of chimeric Casl2a-like nucleases with different gene induction times with different gRNAs is plotted in (8H) galK_l and (81) galK_2.

[0025] FIGS. 9A-9F represents specificity detection of chimeric Casl2a-like variants and enrichment scoring of each PAM site using different guide RNAs. (9A-9F) Round 1 is illustrated of enrichment scores for two rounds of PAM scans. The enrichment score is the frequency change (log2) of each PAM using different gRNA plasmids (on-targeting and non targeting gRNAs).

[0026] FIG. 9G, 9G illustrates an off-target assay for chimeric Casl2a-type variants. 9G represents an individual off-target assay. Nine different off-target spacers were designed as illustrated to test editing efficiency and target recognition, of which 3 were substitutions, 3 were deletions, and 3 were insertions.

6 [0027] FIGS. 10A-10F illustrates in (10 A) a plasmid expressing the M44 (or control) nuclease (with T7 promoter), a single crRNA (with U6 promoter), and GFP were constructed. 10B is a photographic representation of the mammalian cells after transfection. Micrographs were taken under cool white light (left) or fluorescent light (right). An assay was performed as known in the art on cells expressing GFP and isolated by fluorescence activated cell sorting. In this example, ‘Untreated’ as labeled means the PCR products without T7 endonuclease treatment; while ‘Treated’ means the PCR products with T7 endonuclease treatment (10C). 10D is a graphic representation of an indel rate of control versus the chimeric nuclease of certain embodiments disclosed herein. 1 OF is a graphic illustration of editing efficiency of control and the tested chimera Casl2a-like nuclease, of certain embodiments disclosed herein.

[0028] FIGS. 11A-11B illustrates in color coded format crossovers represented in FIG. 7C with 1 crossover library (11 A) and a double crossover library (11B).

[0029] FIGS. 12A and 12B represent a Phylogenetic Tree for the wild-type (WT) Casl2a- type and chimera Casl2a-like gene (Fig. 12A) and amino acid (Fig. 12B) sequences.

[0030] FIGS. 13A and 13 B illustrate methods for screening (13A) and exemplary results of a control (13B) for assessing nuclease gene editing activity of certain embodiments disclosed herein.

[0031] FIG. 14 is an exemplary graph illustrating distribution of functional chimera Casl2a-like nucleases identified using a selection assay of certain embodiments disclosed herein.

[0032] FIG. 15 illustrates a color screening of control versus a chimera Casl2a-like nuclease (e.g. M44) with different gRNAs of certain embodiments disclosed herein.

[0033] FIGS. 16A-16D illustrate exemplary histogram plots that represent transformation efficiency of different Casl2a-like chimera variants using different gRNA of certain embodiments disclosed herein. The gRNA used in the test were (16A) galKl (16B) galK2 (16C) lacZl and (16D) lacZ2.

[0034] FIGS. 17A-17C illustrate genome editing test in the different genomic positions for chimera library variants. 17A illustrates a schematic of targeted genomic positions. 17B illustrates representative plates for colorimetric screening of targeted protein activity with chimera Casl2a-like nuclease variants in different genomic position. 17C illustrates editing efficiency of chimera library variants in different genomic positions of certain embodiments disclosed herein.

7 [0035] FIG. 18 represents an exemplary gene editing assay using a control of a chimera Casl2a-like nuclease for DNA binding assay (dM44) to illustrate behavior of a particular chimera in order to assess any altered activity in genomic editing processes of certain embodiments disclosed herein.

[0036] FIG. 19 represents a histogram plot of binding efficiency of dCasl2a-like chimera nucleases using different guide RNAs of certain embodiments disclosed herein.

[0037] FIGS. 20A-20D represents histogram plots illustrating cutting efficiency assessed by individual verification of unknown PAMs using different nucleases including chimera Casl2a-like nucleases (a)ATTC (b) ATTA (c) GTTA (d) CCTC.

[0038] FIG. 21 represents a color screening of off-target test using of certain embodiments disclosed herein. Sequencing verification of white colony was demonstrated and compared to a wild type spacer design of certain embodiments disclosed herein.

[0039] FIG. 22 represents sequence comparisons of various Casl2a nucleases of use to create chimera Casl2a-like libraries disclosed herein.

DETAILED DESCRIPTION

[0040] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments of the disclosure. It will be obvious to one of skill in the relevant art that practicing the various embodiments does not require the employment of all or even some of the details outlined herein, but rather that concentrations, times and other details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.

[0041] As disclosed herein“modulating” and“manipulating” of genome editing can mean an increase, a decrease, upregulation, downregulation, induction, a change in editing activity, a change in binding, a change cleavage or the like, of one or more of targeted genes or gene clusters of certain embodiments disclosed herein.

[0042] In certain embodiments of the present disclosure, there can be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature and understood by those of skill in the art.

[0043] In certain embodiments of this disclosure, primers used for sequencing and sample preparation per conventional techniques can include sequencing primers and amplification primers. In some embodiments, plasmids and oligomers used per conventional techniques can include synthesized oligomers, oligomer cassettes.

8 [0044] In certain embodiments, designer chimeric Casl2a-like constructs disclosed herein can be created for altered targeting of a gene and/or increased efficiency and/or accuracy of targeted gene editing in a subject.

[0045] In accordance with these embodiments, it is known that Casl2a is a novel single RNA-guided CRISPR/Cas endonuclease capable of genome editing having differing features when compared to Cas9. In certain embodiments, a Casl2a-based system allow fast and reliable introduction of donor DNA into a genome. In addition, Casl2a broadens genome editing. CRISPR/Cas 12a genome editing has been evaluated in human cells as well as other organisms including plants. Several features of the CRISPR/Casl2a system are different when compared to CRISPR/Cas9.

[0046] For example, Casl2a recognizes T-rich protospacer adjacent motif (PAM) sequences (e.g. 5’-TTTN-3’ (AsCasl2a, LbCasl2a) and 5’-TTN-3’ (FnCasl2a); whereas, the comparable sequence for SpCas9 is NGG. The PAM sequence of Casl2a is located at the 5’ end of the target DNA sequence, where it is at the 3’ end for Cas9. In addition, Casl2a is capable of cleaving DNA distal to its PAM around the +18/+23 position of the protospacer. This cleavage creates a staggered DNA overhang (e.g. sticky ends), whereas Cas9 cleaves close to its PAM after the 3’ position of the protospacer at both strands and creates blunt ends. In certain methods, creating altered recognition of Casl2a nucleases can provide an improvement over Cas9 in part due to the creation of sticky ends instead of blunt end cleavages. Further, Casl2a is guided by a single crRNA and does not require a tracrRNA, resulting in a shorter gRNA sequence than the sgRNA used by Cas9.

[0047] It is also known that Casl2a displays additional ribonuclease activity that functions in crRNA processing. This feature may lead to simplified multiplex genome editing. Casl2a is used as an editing tool for different species (e.g. S. cerevisiae ), allowing the use of an alternative PAM sequence compared with the one recognized by CRISPR/Cas9. It also provides an alternative system for multiplex genome editing as compared with Cas9-based multiplex approaches for yeast and can be used as an improved system in mammalian gene editing.

[0048] Casl2a nucleases have emerged as suitable alternatives to Cas9 nucleases, where several nucleases (e.g. Acidaminococcus sp. (AsCasl2a) and Lachnospiraceae bacterium (LbCasl2a)) have now been demonstrated to display comparable genome-editing capability to Cas9 while providing different PAM preferences and increasing the range of targets for gene editing. Casl2a-type, as referenced herein are used to demonstrate recognition of changes in CRISPR-CAS evolutionary classification and naming schemes. As illustrated in

9 the figures, the structures of AsCl2a/LbCasl2a contain a bi-lobed architecture consisting of an a-helical recognition (REC) lobe and a nuclease (NUC) lobe with a positively charged channel between them that binds the crRNA-DNA hybrid. The REC lobe includes REC1 and REC2 domains, and the NUC lobe includes RuvC domain and three additional domains, which are referred to as the WED, PI, and Nuc domains, (See for example, Fig. 7A). The WED domain includes three regions (WED-I, WED-II, and WED-III) in the Casl2a sequence. The REC lobe (REC1 and REC2) is located between the WED-I and WED-II regions, and the PI domain is between the WED-II and WED-III regions. The RuvC domain contains the three motifs (RuvC-I, RuvC-II, and RuvC-III). The bridge helix (BH) is located between the RuvC-I and RuvC-II motifs and connects the REC and NUC lobes, whereas the Nuc domain is inserted between the RuvC-II and RuvC-III motifs.

[0049] Well-known Casl2a protein-RNA complex recognize a T-rich PAM and cleavage leads to a staggered DNA double- stranded break. Casl2a-type nuclease interacts with the pseudoknot structure formed by the 5 '-handle of crRNA. A guide RNA segment, composed of a seed region and the 3' terminus, possesses complementary binding sequences with the target DNA sequences. Casl2a type nucleases characterized to date have been demonstrated to work with a single gRNA and to process gRNA arrays. In addition, when comparing the ratio of total off-target to on-target modification for AsCasl2a and LbCasl2a, it was found that both orthologs demonstrated lower off-target activity than had been observed for SpCas9. While Casl2a-type and Cas9 nuclease systems have proven highly impactful, neither system has been demonstrated to function as predictably as is desired to enable the full range of applications envisioned for gene-editing technologies.

[0050] In the current state, a range of efforts have attempted to engineer improved CRISPR editing systems having increased efficiency and accuracy, which have included engineering of the PAM specificity, stability, and sequence of the gRNA and-or the nuclease. For example, chemical modifications of CRISPR/Cas9 gRNA expected to increase gRNA stability was found to lead to a 3.8-fold higher indel frequencies in human cells. In addition, other studies included structure- guided mutagenesis of Casl2a and screened to identify variants with an increased range of recognized PAM sequences. These engineered AsCasl2a recognized TYCV and TATV PAMs in addition to the established TTTV sequence, with enhanced activities in vitro and in tested human cells. Using the crystal structures of Cas9, rational engineering of the DNA binding region was performed to attempt to decrease binding under the hypothesis that this would result in a lower off target editing rates. These engineered Cas9 nucleases were reported eSpCas9 (1.0) and eSpCas9(l.l) mutants that

10 decreased 50% cleavage with off target sites (<0.2% indel) relative to a wild-type Cas9 using 20-nucleotide RNA guides.

[0051] In certain embodiments, disclosed herein, a platform has been designed for construction of libraries of novel synthetic chimera Casl2a-like nucleases that would span the kinetic space encompassing all potential editing considerations. It was observed that even though Casl2a type nucleases exhibit considerable overall sequence diversity, Casl2a type nucleases also retain several conserved regions. In certain embodiments, conserved regions of Casl2a type nucleases can be used to recombine in a modular fashion one Casl2a type nuclease template to another different Casl2a type nuclease template to produce viable chimeric Casl2a-like nucleases. In some embodiments, engineered chimeric Casl2a-like nucleases exhibit altered kinetic characteristics with desired editing characteristics. In some embodiments, REC1 can be a region of recombination. In other embodiments, other modules or regions of a Casl2a-type nuclease can be recombined in a library at one or more of the identified cross-over regions as illustrated in Fig. 1C. For example, synthetic libraries can be constructed by recombination of one or more Casl2a-type nucleases at cross-over regions as found in Fig. 22. It is understood that other Cas nucleases can be used to generate chimera Cas-like nucleases with improved targeting for gene editing.

[0052] In certain embodiments, designer engineered Casl2a-like chimeric nucleic acid guided nuclease constructs of embodiments disclosed herein enable altered and/or improved CRISPR-Cas editing. In other embodiments, activity of exemplary designer Casl2a-like constructs have been analyzed in E. coli and confirmed to function in yeast as well as mammalian cells, providing diverse applications across multiple species/organisms. In other embodiments, designer chimeric constructs using cross-over technologies disclosed herein can create nucleic acid guided nuclease chimeric constructs including 2 or more nucleic acid fragments derived from 2 or from 3 or from 4 or more Casl2a-type nucleases leading to chimeras with novel PAM recognition sequences that differ from TTTN of wild-type Casl2a nucleases or have similar recognition sequences having improved editing of use in a subject, including humans, pets and livestock for increased accuracy and efficiency of genome editing. Engineered designer Cas 12a- like chimeric nucleases disclosed herein are contemplated of use in bacteria, yeast and other prokaryotes. In other embodiments, engineered designer Casl2a-like chimeric nucleases are contemplated of use in eukaryotes such as mammals as well as of use in birds and fish. In certain embodiments, nucleic acid sequences of chimeric constructs disclosed herein are a combination of nucleic acid sequence fragments of two different starting Cas 12a- type nucleases joined to form a nuclease of a

11 single nucleic acid sequence by recombination event(s). In other embodiments, nucleic acid sequences of chimeric constructs disclosed herein are a combination of nucleic acid sequence fragments of three different starting Casl2a nucleases joined to form a nuclease as a single nucleic acid sequence. In some embodiments, libraries can include a one, two, or three crossover recombination events represented by chimera Casl2a-like nucleases in the library. In accordance with these embodiments, these chimeric constructs are created in order to alter certain features of the wild-type Casl2a sequences that they are derived from; for example, recognize wild-type and/or novel PAM sequences for improved genome editing accuracy and efficiency across multiple species including mammals creating novel and improved Cas l2a- like chimera nucleases.

[0053] In certain embodiments, designer engineered chimeric nucleic acid guided nuclease constructs of embodiments disclosed herein can be created from Casl2as known in the art or not yet discovered and can include, but are not limited to, Succinivibrio dextrinosolvens (SD_Casl2a), Candidatus Methanoplasma termitum (CT_Casl2a), Porphyromonas crevioricanis (PC_Casl2a), Thiomicrospira sp. XS5, (TX_Casl2a), Candidatus

Methanomethylophilus alvus (CA_Casl2a) (TX_Casl2a), Candidatus Roizmanbacteria bacterium GW2011_GWA2_37_7 (CR_Casl2a), Eubacterium rectale,(a positive control is a derivative of this Casl2a), Flavobacterium branchiophilum (FB_Casl2a), and/or a synthetic construct (SC_Ca I2a) or similar. In certain embodiments, chimeric constructs can include using two Casl2as to create a chimera using cross-over technologies. In other embodiments, chimeric constructs can include using three or more Casl2as to create a chimera using cross over technologies. In certain embodiments, chimeric Casl2a constructs can include constructs with reduced off-targeting rates and/or improved editing functions compared to a control or wild-type Casl2a nuclease.

[0054] In some embodiments, a junction region of a Casl2a of use for creating a chimeric construct of certain embodiments disclosed herein can be about 5 to about 25 amino acids in length where at least 2 different Casl2a sequence fragments are represented in the juncture region of the recombination event. In accordance with these embodiments, a junction region of Cas 12a of use for creating a chimeric construct of certain embodiments disclosed herein can be a represented by amino acid: FATSFKDYFKNRAN SEQ ID NO. 149 or mutant or truncation thereof or nucleotide sequence, nt:

TTGCGACTAGCTTTAAAGATTACTTCAAGAACCGTGCAAAT SEQ ID NO.150. or mutant or truncation thereof (e.g. having 80%, 90%, 95% or more sequence homology). In other embodiments, a junction region of a Casl2a of use for creating a chimeric construct of

12 certain embodiments disclosed herein can be represented by amino acid sequence of:

LHKQILCIADTSYE SEQ ID NO.151 or mutant or truncation thereof or nucleotide sequence, nt: CTTCACAAACAGATTCTATGCATTGCGGACACTAGCTATGAG SEQ ID NO. 152. or mutant or truncation thereof (e.g. having 80%, 90%, 95% or more sequence homology). In yet other embodiments, a junction region of a Casl2a of use for creating a chimeric construct of certain embodiments disclosed herein can be represented by amino acid sequence of: VELQGYKIDWTYI SEQ ID NO. 153 or mutant or truncation thereof or nucleotide sequence, nt: gtagagttacaaggttacaagattgattggacatacatt or SEQ ID NO.154 mutant or truncation thereof (e.g. having 80%, 90%, 95% or more sequence homology).

[0055] In some embodiments, off-targeting rates for chimeric constructs disclosed herein can be reduced compared to a control for improved editing. For example, off-targeting rates can be readily tested. In accordance with these embodiments, a wild-type gRNA plasmid can be used to assess baseline off-target editing compared to experimentally designed gRNAs to assess accuracy of chimeric constructs compared to control Casl2a nucleases. In certain methods, spacer mutations can be introduced to a plasmid to test when a substitution gRNA sequence is created or a deletion or insertion mutant. Each of these plasmid constructs can be used to test genome editing accuracy and efficiency, for example, with deletions, substitutions or insertions.

[0056] Alternatively, chimeric constructs created by compositions and methods disclosed herein using two or more Casl2as to create a novel designer chimera can be tested for optimal genome editing time on a select target by observing editing efficiencies over pre determined time periods.

[0057] Examples of target polynucleotides for use of engineered chimeric nucleic acid guided nucleases disclosed herein can include a sequence/gene or gene segment associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Other embodiments contemplated herein concern examples of target polynucleotides related to a disease- associated gene or polynucleotide.

[0058] A "disease- associated" or“disorder-associated” gene or polynucleotide can refer to any gene or polynucleotide which results in a transcription or translation product at an abnormal level compared to a control or results in an abnormal form in cells derived from disease- affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, or where the gene contains one or more mutations and where altered expression or expression directly correlates with the occurrence and/or

13 progression of a health condition or disorder. A disease or disorder-associated gene can refer to a gene possessing mutation(s) or genetic variation that are directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the cause or progression of a disease or disorder. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level

[0059] It is understood by one of skill in the relevant art that examples of disease- associated genes and polynucleotides are available from. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

[0060] Genetic Disorders contemplated herein can include, but are not limited to,

[0061] Neoplasia: Genes linked to this disorder: PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notchl; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF; HIFI a; HIF3a; Met; HRG; Be 12; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL; BRCA1 ; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF Receptor; Igfl (4 variants); Igf2 (3 variants); Igf 1 Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members:l, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Ape

[0062] Age-related Macular Degeneration: Genes linked to these disorders Abcr; Ccl2; Cc2; cp (cemloplasmin); Timp3; cathepsinD; VIdlr; Ccr2

[0063] Schizophrenia Disorders: Genes linked to this disorder: Neuregulinl (Nrgl); Erb4 (receptor for Neuregulin); Complexinl (Cplxl); Tphl Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b

[0064] Trinucleotide Repeat Disorders: Genes linked to this disorder: 5 HTT

(Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Dx); FXN/X25 (Friedrich's Ataxia); ATX3 (Machado-Joseph's Dx); ATXN1 and ATXN2 (spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-l and Atnl (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR (Alzheimer's); Atxn7; AtxnlO

[0065] Fragile X Syndrome: Genes linked to this disorder: FMR2; FXR1; FXR2;

mGLURS

[0066] Secretase Related Disorders: Genes linked to this disorder: APH-l (alpha and beta); Presenil n (Psenl); nicastrin (Ncstn); PEN-2

[0067] Others: Genes linked to this disorder: Nosl ; Paipl; Nati; Nat2

[0068] Prion - related disorders: Gene linked to this disorder: Prp

14 [0069] ALS: Genes linked to this disorder: SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c)

[0070] Drug addiction: Genes linked to this disorder: Prkce (alcohol); Drd2; Drd4;

ABAT (alcohol); GRIA2; GrmS; Grinl; Htrlb; Grin2a; Drd3; Pdyn; Grial (alcohol)

[0071] Autism: Genes linked to this disorder: Mecp2; BZRAP1; MDGA2; SemaSA; Neurexin 1 ; Fragile X (FMR2 (AFF2); FXR1; FXR2; MglurS)

[0072] Alzheimer's Disease Genes linked to this disorder: El; CHIP; UCH; UBB; Tau; FRP; PICAFM; Clusterin; PS1 ; SORF1 ; CR1 ; VIdlr; Ubal; Uba3; CHIP28 (Aqpl,

Aquaporin 1); Uchll; Uchl3; APP

[0073] Inflammation and Immune-related disorders Genes linked to this disorder: IF- 10; IF-l (IF-la; IF- lb); IF-13; IF-17 (IF-17a (CTFA8); IF-17b; IF-17c; IF-17d; IF-17f); 11- 23; Cx3crl; ptpn22; TNFa; NOD2/CARD15 for IBD; IF-6; IF-12 (IF-12a; IF-12b); CTFA4; Cx3cl l, AAT deficiency/mutations, AIDS (KIR3DF1, NKAT3, NKB1, ANIB11, KIR3DS1, IFNG, CXCF12, SDF1); Autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, AFPS1A); Combined immunodeficiency, (IF2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCF5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IF10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40FG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI); Inflammation (IL- 10, IF-l (IF-la, IF-lb), IF-13, IF-17 (IF-l7a (CTFA8), IF-l7b, IF-l7c, IF-l7d, IF-l7f), 11- 23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IF-6, IF-12 (IF-12a, IF-12b), CTFA4, Cx3cl l); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKE, DCFRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, FCA, IF7R, CD3D, T3D, IF2RG, SCIDX 1, SCIDX, IMD4).

[0074] Parkinson's, Genes linked to this disorder: x-Synuclein; DJ-l; FRRK2; Parkin; PINK1

[0075] Blood and coagulation disorders: Genes linked to these disorders: Anemia (CDAN1, CDA1, RPS19, DBA, PKFR, PK1, NT5C3, UMPH I, PSN1, RHAG, RH50A, NRAMP2, SPTB, AFAS2, ANH I, ASB, ABCB7, ABC7, ASAT); Bare lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RINGI 1, MHC2TA, C2TA, RFX5, RFXAP, RFX5), Bleeding disorders (TBXA2R, P2RX I, P2X I); Factor H and factor H-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VII deficiency (F7); Factor X deficiency (F10); Factor XI deficiency (Fl 1); Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A); Factor XIIIB deficiency (F13B); Fanconi anemia

15 (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FANCM, ICIAA1596); Hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB), Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies and disorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia (HBA2, HBB, HBD, LCRB, HBA1).

[0076] Cell dysregulation and oncology disorders: Genes linked to these disorders: B- cell non-Hodgkin lymphoma (BCL7A, BCL7); Leukemia (TALI TCL5, SCL, TAL2, FLT3, NBS 1 , NBS, ZNFNIAI, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEFI2, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN,

RUNX 1 , CBFA2, AML1, WHSC 1 LI , NSD3, FLT3, AF1Q, NPM 1 , NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AFI 0, CALM, CLTH, ARLI 1, ARLTS1, P2RX7, P2X7,

BCR, CML, PHL, ALL, GRAF, NFI, VRNF, WSS, NFNS, PTPNI 1, PTP2C, SHP2, NS 1 , BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQOl,

DIA4, NMOR1, NUP2I4, D9S46E, CAN, CAIN).

[0077] Metabolic, liver, kidney disorders: Genes linked to these disorders: Amyloid neuropathy (TTR, PALS); Amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, UR, PALS); Cirrhosis (KATI 8, KRT8, CaHlA, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPS, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1, SCOl), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHDl, ARPKD, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).

[0078] Muscular/Skeletal Disorders: Genes linked to these disorders: Becker muscular dystrophy (DMD, BMD, MYF6), Duchenne Muscular Dystrophy (DMD, BMD); Emery- Dreifuss muscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy

16 (FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LAPS, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, 0C116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1).

[0079] Neurological and Neuronal disorders: Genes linked to these disorders: ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c); Alzheimer disease (APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PACIP1, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP I, MDGA2, Sema5A, Neurex 1, GLOl, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARKS, PINK1, PARK6, UCHL1, PARKS, SNCA, NACP, PARK1 , PARK4, PRKN, PARK-2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-l); Schizophrenia (Neuregulinl (Nrgl ), Erb4 (receptor for Neuregulin), Complexinl (Cplxl), Tphl Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1,

GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd la), SLC6A3, DAOA, DTNBP1, Dao (Daol)); Secretase Related Disorders (APH-l (alpha and beta), Preseni I in (Psenl ), nicastrin, (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2); Trinucleotide Repeat Disorders (HTT (Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-l and Atnl (DRPLA Dx), CBP (Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7, AtxnlO).

[0080] Occular-related disorders: Genes linked to these disorders: Age-related macular degeneration (Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsinD, Vldlr, Ccr2);

Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA,

17 CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPAL, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

[0081] P13K/AKT Cellular Signaling disorders: Genes linked to these disorders:

PRKCE; ITGAM; ITGA5 ; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1 ; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2;

PIK3CB; PPP2R1A; MAPK8; BCL2L1 ; MAPK3 ; TSC2; ITGA1; KRAS; EIF4EBP1 ; RELA; PRKCD; NOS3; PRKAA1 ; MAPK9; CDK2; PPP2CA; PIM1 ; ITGB7; YWHAZ; ILK; TP53; RAF1 ; IKBKG; RELB; DYRK1A; CDKN1A; ITGB1 ; MAP2K2; JAK1 ; AKT1 ; JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3;

CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK; CSNK1A1 ; BRAF; GSK3B; AKT3;

FOXOl; SOK; HS P90AA1; RP S 6KB 1

[0082] ERK/MAPK Cellular Signaling disorders: Genes linked to these disorders: PRKCE; ITGAM; ITGA5 ; HSPB1; IRAK1 ; PRKAA2; EIF2AK2; RAC1 ; RAP1A; TLN1 ; EIF4E; ELK1; GRK6; MAPK1 ; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETS1 ; KRAS; MYCN; EIF4EBP1 ; PPARG; PRKCD; PRKAA1 ; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1 ; PXN; RAF1; FYN; DYRK1A;

ITGB1 ; MAP2K2; PAK4; PIK3R1 ; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1 ; ITGA2; MYC; TTK; CSNK1A1 ; CRKL; BRAE; ATF4; PRKCA; SRF; STAT1 ; SGK

[0083] Glucocorticoid Receptor Cellular Signaling disorders: Genes linked to these disorders: RAC1 ; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1 ; MAPK1 ; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1 ; FOS; HSPA5; NFKB2; BCL2;

MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1 ; MAPK3 ; TSC22D3; MAPK10; NRIP1 ; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1 ; NR3C1; PIK3C2A;

18 CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7;

CREBBP; CDKN1A; MAP2K2; JAK1 ; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK;

STAT3; MAP2K1 ; NFKB1; TGFBR1 ; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP 1 ; STAT1; IL6; HSP90AA1

[0084] Axonal Guidance Cellular Signaling disorders: Genes linked to these disorders: PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM 12; IGF1; RAC1; RAP1A; El F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1 ; PGF; RAC2; PTPN11 ; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1 ; GNAQ; PIK3CB; CXCL12;

PIK3C3; WNT11 ; PRKD1 ; GNB2L1 ; ABL1 ; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1 ; GUI; WNT5A; ADAM10; MAP2K1 ; PAK3 ; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA

[0085] Ephrin Recptor Cellular Signaling disorders: Genes linked to these disorders: PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1 ; PRKAA2; EIF2AK2; RAC1 ; RAP1A; GRK6; ROCK2; MAPK1 ; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; CDK8; CREB1 ; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1 ; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1 ; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1 ; JAK2; STAT3; ADAM10;

MAP2K1 ; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK

[0086] Actin Cytoskeleton Cellular Signaling disorders: Genes linked to these disorders: ACTN4; PRKCE; ITGAM; ROCK1 ; ITGA5; IRAK1; PRKAA2; EIF2AK2;

RAC1; INS; ARHGEF7 ; GRK6; ROCK2; MAPK1 ; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1;

ITGA1; KRAS; RHOA; PRKCD; PRKAA1 ; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7 ; PPP1CC; PXN; VIL2; RAF1 ; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A;

PIK3R1; MAP2K1 ; PAK3; ITGB3 ; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK

[0087] Huntington’s Disease Cellular Signaling disorders: Genes linked to these disorders: PRKCE; IGF1 ; EP300; RCOR1; PRKCZ; HDAC4; TGM2; MAPK1 ; CAPNS1 ; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKC1; HS PA5 ;

REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3 ; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A;

19 HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1 ; PDPK1 ; CASP1; APAF1 ; FRAP1 ; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CFTC; SGK; HDAC6; CASP3

[0088] Apoptosis Cellular Signaling disorders: Genes linked to these disorders:

PRKCE; ROCK1 ; BID; IRAK1 ; PRKAA2; EIF2AK2; BAK1 ; BIRC4; GRK6; MAPK1 ; CAPNS1 ; PEK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCF2; MAP3K14;

MAPK8; BCF2F1; CAPN1; MAPK3 ; CASP8; KRAS; REFA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1 ; TP53; TNF; RAF1; IKBKG; REFB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1 ; MAP2K1 ; NFKB1 ; PAK3; FMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3 : BTRC3: PARPI

[0089] B Cell Receptor Cellular Signaling disorders: Genes linked to these disorders: RAC1; PTEN; FYN; EFK1 ; MAPK1 ; RAC2; PTPN11 ; AKT2; IKBKB; PIK3CA; CREB1 ; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCF2F1 ; ABF1;

MAPK3 ; ETS1; KRAS; MAPK13; REFA; PTPN6; MAPK9; EGR1 ; PIK3C2A; BTK;

MAPK14; RAF1; IKBKG; REFB; MAP3K7; MAP2K2; AKT1; PIK3R1 ; CHUK; MAP2K1 ; NFKB1; CDC42; GSK3A; FRAP1 ; BCF6; BCF10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1

[0090] Leukocyte Extravasation Cellular Signaling disorders: Genes linked to these disorders: ACTN4; CD44; PRKCE; ITGAM; ROCK1 ; CXCR4; CYBA; RAC1 ; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11 ; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1 ; PIK3R1; CTNNB1 ; CLDN1 ; CDC42; FUR; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9

[0091] Integrin Cellular Signaling disorders: Genes linked to these disorders: ACTN4; ITGAM; ROCK1 ; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1 ; RAC2; CAPNS1 ; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1 ; MAPK3 ; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1 ; MAP2K2; PAK4; AKT1; PIK3R1 ; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3

[0092] Acute Phase Response Cellular Signaling disorders: Genes linked to these disorders: IRAK1 ; SOD2; MYD88; TRAF6; ELK1; MAPK1 ; PTPN11 ; AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14;

20 TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1 ; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6

[0093] PTEN Cellular Signaling disorders: Genes linked to these disorders: ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7 ; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1 ; MAP2K2; AKT1; PIK3R1 ; CHUK; PDGFRA; PDPK1 ; MAP2K1 ; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXOl; CASP3 ;

[0094] p53 Cellular Signaling disorders: Genes linked to these disorders: RPS6KB1

PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5 ; AKT2; PIK3CA;

CHEK1; TP53INP1 ; BCL2; PIK3CB; PIK3C3; MAPK8; THBS 1; ATR; BCL2L1 ; E2F1; PMAIP1; CHEK2; TNFASF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; EPRK2; AKT1 ; PIK3R1; RAM2B; APAF1 ; CTNNB1; SIRT1 ; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3

[0095] Aryl Hydrocarbon Receptor Cellular Signaling disorders: Genes linked to these disorders: HSPB1 ; EP300; FASN; TGM2; RXRA; MAPK1 ; NQOl ; NCOR2; SP1 ; ARNT; CDKN1B; FOS; CHEK1 ; SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1 ; MAPK3 ; NRIP1 ; CHEK2; RELA; TP73; GSTP1 ; RB1 ; SRC; CDK2; AHR; NFE2L2;

NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1 ; NFKB1; CCND1; ATM; ESR1;

CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1

[0096] Xenobiotic Metabolism Cellular Signaling disorders: Genes linked to these disorders: PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQOl ; NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1 ; ALDH1A1 ; MAPK3 ; NRIP1 ; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A; ABCB1 ; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP;

MAP2K2; PIK3R1 ; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6;

CYP1B1; HSP90AA1

[0097] SAPL/JNK Cellular Signaling disorders: Genes linked to these disorders:

PRKCE; IRAK1 ; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1 ; GADD45A; RAC2; PLK1 ; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1 ; IRS1 ; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1 ; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1 ; MAP2K1 ; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK

21 [0098] PPAr/RXR Cellular Signaling disorders: Genes linked to these disorders:

PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1 ; SMAD3 ;

GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IASI; MAPK3 ; KRAS; REFA; PRKAA1 ; PPARGC1A; NCOA3; MAPK14; INSR; RAF1 ;

IKBKG; REFB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1 ; NFKB 1 ;

TGFBA1; SMAD4; JUN; IE1R1; PRKCA; IE6; HSP90AA1; ADIPOO

[0099] NF-KB Cellular Signaling disorders: Genes linked to these disorders: IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB;

PIK3CA; BTRC; NFKB 2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; REFA; PIK3C2A; TRAF2; TFR4: PDGFRB; TNF; INSR; FCK; IKBKG; REFB; MAP3K7; CREBBP; AKT1; PIK3R1 ; CHUK; PDGFRA; NFKB1; TER2; BCE10; GSK3B; AKT3; TNFAIP3; IE1R1

[00100] Neuregulin Cellular Signaling disorders: Genes linked to these disorders:

ERBB4; PRKCE; ITGAM; ITGA5 : PTEN; PRKCZ; ELK1 ; MAPK1 ; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1 ; MAPK3; ITGA1 ; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM 17; AKT1; PIK3R1; PDPK1 ; MAP2K1 ; ITGB3; EREG; FRAP1; PSEN1 ; ITGA2; MYC; NRG1 ; CRKL; AKT3; PRKCA; HS P90AA1; RPS6KB1

[00101] Wnt and Beta catenin Cellular Signaling disorders: Genes linked to these disorders: CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO;

[00102] AKT2; PIN1 ; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1 ; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LAPS; CTNNB1; TGFBR1 ; CCND1; GSK3A; DVL1 ; APC; CDKN2A; MYC; CSNK1A1 ; GSK3B; AKT3; SOX2

[00103] Insulin Receptor Signaling disorders: Genes linked to these disorders: PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1 ; TSC1; PTPN11 ; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IASI; MAPK3; TSC2; KRAS; EIF4EBP1 ; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1 ; AKT1; JAK2; PIK3R1; PDPK1 ; MAP2K1 ; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXOl ; SGK; RPS6KB1

[00104] IL-6 Cellular Signaling disorders: Genes linked to these disorders: HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1 ; PTPN11; IKBKB; FOS; NFKB2: MAP3K14;

MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1 ; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1 ; NFKB 1 ; CEBPB; JUN; IL1R1; SRF; IL6

22 [00105] Hepatic Cholestasis Cellular Signaling disorders: Genes linked to these disorders: PRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10; REFA; PRKCD; MAPK9;

ABCB1; TRAF2; TFR4; TNF; INSR; IKBKG; REFB; MAP3K7; IE8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1 ; FGFR4; JUN; IE1R1 ; PRKCA; IE6

[00106] IGF-1 Cellular Signaling disorders: Genes linked to these disorders: IGF1 ;

PRKCZ; EEK1; MAPK1; PTPN11 ; NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS;

PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1 ; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1 ; PDPK1 ; MAP2K1 ; IGFBP2; SFN; JUN; CYR61 ; AKT3; FOXOl; SRF; CTGF; RPS6KB1

[00107] NRF2-mediated Oxidative Stress Response Signaling disorders: Genes linked to these disorders: PRKCE; EP300; SOD2; PRKCZ; MAPK1 ; SQSTM1; NQOl; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; PRKD1 ; MAPK3; KRAS; PRKCD; GSTP1 ; MAPK9; FTF; NFE2F2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1 ; MAP2K1 ; PPIB; JUN; KEAP1 ; GSK3B; ATF4; PRKCA; EIF2AK3;

HSP90AA1

[00108] Hepatic Fibrosis/Hepatic Stellate Cell Activation Signaling disorders: Genes linked to these disorders: EDN1 ; IGF1 ; KDR; FFT1; SMAD2; FGFR1 ; MET; PGF; SMAD3; EGFR; FAS; CSF1 ; NFKB2; BCF2; MYH9; IGF1R; IF6R; REFA; TFR4; PDGFRB; TNF; REFB; IF8; PDGFRA; NFKB1 ; TGFBR1; SMAD4; VEGFA; BAX; IF1R1; CCF2; HGF; MMP1; STAT1 ; IF6; CTGF; MMP9

[00109] PPAR Signaling disorders: Genes linked to these disorders: EP300; INS; TRAF6; PPARA; RXRA; MAPK1 ; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1 ; KRAS; PPARG; REFA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1 ; IKBKG; REFB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;

NFKB1; JUN; IF1R1; HSP90AA1

[00110] Fc Epsilon RI Signaling disorders: Genes linked to these disorders: PRKCE; RAC1; PRKCZ; FYN; MAPK1 ; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3 ; MAPK10; KRAS; MAPK13; PRKCD; MAPK9;

PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1 ; PIK3R1; PDPK1 ;

MAP2K1 ; AKT3; VAV3; PRKCA

[00111] G-Protein Coupled Receptor Signaling disorders: Genes linked to these disorders: PRKCE; RAP1A; RGS16; MAPK1 ; GNAS; AKT2; IKBKB; PIK3CA; CREB1 ; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; REFA; SRC; PIK3C2A;

23 RAF1 ; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1 ; CHUK; PDPK1; S TAT3 ; MAP2K1 ; NFKB1; BRAF; ATF4; AKT3; PRKCA

[00112] Inositol Phosphate Metabolism Signaling disorders: Genes linked to these disorders: PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MAPK1 ; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; PIM1 ; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK

[00113] PDGF Signaling disorders: Genes linked to these disorders: EIF2AK2; ELK1; ABL2; MAPK1 ; PIK3CA; FOS; PIK3CB; P IK3 C3 ; MAPK8; CAV1 ; ABL1 ; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF Signaling disorders: Genes linked to these disorders: ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1 ; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3;

BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1 ; MAP2K2; ELAVL1 ; AKT1; PIK3R1 ; MAP2K1 ; SFN; VEGFA; AKT3; FOXOl; PRKCA

[00114] Natural Killer Cell Signaling disorders: Genes linked to these disorders: PRKCE; RAC1; PRKCZ; MAPK1 ; RAC2; PTPN11 ; KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1 ; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1 ; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1 ; PAK3; AKT3; VAV3; PRKCA

[00115] Cell Cycle: Gl/S Checkpoint Regulation Signaling disorders: Genes linked to these disorders: HDAC4; SMAD3 ; SUV39H1; HDAC5; CDKN1B; BTRC; ATR; ABL1 ; E2F1; HDAC2; HDAC7A; RB1; HD AC 11 ; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1 ; GSK3B; RBL1 ; HDAC6

[00116] T Cell Receptor Signaling disorders: Genes linked to these disorders: RAC1; ELK1 ; MAPK1 ; IKBKB; CBL; PIK3CA; FOS; NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3 ; KRAS; RELA, PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1 ; NFKB 1 ; ITK; BCL10; JUN; VAV3

[00117] Death Receptor disorders: Genes linked to these disorders: CRADD; HSPB1 ; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1 ; NFKB1; CASP2; BIRC2; CASP3; BIRC3

[00118] FGF Cell Signaling disorders: Genes linked to these disorders: RAC1 ; FGFR1; MET; MAPKAPK2; MAPK1 ; PTPN11; AKT2;PIK3CA; CREB1 ; PIK3CB; PIK3C3;

24 MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1 ; AKT1; PIK3R1;

STAT3; MAP2K1 ; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF

[00119] GM-CSF Cell Signaling disorders: Genes linked to these disorders: LYN; ELK1; MAPK1 ; PTPN11 ; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1 ; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; PIM1 ; PIK3C2A; RAF1; MAP2K2; AKT1 ; JAK2; PIK3R1 ; STAT3; MAP2K1 ; CCND1; AKT3; STAT1

[00120] Amyotrophic Lateral Sclerosis Cell Signaling disorders: Genes linked to these disorders: BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1 ; CAPN1 ; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1 ; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 PTPN1; MAPK1 ; PTPN11 ; AKT2;

PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3 ; KRAS; SOCS1 ; STAT5A; PTPN6;

PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1 ; FRAP1; AKT3; STAT1

[00121] JAK/Stat Cell Signaling disorders: Genes linked to these disorders: PTPN1 ; MAPK1 ; PTPN11 ; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1 ; STAT5A; PTPN6; PIK3C2A; RAF1 ; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1 ; FRAP1 ; AKT3; STAT1

[00122] Nicotinate and Nicotinamide Metabolism Cell Signaling disorders: Genes linked to these disorders: PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1 ; PLK1 ; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1 ; PBEF1; MAPK9; CDK2; PIM1 ; DYRK1A; MAP2K2; MAP2K1 ; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK

[00123] Chemokine Cell Signaling disorders: Genes linked to these disorders: CXCR4; ROCK2; MAPK1 ; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1 ; JUN; CCL2; PRKCA

[00124] IL-2 Cell Signaling disorders: Genes linked to these disorders: ELK1; MAPK1 ; PTPN11 ; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1 ; MAP2K2; JAK1 ; AKT1; PIK3R1; MAP2K1 ; JUN; AKT3

[00125] Synaptic Long Term Depression Signaling disorders: Genes linked to these disorders: PRKCE; IGF1 ; PRKCZ; PRDX6; LYN; MAPK1 ; GNAS; PRKCI; GNAQ;

PPP2R1A; IGF1R; PRKD1 ; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1 ; MAP2K2; PPP2R5C; MAP2K1 ; PRKCA

25 [00126] Estrogen Receptor Cell Signaling disorders: Genes linked to these disorders: TAF4B; EP300; CARM1; PCAF; MAPK1 ; NCOR2; SMARCA4; MAPK3 ; NRIP1 ; KRAS; SRC; NR3C1 ; HDAC3; PPARGC1A; RBM9; NCOA3 ; RAF1 ; CREBBP; MAP2K2;

NCOA2; MAP2K1 ; PRKDC; ESR1 ; ESR2

[00127] Protein Ubiquitination Pathway Cell Signaling disorders: Genes linked to these disorders: TRAF6; SMURF 1; BIRC4; BRCA1; UCHL1; NEDD4; CBL; UBE2I; BTRC; HSPA5 ; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1 ; BIRC3

[00128] IL-10 Cell Signaling disorders: Genes linked to these disorders: TRAF6; CCR1; ELK1 ; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14;

TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1 ; IL6

[00129] VDR/RXR Activation Signaling disorders: Genes linked to these disorders: PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3 ; CDKN1A; NCOA2; SPP1; LAPS;

CEBPB; FOXOl; PRKCA

[00130] TGF-beta Cell Signaling disorders: Genes linked to these disorders: EP300; SMAD2; SMURF1 ; MAPK1 ; SMAD3; SMAD1; FOS; MAPK8; MAPK3 ; KRAS; MAPK9; RUNX2; SERPINE1 ; RAF1 ; MAP3K7 ; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5

[00131] Toll-like Receptor Cell Signaling disorders: Genes linked to these disorders: IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN

[00132] p38 MAPK Cell Signaling disorders: Genes linked to these disorders: HSPB1 ; IRAK1; TRAF6; MAPKAPK2; ELK1 ; FADD; FAS; CREB1; DDIT3 ; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF;

STAT1

[00133] Neurolrophin/TRK Cell Signaling disorders: Genes linked to these disorders: NTRK2; MAPK1 ; PTPN11; PIK3CA; CREB1 ; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3 ; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1 ; PDPK1 ; MAP2K1 ; CDC42; JUN; ATF4

[00134] Other cellular dysfunction disorders linked to a genetic modification are contemplated herein for example, FXR/RXR Activation, Synaptic Long Term Potentiation, Calcium Signaling EGF Signaling, Hypoxia Signaling in the Cardiovascular System, LPS/IL-

26 1 Mediated Inhibition of RXR Function LXR/RXR Activation, Amyloid Processing, IL-4 Signaling, Cell Cycle: G2/M DNA Damage Checkpoint Regulation, Nitric Oxide Signaling in the Cardiovascular System Purine Metabolism, cAMP-mediated Signaling, Mitochondrial Dysfunction Notch Signaling Endoplasmic Reticulum Stress Pathway Pyrimidine

Metabolism, Parkinson's Signaling Cardiac & Beta Adrenergic Signaling Glycolysis/ Gluconeogenesis Interferon Signaling Sonic Hedgehog Signaling Glycerophospholipid Metabolism, Phospholipid Degradation, Tryptophan Metabolism Lysine Degradation Nucleotide Excision Repair Pathway, Starch and Sucrose Metabolism, Aminosugars Metabolism Arachidonic Acid Metabolism, Circadian Rhythm Signaling, Coagulation System Dopamine Receptor Signaling, Glutathione Metabolism Glycerolipid Metabolism Linoleic Acid Metabolism Methionine Metabolism Pyruvate Metabolism Arginine and Praline Metabolism, Eicosanoid Signaling Fructose and Mannose Metabolism, Galactose Metabolism Stilbene, Coumarine and Lignin Biosynthesis Antigen Presentation Pathway, Biosynthesis of Steroids Butanoate Metabolism Citrate Cycle Fatty Acid Metabolism

Glycerophosphol ipid Metabolism, Histidine Metabolism Inositol Metabolism Metabolism of Xenobiotics by Cytochrome p450, Methane Metabolism, Phenylalanine Metabolism, Propanoate Metabolism Selenoamino Acid Metabolism Sphingolipid Metabolism

Aminophosphonate Metabolism, Androgen and Estrogen Metabolism Ascorbate and Aldarate Metabolism, Bile Acid Biosynthesis Cysteine Metabolism Fatty Acid Biosynthesis Glutamate Receptor Signaling, NRF2-mediated, Oxidative Stress Response Pentose Phosphate Pathway, Pentose and Glucuronate Interconversions, Retinol Metabolism Riboflavin Metabolism Tyrosine Metabolism Ubiquinone Biosynthesis Valine, Leucine and Isoleucine Degradation Glycine, Serine and Threonine Metabolism Lysine Degradation Pain/Taste, or Mitochondrial Function Developmental Neurology or combinations thereof.

[00135] In certain embodiments, compositions and methods of modifying a target polynucleotide in a eukaryotic cell are disclosed. In accordance with these embodiments, engineered chimeric nucleic acid guided nucleases bind to a target polynucleotide to effect cleavage of the target polynucleotide thereby modifying the target polynucleotide, wherein the engineered chimeric nucleic acid guided nuclease system comprises an engineered chimeric nucleic acid guided nuclease complexed with a guide sequence (gRNA) hybridized to a target sequence within the target polynucleotide for improved targeting and editing of the polynucleotide.

[00136] In another aspect disclosed herein, methods and compositions are provided for modifying expression of a polynucleotide in a eukaryotic cell of a subject. In some

27 embodiments, compositions and methods include an engineered chimeric nucleic acid guided nuclease system complex capable of binding a target polynucleotide such that binding leads to an in increased or decreased expression of the targeted polynucleotide; wherein the engineered chimeric nucleic acid guided nuclease system complex comprises an engineered chimeric nucleic acid guided nuclease complexed with a guide sequence (gRNA) hybridized to a target sequence within the targeted polynucleotide, wherein the complex is capable of altering expression of the targeted polynucleotide.

[00137] In some embodiments, a target polynucleotide of an engineered chimeric nucleic acid guided nuclease system complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell or other cell. In accordance with these embodiments, the target polynucleotide can be a polynucleotide located in the nucleus of the eukaryotic cell. In certain embodiments, the target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). In other embodiments, the target sequence is associated with a PAM (protospacer adjacent motif). A PAM is, a short sequence recognized by the engineered chimeric nucleic acid guided nuclease. Sequences and lengths for PAM differ depending on the engineered chimeric nucleic acid guided nuclease used, but PAMs can be 2-5 base pair sequences adjacent a protospacer (that is, the target sequence. Examples of PAM sequences provided herein and in the examples section below. One of skill in the art will be able to identify further PAM sequences for use with a given engineered chimeric nucleic acid guided nuclease of the instant application using known methods.

[00138] In certain embodiments, a targeted gene of a genetic disorder can include a genetic disorder of a human or other mammal such as a pet, livestock or other animal. In yet other embodiments, a targeted gene of a genetic disorder can include a genetic plant disorder.

[00139] With advances in crop genomics, the ability to use gene-editing systems to perform efficient and cost effective gene editing and manipulation can allow rapid selection and comparison of single and multiplexed genetic manipulations to transform such genomes for improved production and enhanced traits such as drought resistance and resistance to infection, for example.

[00140] Some embodiments disclosed herein relate to use of an engineered chimeric nucleic acid guided nuclease system disclosed herein; for example, in order to target and knock out genes, amplify genes and/or repair particular mutations associated with DNA repeat instability and a medical disorder. This chimeric nuclease system may be used to harness and to correct these defects of genomic instability. In other embodiments, engineered

28 chimeric nucleic acid guided nuclease systems disclosed herein can be used for correcting defects in the genes associated with Lafora disease. Lafora disease is an autosomal recessive condition which is characterized by progressive myoclonus epilepsy which may start as epileptic seizures in adolescence. This condition causes seizures, muscle spasms, difficulty walking, dementia, and eventually death.

[00141] In yet another aspect of the invention, the engineered chimeric nucleic acid guided nuclease system can be used to correct genetic-eye disorders that arise from several genetic mutations further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

[00142] Several further aspects of the invention relate to correcting defects associated with a wide range of genetic diseases which are further described on the website of the National Institutes of Health under the topic subsection Genetic Disorders. Certain genetic disorders of the brain can include, but are not limited to, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi Syndrome, Alpers' Disease, glioblastoma, Alzheimer's, Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration, Fabry's Disease, Gerstmann-Straussler- Schei-nker Disease, Huntington's Disease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-Nyhan Syndrome, Menkes Disease, Mitochondrial Myopathies and NINDS Colpocephaly or other brain disorder contributed to by genetic ally- linked causation.

[00143] In some embodiments, a genetically-linked disorder can be a neoplasia. In some embodiments, where the condition is neoplasia, targeted genes can include one or more genes listed above. In some embodiments, a health condition contemplated herein can be Age- related Macular Degeneration or a Schizophrenic-related Disorder. In other embodiments, the condition may be a Trinucleotide Repeat disorder or Fragile X Syndrome. In other embodiments, the condition may be a Secretase-related disorder. In some embodiments, the condition may be a Prion-related disorder. In some embodiments, the condition may be ALS. In some embodiments, the condition may be a drug addiction related to prescription or illegal substances. In accordance with these embodiments, addiction-related proteins may include ABAT for example.

[00144] In some embodiments, the condition may be Autism. In some embodiments, the health condition may be an inflammatory-related condition, for example, over-expression of a pro-inflammatory cytokine. Other inflammatory condition-related proteins can include one or more of monocyte chemoattractant protein- 1 (MCP1) encoded by the Ccr2 gene, the C C chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgG receptor IIB

29 (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, or the Fc epsilon Rlg (FCERlg) protein encoded by the Fcerlg gene, or other protein having a genetic-link to these conditions.

[00145] In some embodiments, the condition may be Parkinson's Disease. In accordance with these embodiments, proteins associated with Parkinson's disease can include, but are not limited to, a-synuclein, DJ-l, LRRK2, PINK1, Parkin, UCHL1, Synphilin-l, and NURR1.

[00146] Cardiovascular- associated proteins that contribute to a cardiac disorder, can include, but are not limited to, IIAb (interleukin l-beta), XDH (xanthine dehy-drogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB

(myoglobin), IL4 (interleu-kin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cas- sette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), or other known contributors to these conditions.

[00147] In some embodiments, the condition may be Alzheimer's disease. In accordance with these embodiments, Alzheimer's disease associated proteins may include very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or for example, NEDD8- activating enzyme El catalytic subunit protein (UBE1C) encoded by the UBA3 gene or other genetically-related contributor.

[00148] In some embodiments, the condition may be an Autism Spectrum Disorder. In accordance with these embodiments, proteins associated Autism Spectrum Disorders can include the benzodiazapine receptor (peripheral) associated protein 1 (BZRAP1) encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2) encoded by the AFF2 gene (also termed MFR2), the fragile X mental retardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or the fragile X mental retardation autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene, or other genetically-related contributor.

[00149] In some embodiments, the condition may be Macular Degeneration. In accordance with these embodiments, proteins associated with Macular Degeneration can include, but are not limited to, the ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded by the APOE gene, or the chemokine (CC motif) Llg and 2 protein (CCL2) encoded by the CCL2 gene, or other genetically-related contributor.

[00150] In some embodiments, the condition may be Schizophrenia. In accordance with these embodiments, proteins associated with Schizophrenia In accordance with these

30 embodiments, proteins associated with Schizophrenia y include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISCI, GSK3B, and combinations thereof.

[00151] In some embodiments, the condition may be tumor suppression. In accordance with these embodiments, proteins associated with tumor suppression can include ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene ho mo log 4), Notch 1, Notch2, Notch 3, or Notch 4 or other genetically-related contributor.

[00152] In some embodiments, the condition may be a secretase disorder. In accordance with these embodiments, proteins associated with a secretase disorder can include PSENEN (presenilin enhancer 2 ho mo log (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B (anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), or BACE1 (beta-site APP- cleaving enzyme 1), or other genetically-related contributor.

[00153] In some embodiments, the condition may be Amyotrophic Lateral Sclerosis. In accordance with these embodiments, proteins associated with can include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof or other genetically-related contributor.

[00154] In some embodiments, the condition may be a prion disease disorder. In accordance with these embodiments, proteins associated with a prion diseases disorder can include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof or other genetically-related contributor.

Examples of proteins related to neurodegenerative conditions in prion disorders can include A2M (Alpha-2-Macro-globulin), AATF (Apoptosis antagonizing transcription factor), ACPP (Acid phosphatase prostate), ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidase domain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha- 1D adrenergic receptor for Alpha- 1D adrenoreceptor), or other genetically-related contributor.

31 [00155] In some embodiments, the condition may be an immunodeficiency disorder. In accordance with these embodiments, proteins associated with an immunodeficiency disorder can include A2M [alpha-2-macro globulin]; AANAT [aryla-lkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2 [ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3 [ATP-binding cassette, sub-family A (ABC 1 ), member 3]; or other genetically-related contributor.

[00156] In some embodiments, the condition may be an immunodeficiency disorder. In accordance with these embodiments, proteins associated with an immunodeficiency disorder can include Trinucleotide Repeat Disorders include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin), or DMPK (dystro-phia myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), or other genetically-related contributor.

[00157] In some embodiments, the condition may be a Neuro transmission Disorders. In accordance with these embodiments, proteins associated with a Neurotransmission Disorders can include SST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A (adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-, receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydrox-ytryptamine (serotonin) receptor 2C), or other genetically-related contributor. In other embodiments, neurodevelopmental-associated sequences can include, but are not limited to, A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4- aminobutyrate aminotrans- ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member 13], or other genetically-related contributor.

[00158] In yet other embodiments, genetic health conditions can include, but are not limited to Aicardi-Goutieres Syndrome; Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-Related Disorders; Alpha-Mannosidosis (Type II and III); Alstrom Syndrome;

Angelman; Syndrome; Ataxia-Telangiectasia; Neuronal Ceroid-Lipofuscinoses; Beta- Thalassemia; Bilateral Optic Atrophy and (Infantile) 3 Optic Atrophy Type 1;

Retinoblastoma (bilateral); Canavan Disease; Cerebrooculofacioskeletal Syndrome 1

[COFS1]; Cerebrotendinous Xanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders; Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial Alzheimer Disease; 4 Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; Fukuyama Congenital Muscular Dystrophy; Galactosialido-sis; Gaucher Disease; Organic Acidemias;

Hemophagocytic Lymphohistiocytosis; Hutchinson-Gilford Progeria Syndrome;

Mucolipidosis II; Infantile Free Sialic Acid Storage 4 Disease; PLA2G6- Associated

32 Neurodegeneration; Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa; Huntington Disease; Krabbe Disease (Infantile); Mitochondrial DNA- Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome; LIST- Associated Lissen- 5 cephaly; Lowe Syndrome; Maple Syrup Urine Disease; MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders; LAMA2-Related Muscular Dystrophy; Arylsulfatase A

Deficiency; Mucopolysaccharidosis Types I, II or III; Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum; Neurodegeneration with Brain Iron Accu-mulation

Disorders; Acid Sphingomyelinase Deficiency; Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-Related Disorders; Urea Cycle Disorders; COLlAl/2-Related Osteogenesis Imperfecta; Mitochondrial DNA Deletion Syndromes; PLP1 -Related Disorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen Storage Disease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders; MECP2-Related Disorders; Rhizomelic Chondrodys-plasia Punctata Type 1; Roberts Syndrome; Sandhoff Disease; Schindler Disease Type 1 ; Adenosine Deaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal Muscular Atrophy; Infantile-Onset Spinocerebellar Ataxia; Hex-osaminidase A Deficiency;

Thanatophoric Dysplasia Type 1 ; Collagen Type VI-Related Disorders; Usher Syndrome Type I; Congenital Muscular Dystrophy; Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; and Xeroderma Pigmentosum.

[00159] In other embodiments, genetic disorders in animals targeted by editing systems disclosed herein can include, but are not limited to, Hip Dysplasia, Urinary Bladder conditions, epilepsy, cardiac disorders, Degenerative Myelopathy, Brachycephalic Syndrome, Glycogen Branching Enzyme Deficiency (GBED), Hereditary Equine Regional Dermal Asthenia (HERD A), Hyperkalemic Periodic Paralysis Disease (HYPP), Malignant

Hyperthermia (MH), Polysaccharide Storage Myopathy - Type 1 (PSSM1), junctional epdiermolysis bullosa, cerebellar abiotrophy, lavender foal syndrome, fatal familial insomnia, or other animal-related genetic disorder.

[00160] In certain embodiments disclosed herein can include engineered chimeric nucleic acid guided nuclease construct libraries having a first module from at least a first Casl2a-like nuclease; and at least a second module from at least a second Casl2a-like nuclease, wherein the first nucleotide module and the second module form chimeric nucleases. In accordance with these embodiments, the engineered chimeric construct nuclease library can recognize a protospacer adjacent motif (PAM) sequence other than TTTN or in addition to TTTN. In other embodiments, engineered chimeric nucleases of libraries disclosed herein can be further mutated to improve targeting efficiency or can be selected from a library for particular

33 targeted features. Certain engineered chimeric Casl2a-like nuclease constructs are generated by a cross-over of about five to about thirty- five amino acids in length, located between various modules described in certain embodiments herein. Other embodiments disclosed herein concern vectors comprising constructs of libraries disclosed herein of use for further analysis and to select for improved genome editing features.

[00161] Other embodiments include kits for packaging and transporting whole libraries or individual or multiple chimera Casl2a-like nucleases disclosed herein and further include at least one container.

[00162] As will be apparent, it is envisaged that the present system can be used to target any polynucleotide sequence of interest. Some examples of conditions or diseases that might be use fully treated using the present system are included in the Tables above and examples of genes currently associated with those conditions are also provided there. However, the genes exemplified are not exhaustive. Additional objects, advantages, and novel features of this disclosure will become apparent to those skilled in the art upon review of the following examples in light of this disclosure. The following examples are not intended to be limiting. EXAMPLES

[00163] The following examples are included to illustrate various embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practice of the claimed methods, compositions and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[00164] Instead of relying on identification, isolation and characterization of new Casl2a- type nucleases that occur in nature, the generation of chimeric sequences from known orthologs could more effectively expand the search space for the selection of novel functions that are non-naturally occurring and possess improved features. In addition, the module-like nature of CRISPR nucleases would facilitate domain recombination strategies for chimera generation. In certain exemplary methods, nine Casl2a-type gene sequences (Fig. 11 A and 11B) were selected as a pool spanning a broad sequence number (e.g. 560 variants). Multiple variants were tested for editing efficiency in E. coli (see for example, Fig. 7B). All of the tested nucleases were functional in E. coli using the galK inactivation assay, and the transformation efficiencies (CFU/ug) of all the nucleases were at or higher than 10⁵ (see for example Fig. 7B).

34 [00165] In certain exemplary methods, a structure-guided design approach was used to build a chimeric nuclease library. The Casl2a-type nuclease sequences were aligned with the AsCasl2a and LbCasl2a sequences (Fig. 22 and Figs. 8A-8I) and observed a relatively low level (33.8% ~ 42.99%) of sequence identity (Fig. 22), which limits homology driven strategies and constraining the mutational space capable of exploring via chimeragenesis. Therefore, modules based on known structures from AsCl2a/LbCasl2a designer junctions points were identified for crossover. Based on the sequence alignment results, six crossover points were identified for chimeragenesis (Fig. 7C, Fig 22). Regions that spanned at least 500 bp and contained one or more functional domains were selected. Each cross over point had higher amino acid conservation across orthologs than the surrounding sequence (Fig. 22). These crossover points were designed to include the following chimeric domains: 1) REC1; 2) REC1/REC2; 3) REC 1 /REC2/WED-IFPI; 4) WED-III/RuvC-I/BH/RuvC-II/Nuc/RuvC- III; 5) RuvC-I/BH/RuvC-II/Nuc/RuvC-III; and/or 6) Nuc/RuvC-III. Libraries were constructed in parallel (Fig. 22 and additional data not shown), and the distribution of library variants was not higher than 25% and 15% in 1 and 2 crossover library constructions, respectively (Fig. 7D).

[00166] A bacterial test system was designed to assess genetic editing capabilities (e.g. Escherichia coli) in a Casl2a-like chimera nuclease (and non-chimera Casl2a nuclease system) library with 560 mutants that combined up to six conserved regions from a diverse starting pool of Casl2a like nucleases. Then the library of mutants was selected and screened for functional chimera Casl2a-type nucleases. To demonstrate efficacy and use of this strategy as a platform for rapid generation of novel nucleases, several of the most active chimeras were further tested that demonstrated altered PAM preferences and on- vs off- targeting. As disclosed herein, it was observed that these chimera Casl2a-type nuclease mutant libraries contain novel nucleases that are capable of editing bacteria, yeast (e.g. Saccharomyces cerevisiae) and human cells (e.g. HEK293T). These strategies provide for rapidly building and selecting novel and targeted synthetic nucleases across a broad range of applications from prokaryotic to eukaryotic systems.

Example 1

[00167] In one exemplary method, several different wild-type Casl2as were used to generate chimeras of the instantly claimed inventions. For example, Casl2a nucleases have different lobes REC1, REC2, WED-I, NuvC, RuvC-I, RuvC-II, etc. In certain methods, many different Casl2a nucleases (e.g. nine different Casl2a nucleases) can be used as templates for constructing chimeras. Any Casl2a nuclease is contemplated for use in systems and methods

35 disclosed herein. In this example, Casl2a nucleases were cleaved 5’ of these recognition sites in certain exemplary methods to construct designer non-naturally occurring chimeric Casl2a constructs with conserved genome editing capabilities. For example, Casl2as were separated into DNA fragments by the above lobes based on protein sequence alignment. Chimera Cas 12 as were constructed by for example, a Gibson assembly method to recombine the DNA fragments to the chimera Cas 12a- like nucleases. The overlap region was designed of about 5 to about 50 bps (e.g. ~40 base pairs) among the fragments which were lobes adjacent to one another. Gene fragments with overlap region (data not shown) were obtained from DNA synthesis. In some examples, a NEBuilder HiFi DNA Assembly Cloning Kit was used to process construction of the exemplary Casl2a plasmids.

[00168] In other methods, a control Casl2a was used to assess Casl2a genome editing capabilities of the designer chimeric constructs. A schematic representation is illustrated in Fig. 1A. The control was used as a comparison template where one and in some cases two cleavages were made in the control sequence. For example, a Cas 12a chimeric construct was introduced into a plasmid having lambda red proteins. The exemplary Casl2a nuclease plasmid contains a temperature sensitive inducible promoter. The lambda red proteins of the plasmid used in these recombineering techniques have an arabinose inducible promoter.

Then, the designer chimera Cas 12a nucleases were introduced into a plasmid library in a bacterial culture (e.g., E. coli strain MG1655). See Fig. 1B for a schematic of exemplary Casl2a-like chimera nucleases. Following this process, a second plasmid (e.g., gRNA plasmid) was introduced to the bacterial culture. This second plasmid targets the galK gene (e.g. knocking out this galK gene) on the E. coli genome. (See for example the schematic of Fig. 2) It was demonstrated that the designer chimeric constructs in the tested bacterial cultures created two phenotypes when the strain contained a chimera having genome editing capabilities :l) the E. coli is capable of growing on the 2-DOG media; and 2) the E. coli colony is white in color on MacConkey agar. It was demonstrated that these chimeric constructs in the tested bacterial cultures created two phenotypes when the strain contained a chimera not having genome editing capabilities:!) the E. coli is unable to grow on the 2-DOG media and 2) the E. coli colony is red in color on the MacConkey agar. Therefore, these easily distinguishable phenotypes were used to demonstrate E. coli having editing or not having editing capabilities, for screening and selecting for genome-editing/functional chimera Casl2a constructs.

[00169] In certain methods, these 2-DOG selection methods were used to readily identify genome-editing/functional chimera Casl2a constructs. With these methods, a gal-off color

36 screening method (on the MacConkey agar) was used wherein editing efficiency of chimera Casl2a construct was calculated.

[00170] In certain exemplary methods, verification of functional chimera library variants. (Fig. 1A) illustrates exemplary workflow to verify positive variants. Two plasmid systems for genome editing were constructed: in certain methods, one plasmid can express a Cas protein as well as for example, lambda red proteins (exo, bet, and gam); a second plasmid can express a single crRNA (with J23119 promoter) targeting the galK gene and a homology arm (HM) containing a galK-inactivating mutation as a template for recombineering. Strains with GalK inactivation are able to grow in media supplemented with 2-DOG (demonstrating editing), while any unedited strains will not grow in 2-DOG, eliminating the need for further screening. In addition, positive mutants were evaluated for editing efficiency in E. coli using a color screening method based on GalK inactivation. In this assay, colonies with active GalK are red and colonies with inactive (edited) GalK are white. (1B) The editing and

transformation efficiency test of library variants. Transformation efficiency is defined as the number of colony forming units (cfu) per pg of gRNA plasmid. (1C) The diagram of positive chimera variants based on the Casl2a structure. (The structure of AsCasl2a used as the template).

Example 2

[00171] In other exemplary methods, kanamycin-containing plasmid constructs containing PAM_testing cassettes libraries were created for assessing genome editing specificity and efficiency. For these libraries, each plasmid contained the same spacer but different PAM sites for Casl2a. The designer chimeric Casl2a constructs were introduced to test genome editing capabilities of the constructs when in the presence of the gRNA targeting having the same spacer as the PAM_testing cassettes library. In these experiments, if the E. coli cells cannot grow on a kanamycin-containing media, then the PAM on the kanamycin plasmid is a functional PAM, recognized by the designer chimeric Casl2a-like construct. Alternatively, if the E. coli cells can grow on the kanamycin media, then the PAM on the kanamycin plasmid is a non- functional PAM and the designer chimeric Cas 12a- like construct is incapable of performing Cas 12a genome editing by lack of recognition of the PAM.

[00172] In certain methods, chimeric constructs created by strategies disclosed herein were selected based on criteria referenced above where the chimeric construct created grew on 2- DOG media but was white in color on MacConkey agar. These designer chimeric Casl2a- like nucleases were selected and further analyzed for improved editing, for example, reduced off-targeting rates and PAM recognition criteria.

37 Structure-guided design of Casl2a-type chimera library

Example 3

Isolation of functional Casl2a-type chimeras

[00173] In certain exemplary methods, functional chimeras were selected using both a galK based growth selection and galK colorimetric screen ((Fig. 1A and Figs. 13A and 13B). Selected variants were then characterized by sequencing of the plasmid insert region containing the chimeric gene. In this example, it was expected that 1) a small number of chimeric Casl2a-like protein sequences retain specific modules to produce a spectrum of activities, or 2) a large number of overall chimeric Casl2a-like protein sequences have a small number specific recombined domains. Sequencing of certain selected chimeric Casl2a- like nucleases identified 24 chimeric sequences after selection, eight of which were identified more than once in the sequenced pool (See for example Fig. 14). All eight chimeric Casl2a- like nuclease variants were characterized by recombination at the REC1 lobe, which has not been previously reported. In addition, one crossover at position 6 that defines the boundary between RuvC-II and the NUC domains was also identified. All eight chimeric Casl2a-like nuclease variants exhibited reasonable editing efficiency, with two (e.g. M44, M21) exhibiting editing efficiency approaching that of the best WT nuclease sequence (Fig. 7B compared to Fig. 1A-1C) when confirmed by the galK based colorimetric screen (Fig. 1A- 1B). A second crossover at position 6 in M22 significantly decreased the editing efficiency compared to M44, which suggests REC1 domain had potential flexibility compared to other domains. These data provide evidence that this method for chimeragensis is a viable approach for the generation of novel chimeric Casl2a-like nuclease sequences allowing for selectivity of improved traits depending on the target for genetic editing.

Example 4

Verification of functional Casl2a-type variants

[00174] In another exemplary method, activity of selected Casl2a-like chimera nuclease were analyzed using several additional editing assays and a positive constrol nuclease for comparison. Five additional inactivating mutations were selected positioned in the galK and lacZ genes that could be used to measure nuclease mediated cell killing (gRNA directed cutting only) (Fig. 8A-8I) and nuclease mediated editing via color screening (Fig. 8C and Fig. 15). For these examples, mutants M44 and M21 (which is identical to M44 except for a single G218A substitution), displayed the same cutting efficiency (100%) with wild-type control using 4 of 6 designed gRNAs targeted on galK and laz genes (Fig. 8B). However, not every gRNA elicits cleavage although the gRNAs were targeted in the same gene. 4 gRNAs with

38 high cutting efficiency were identified and used for editing constructs as a representative sample for testing the inactivation editing at different position of galK and lacZ genes. Editing efficiencies of control, M44, and M21 were not as high as their cutting efficiencies. Using galK2 and lacZl gRNAs, the editing efficiencies of M21 and M44 were 12.5% and 38% higher than control. Using galKl and lacZ2, the editing efficiency of the control was 60% higher than M44 (mutants with highest editing efficiency) (Fig. 8C). These results indicated that there were more factors affecting the recombineering using Casl2a-like chimeras. For example, transformation efficiency for all the Casl2a-type chimeras were higher than the control (see for example Figs. 16A-16D).

[00175] After testing different targets at the same position on the genome, the editing capability of chimeras was assessed for change as a function of targeting different positions in the genome. Five distinct“safe sites” were targeted which are non-essential sites in E. coli BW25113 genome chosen for integration of heterologous genes with minimal predicted side effects. For these exemplary methods, wild-type galK gene was deleted, and then the galK gene was integrated with a strong constitutive promoter J23119 into the five chosen safe sites (Fig. 17A). Editing efficiency of exemplary chimeric Casl2a-like nucleases (e.g. M44, M21, and M38) was confirmed (Fig. 17A-17C). In addition, a positional dependency conserved across all testing nucleases was identified with higher editing efficiency observed closer to the origin of replication (Fig. 17B-17C).

Example 5

Increased expression increases editing efficiency

[00176] The editing efficiency of the characterized chimeric Casl2a-like nucleases spanned a range of 5-95% depending on the specific PAM targeted (galk, lacZ) or the specific loci in the genome targeted. This broad range is consistent with the understanding that chimeric sequences would not only be functional but would provide a range of kinetic capabilities that can be used to create a designer nuclease for desired performance (e.g. on vs off targeting). It was further explored that chimeric Casl2a-like nucleases could be less stable than wild-type nucleases for example, because they are not naturally-occurring or selected for in nature. Fowered stability could affect function broadly, including altered on-/off- targeting and cleavage kinetic constants, or overall concentrations due to increased degradation in vivo.

[00177] To investigate this consideration, in other exemplary methods, certain chimeric Casl2a-like nucleases were examined through a process of CRISPR editing, included on- targeting binding, cutting, and editing associated with recombineering proteins. Initially, a

39 dCasl2a (or Casl2a with greatly reduced activity) was designed in a protein binding assay that allowed the on-target and off-target status to be monitored by antibiotic selections in E. coli (Fig. 8D). Three plasmid system that expresses dCasl2a (or Casl2a with greatly reduced activity) using a arabinose inducible promoter, a single crRNA (with J23119 promoter) targeting kanR gene, and a kanamycin resistance protein (encoded by kanR gene) using a constitutive promoter containing a fully complementary (on-target) crRNA binding site as well as a nitroreductase (encoded by nfsl gene) which conferred the cells sensitive to metronidazole (Fig. 8D and Fig. 18). We introduced two tested on-target crRNA (galKl and galK2) binding sites in the upstream of kanR gene individually. Using dCasl2a:crRNA as a transcriptional repressor, the cells cannot grow with unexpressed kanamycin, and expressed nitroreductase also repress the cell growth with metronidazole (Fig. 8D).

[00178] The decreased cell grown under antibiotic selection means the dCasl2a:crRNA repressed the transcription of kanamycin resistance protein. However, repression levels of chimeras were 5%-60% lower than the wild type control nuclease (Fig. 19). It is understood by those of skill in the art that DNA target binding by wild-type CRISPR-Casl2a is rate limiting for DNA cleavage, and the maximal rate constant Umax) for the targeted DNA binding of (AsCasl2a) is 0.13 ± 0.01 s ¹ which is orders of magnitude slower than the DNA cleavage. In light of the DNA binding assay data disclosed herein, it is thought that chimeric Casl2a-like nucleases were less stable than the wild-type Casl2a-type nucleases under the same expression level, and increased chimera degradation may limit the overall DNA binding rate.

[00179] In other exemplary methods, chimeric Casl2a-like nucleases were introduced into three plasmid systems to replace the dCasl2a, and test the cutting efficiency with the different expression level of chimeric Casl2a-like nucleases by controlling the induction time. Prolonged induction increases the chimeric Casl2a-like nuclease expression level, which resulted in increased cutting efficiency of chimeric Casl2a-like nucleases (Fig. 8E- 8F). Additionally, genome editing under this inducible system was tested (Fig. 8G). It was demonstrated that editing efficiency of chimeric Casl2a-like nucleases were further improved with longer induction time, and the editing efficiency of chimeric Casl2a-like nucleases (e.g. M44 and M21) were similar to a wild-type Casl2a-type nuclease after 2 hours induction (Fig. 8H-8I). Therefore, it was demonstrated that chimeric Casl2a-like nucleases can be less stable than wild-type sequences and that this effect can be mitigated by increased expression of the chimeric Casl2a-like nucleases having modified editing effects. These exemplary methods demonstrate that chimeric Casl2a-like nucleases (or other Cas systems) can be designed

40 using recombination libraries and selection for development of editing systems that span a broad kinetic landscapes. In certain exemplary methods, reduced editing efficiency or increased editing efficiency and/or accuracy may be desired depending on the targeted gene or pathway.

Casl2a-type chimeras display altered PAM preferences and off- target editing rates

[00180] In another exemplary method, PAM preferences were tested of three chimeric Casl2a-like nucleases. To elucidate functional PAM sequences, a high-throughput in vivo screen was developed with two features: applicability across PAM-dependent CRISPR-Cas systems and the generation of a distinct signal for functional PAMs. More recent efforts have developed high-throughput experimental screens to determine functional PAMs based on the depletion of a target plasmid or on the introduction of a double-stranded break in vitro. In these experiments, a Casl2a binding assay described above to generate a comprehensive screen to elucidate the complete landscape of functional PAM sequences was developed (see for example, Fig. 8D). A reporter plasmid containing KanR gene was selected encoding for kanamycin resistance and a functional protospacer with an NNNN PAM library. Chimeric Casl2a-like nucleases and two equivalent gRNA plasmids were transformed individually into the E. coli MG1655. One gRNA design is targeted on the KanR gene, and another gRNA is a non-targeting control. Cells grown on kanamycin media were collected using different gRNA plasmids, and then the region of the PAM library was amplified from the reported plasmid for the high throughput sequencing.

[00181] A PAM enrichment score revealed that PAM preferences appeared to differ among the chimeric Casl2a-like nucleases tested (Figs. 9A-9F and additional data not shown). It is noted that the TTTC PAM remained the top sequence for these tested chimeric Casl2a-like nucleases and wild-type Casl2a-type proteins (except for TX_Casl2a) (Figs. 9A-9F). It is noted that CTTT PAM had the lowest enrichment score for all known PAMs (Figs. 9A-9F and additional data not shown). In order to further evaluate the high-throughput observations, several novel PAMs were examined individually. In thisexemplary method, there was varying PAM specificity among different chimeric Casl2a-like nucleases (data not shown).

[00182] It is understood that off-target mutations observed at frequencies greater than desired remain a major concern when applying CRISPR systems to biomedical and clinical application. Several prior studies have engineered altered off-target rates by site-directed and random mutagenesis on CRISPR nucleases to decrease non-specific interactions with target DNA. It was thought that this disclosed chimera strategy for developing nuclease libraries may affect the non-specific interactions with the target DNA site. Therefore in another

41 exemplary method, off-targeting library test was carried out in which effects of systematically mismatching various positions within gRNAs were tested and observed. In these methods, nine off-target cassettes were designed, including 3 each with substitutions, insertions, or deletions in different positions (Fig. 9G). It was observed that there was a potential for off-target activity in both LbCasl2a and the positive control, only a single potential off-target for AsCasl2a, and no off-targeting for the a tested chimera Casl2a-like nuclease (M44, color screening assay) (see for example, Fig. 5G, 4B and 21). To further assess this important observation, these studies were expanded with a comprehensive and sensitive genome wide off-target assay (CIRCLE-seq). Using a bacterial genome (e.g. E. coli MG1655 genome) as targeting DNA, 2 gRNAs (galKl and lacZ2) were tested using AsCasl2a, LbCasl2a, a positive control and chimera Casl2a-like nuclease (M44) (data not shown but incorporated by reference in the inventors authored manuscript to be disclosed). The target sequence is shown at the top of the figure (data not shown) and off-target sequences are shown below. The read counts for each sequence are shown to the right and represent a measure of cleavage efficiency at a given site. For each gRNA, the target sequence is shown at the top of the figure (data not shown) and off-target sequences are shown below (data not shown). The read counts for each sequence are shown to the right and represent a measure of cleavage efficiency at a given site. The results generally confirmed the results obtained using our designer off-target assay (data not shown), control and LbCasl2a had substantially higher off- targeting relative to AsCasl2a with the chimeric M44 demonstrating the lowest levels of off-targeting.

Example 6

Chimeric nucleases enable genome editing in eukaryotic cell

[00183] Targeted genome editing with the promise of treating and curing both human, animal and other genetic conditions has always been a major goal in the field of biology. Therefore, chimeric Casl2a-like nucleases for genome editing in mammalian cells was tested. In these exemplary methods, a plasmid expressing a chimera Casl2a-like nuclease (M44) (with T7 promoter), a single crRNA (with U6 promoter), and GFP as a transfection control were created (see Fig. 10A). Transfected cells were isolated by FACs (Fig. 10B), cell lysate harvested 72 hours post-transfection, and indel detection was performed using T7E1 assay (Fig. 10B and 10C). These observations demonstrate that this exemplary chimera Casl2a-like nuclease is fully functional in mammalian gene editing experiments (Fig. 10D).

[00184] In addition to mammalian cells, a chimeric Casl2a-like nuclease was examined for genome editing in yeast (e.g. S. cerevisiae) (Fig. 10E). Yeast cells have long been the most

42 tractable organism for eukaryotic cell biology, owing to its genetic malleability, greatly facilitated by a preference for homologous recombination (HR) over non-homologous end joining (NHEJ) for double stranded break (DSB) repair. To examine a chimeric Casl2a-like nuclease activity in yeast, gRNAs were designed to target the endogenous genomic negative selectable marker CAN1, where its null mutation can be selected with media containing canavanine (a toxic arginine analogue, by methods known in the art). It was observed that the chimera Casl2a-like nuclease (e.g. M44) edited at efficiencies greater than 40% (Fig. 10F). Collectively, these results demonstrated that the chimeragenesis strategy reported herein can be used to rapidly develop novel synthetic nucleases functional across multiple species and organisms of use in mammals, bacteria and yeast and likely birds, plants and fish, etc.

[00185] Structure guided chimeragenesis is an effective way to generate synthetic protein families with broad sequence diversity while maintaining a relatively high percentage of folded and functional proteins. Furthermore, the proportion of folded variants can be increased through simple solutions such as utilizing stabilized parental sequences. Farge datasets are generated by characterizing these libraries, and, unlike natural protein families, these sets include both functional and nonfunctional sequences that can be queried for specific properties in high throughput formats as designed herein. There are abundant Casl2a-type family nucleases in the database. However, not all the characterized Casl2a- type family nucleases are efficient in the model systems, such as E. coli, yeast, and mammalian cells, emphasizing the need to improve existing nuclease classifications. While many Casl2a-type family nuclease sequences are easily identified, predicting which ones are functional and what are the preferred PAM designs and gRNA designs remains intractable. As presented herein, a chimera Casl2a-like nuclease library with 560 mutants using domain recombination points based on the homology substructure of 9 different Casl2as was designed and is applicable for other Cas proteins. In order to account for lack of predictability, selection/screening systems were designed to identify functional Casl2a-type chimeras. It was observed that about 30% of the sequences of the positive variants do not align to the wild-type Casl2a-type proteins. In addition, the PAM specificity and off- targeting characteristics were different among the characterized chimera mutants, which not only emphasizes the unique status of these chimera Cas 12a- like nuclease but also the potential of this strategy for generating nucleases fit for a particular application.

[00186] Methods, compositions and systems designed herein have uncovered several chimeric Casl2a-like nuclease variants with substantially eliminated off-target activity and well-preserved on-target activity. It has further been demonstrated that these novel chimeric

43 Casl2a-like nuclease variants facilitate genome editing in E. coli, yeast, and mammalian cells, opening up this strategy to a wide range of downstream applications ranging from human health to industrial biotechnology to plant biology and veterinary health, etc.

[00187] Engineering chimeric Casl2a-like nuclease variants based on structural information expands the current genome editing possibilities and solutions. For example, future efforts to further alter PAM preference or on/off target specificity could entail the evaluation of a much larger collection of REC1 domains (e.g. recombinations) or by saturation/random mutagenesis of specific regions of chimeric Casl2a-like nuclease variants, among other well established directed evolution approaches. These strategies can be used to engineer additional homologous and non-homologous RNA-guided endonucleases. Finally, the novel screening platform could be applied for the development of chimeric Casl2a-like nuclease variants tailored to a range of specific functional objectives, such as optimization of editing at a specific loci or in a targeted cell line, among others. The vast collection of already identified nucleases in combination with a rapid approach for generating large combinations thereof permits generation of specialized synthetic nucleases tailor made for a range of applications.

Additional Data Description

[00188] Figs. 7A-7D: (A) The Casl2a like protein structure analysis based on AsCasl2a (PDB:5B43) (B) The editing and transformation efficiencies of the Casl2a like nucleases used in this study. The Casl2a like nucleases, used in this study, are SD_Casl2a (Succinivibrio dextrinosolvens), CT_Casl2a ( Candidatus Methanoplasma termitum), TX_Casl2a ( Thiomicrospira sp. XS5), CA_Casl2a ( Candidatus Methanomethylophilus alvus), PC_Casl2a ( Porphyromonas crevioricanis ), FB_Casl2a ( Flavobacterium branchiophilum ), CR_Casl2a ( Candidatus Roizmanbacteria bacterium

GW20l l_GWA2_37_7), SC_Casl2a (synthetic construct of AsCasl2a), and MAD7. The editing efficiency, as determined by galK inactivation assay, was shown in blue, and the transformation efficiency was shown in green. (7C) The domains were separated by the 6 crossover points, which were WED-I&REC1, REC2, WED-II&PI, WED-III, RuvC- l&BH&RUVC-II, and Nuc&RuvC-III. (d) The distribution of the chimera library variants. Casl2a-type libraries were made as described in the online methods. The numbers to the left of variants were the crossover points shown in 1C. The swapped regions are shown by different colors; the colors are the same as in 7B.

[00189] Figs. 8A-8I: Genome editing test with different gRNAs for chimera library variants in bacteria (e.g. E .coli ) (8 A) Editing (cutting) efficiency test using gRNA targeting galK or

44 lacZ genes. In certain exemplary methods two plasmid system constructs were created for genome editing: one plasmid expresses a Cas protein as well as lambda red proteins (exo, bet, and gam)⁶⁶; a second plasmid expresses a single crRNA (with J23119 promoter) targeting the galK or lacZ gene and a homology arm (HM) containing a gene-inactivating mutation. For cutting, there were no lambda red proteins or homology arm in the system. (8B) illustrates a histogram plot of cutting efficiency of chimeric Cas 12a like proteins using 6 different gRNA plasmids. In this example, gRNA plasmids galKl, galK2, and galK3 targeted different positions in the galK gene. Further, gRNA plasmids lacZl, lacZ2, and lacZ3 targeted different positions in the lacZ gene. In 8C, editing efficiency of chimera library variants with different gRNAs was examined. In these examples, the gRNAs used in the test were galKl, galK2, lacZl, and lacZ2. Editing efficiency can be determined by color screening for quick analysis, for example red/white for GalK or blue/white for LacZ. A subset of colonies were sequenced to verify that the edit took place and to assess editing. In 8D, dCasl2a (or Casl2a with reduced activity) was evaluated in a protein binding assay. In this exemplary method, three plasmid systems were designed: one plasmid expresses dCasl2a (or Casl2a with reduced activity) using an arabinose inducible promoter (pBAD); a second plasmid expresses a single crRNA (with J23119 promoter) targeting the kanR gene; and a third plasmid expresses the kanamycin resistance protein (encoded by kanR gene) using a constitutive promoter containing a fully complementary (on-target) crRNA binding site as well as a nitroreductase (encoded by nfsl gene) which makes the cells sensitive to metronidazole. (8E and 8F) Cutting efficiency of chimeric Cas 12a like nucleases with different arabinose induction times using different gRNA were analyzed. (8E) galK_l and (8F) galK_2. 8G represents a schematic of the system used for testing various Casl2a-like chimera nucleases and controls. In certain methods, an arabinose inducible system for chimeric Cas 12a- like proteins was used. In this example, three novel plasmid systems were created for testing genome editing: one plasmid expresses a Cas 12a- like protein using an arabinose inducible promoter; a second plasmid expresses lambda red proteins (exo, bet, and gam) using a temperature- inducible promoter (pL); and a third plasmid expresses a single crRNA (with J23119 promoter) targeting the galK gene with homology arm (HM) containing a galK- inactivating mutation as a template for recombineering. (8H and 81) Editing efficiency of chimeric Casl2a like nucleases with different arabinose induction times using different gRNA were analyzed and are represented by 8H: galK_l and 81: galK_2.

[00190] Figs. 9A-9F represents specificity detection of chimeric Casl2a-type variants and enrichment scoring of each PAM site using different guide RNAs. (9A-9F) Round 1 is

45 illustrated of enrichment scores for two rounds of PAM scans. The enrichment score is the frequency change (log2) of each PAM using different gRNA plasmids (on-targeting and non targeting gRNAs). (9A) AsCasl2a (9B) LbCasl2a (9C) TX_Casl2a (9D) Control (9E) M44 (9F) M21.

[00191] Fig. 9G illustrates an off-target assay for chimeric Casl2a-type variants. 9G represents an individual off-target assay. 9 different off-target spacers were designed as illustrated to test editing efficiency and target recognition, of which 3 were substitutions, 3 were deletions, and 3 were insertions (data not shown) Genome-wide off-target analysis was done using one method referenced as the CIRCFE-seq method. gRNA targeting the galKl site and gRNA targeting the lacZ2 site were assessed (data not shown). Positions with mismatches to the target sequences, i.e. off-target sites, are highlighted in color. CIRCFE- seq read counts are shown to the right of the on- and off-target sequences and represent a measure of cleavage efficiency at a given site. The on/off-target reads shown in the figure were higher than 10.

[00192] Figs. 10A-10F In certain exemplary methods, chimeric Casl2a-like nucleases disclosed herein are capable of genome editing in eukaryotic cells. In one method, genome editing in mammalian cells (e.g. HEK293T) were analyzed using chimeric Casl2a-like variants disclosed in certain embodiments herein. A plasmid expressing the M44 (or control) nuclease (with T7 promoter), a single crRNA (with U6 promoter), and GFP were constructed (10A). Fig. 10B is a photographic representation of the mammalian cells after transfection. The mammalian cells were transfected with the plasmid containing the chimeric Casl2a (e.g. M44) nuclease and GFP. Micrographs were taken under cool white light (left) or fluorescent light (right). The T7E1 assay was performed as known in the art on cells expressing GFP and isolated by fluorescence activated cell sorting. In this example,‘Untreated’ as labeled means the PCR products without T7 endonuclease treatment; while ‘Treated’ means the PCR products with T7 endonuclease treatment (10C). 10D is a graphic representation of an indel rate of control versus the chimeric nuclease, M44. This calculation was made using the formula illustrated in the methods section. 10E represents assessment of genome editing in yeast ( S . cerevisiae BY4741) using chimeric Casl2a-type variants as another example of the diversity of organism applicability. In this example, a plasmid was constructed containing the M44 (or control) nuclease (with TEFlp promoter), a single crRNA (SNR52p promoter) targeting the CAN1 gene and a homology arm (HM) containing a CAN1 -inactivating mutation as a template for recombineering. Only colonies with an inactivated CAN1 gene can grow on a -i-can plate. 10F is a graphic illustration of editing efficiency of control and the tested

46 chimera Casl2a-like nuclease, M44. The editing efficiency was calculated by determining the ratio of colonies on plates +/-can. Editing was also confirmed by sequencing 20 colonies from +can plates.

[00193] FIGS. 11A-11B: In certain methods, library of chimeric constructs was generated. It was assessed that a previous construct had the highest editing efficiency in the initial test (1 IB).

Therefore, this nuclease was used as a positive control (in black color) as a template and this nuclease was also used as an original plasmid (in dark green color) as backbone. Using crossover points in Fig 7C, a chimera library of nucleic acid sequences was designed (Fig. 22) using a ~40bp homology arm with the control nuclease and its plasmid. The chimera sequences were illustrated in different colors except for black and dark green. Homology arms were illustrated in black and dark green, and linked to the chimera sequences. The library construction for crossover 11A and 11B were created using, for example, a Gibson assembly method. 11A is a library construction used 1 crossover. The library variants for 1 crossover should be 48 (8 chimera sequences x 6 positions) theoretically. 1 IB is a library construction using 2 crossovers. The library variants for 2 crossovers should be 512 (8 chimera sequences x 8 chimera sequences x 8 combinations) theoretically.

Therefore, combining a single and double crossover library, the total number of variants should be 560 different combinations.

[00194] In one exemplary method, a Phylogenetic Tree for the wild-type (WT) Casl2a-type and chimera Casl2a-like gene (Fig. 12A) and amino acid (Fig. 12B) sequences were generated. A Clustal Omega system for the data analysis and figure generation was used. As_Casl2a and Lb_Casl2a were previously reported. M44, M21 , M38, M43, and M8, chimera Casl2a-like nuclease are demonstrated. The numbers identified in these figures represent the distance values which show the number of substitutions (nucleotides or amino acid residues) as a proportion of the length of the alignment (excluding gaps).

[00195] In another exemplary method, a galK based growth selection and colorimetric screen for Casl2a-type chimera library was designed. Fig. 13 A illustrates the design and potential outcome illustrating potential results using 2-DOG selection and a MacConkey agar color screening. Both of these exemplary methods were used to identify function control- based library variants. Fig. 13B represents some exemplary plates illustrating results of 2- DOG selection and MacConkey color screening test using WT control (top) or dysfunctional control screening (bottom).

[00196] FIG. 14 is an exemplary graph illustrating distribution of functional chimera Casl2a-like nucleases identified using a selection assay (e.g. 2-DOG) of certain embodiments disclosed herein.

47 [00197] FIG. 15 illustrates a color screening of control versus a chimera Casl2a-like nuclease (e.g. M44) with different gRNAs. The edited cells in the galK/lacZ color screening should be shown as white color. The unedited cells in the galK/lacZ color screening should be shown as red color.

[00198] FIGS. 16A-16D illustrate exemplary histogram plots that represent transformation efficiency of different Casl2a-like chimera variants using different gRNA. The gRNA used in the test were (16A) galKl (16B) galK2 (16C) lacZl and (16D) lacZ2. Transformation efficiency is defined as the number of colony forming units (cfu) per pg of gRNA plasmid.

[00199] FIGS 17A-17C illustrate genome editing tests in the different genomic positions for chimera Casl2a-like library variants. 17A illustrates a schematic of targeted genomic position. galK gene was integrated individually in the different genomic position (SS1, SS3, SS5, SS7, and SS9) of MGl655AgalK. 17B illustrates representative plates for colorimetric screening of GalK activity with chimera nuclease variants M44 and M38 in different genomic position. 17C illustrates editing efficiency of chimera library variants in different genomic positions.

[00200] FIG. 18 represents an exemplary gene editing assay using a negative control of a chimera Casl2a-like nuclease for DNA binding assay (dM44) to illustrate behavior of a particular chimera in order to assess any altered activity in genomic editing processes. To access behavior of chimera dCasl2a variants, an assay was set up to assess whether activity is altered with an outcome of 1) Cutting: cells cannot grow with kanamycin, but cells can grow with metronidazole; 2) DNA binding: cells cannot grow with kanamycin or metronidazole; 3) No DNA binding: cells cannot grow with metronidazole, but cells can grow with kanamycin. The results are illustrated in for example, FIG. 18. These assays can be used to test control as well as mutant constructs for directed altering of activity of a particular Casl2a-like chimera nuclease of certain embodiments disclosed herein.

[00201] FIG. 19 represents a histogram plot of binding efficiency of dCasl2a using different guide RNAs (e.g. galK_l and galK_2). The binding efficiency was calculated by the following formula.

[00202] In certain methods, PAM scan methods were designed to assess on and off- targeting rates. Reporter plasmids were constructed containing KanR gene encoding kanamycin resistance and the functional protospacer with NNNN PAM library. The chimera

48 Casl2a-like proteins were transformed and one of two gRNA plasmids were also transformed individually into the E. coli MG1655. One gRNA design is targeted on the KanR gene, and another gRNA plasmid is non-targeting control. These two gRNA plasmids were equivalent amount for the transformation. Cells grown on kanamycin media were collected using different gRNA plasmids, and amplified the region of the PAM library from the reported plasmid for the high throughput sequencing. The enrichment score of PAM and accompanying sequence logo for one of two library replicates revealed the PAM specificity among different chimera Casl2a like proteins. A first round PAM scan tests different variants (b) AsCasl2a (c) LbCasl2a (d) TX_Casl2a (e) MAD7 (f) M44 (g) M21 (h) M38 and then plotted where the X- and Y- axis were normalized reads frequency (data not shown).

[00203] FIGS. 20A-20D In certain experiments, cutting efficiency is assessed by individual verification of unknown PAMs using different nucleases including chimera Casl2a-like nucleases. (a)ATTC (b) ATTA (c) GTTA (d) CCTC.

[00204] FIG. 21 The off-targeting test of control versus a Casl2a-like chimera nuclease using 9 plasmids with off-targeting design (data not shown) Color screening of off-target test using design 5. Sequencing verification of white colony was demonstrated and compared to a wild type spacer design.

Materials and Methods

[00205] In certain methods chimeric constructs were created by strategies disclosed herein using at least two Casl2a nuclease molecules to create a chimeric Casl2a nuclease. For example, certain chimeric constructs created by methods disclosed herein are referred to as CU_CHl (M6), CU_CH2 (M7), CU_CH3 (M8), CU_CH4 (M13), CU_CH5 (M21),

CU_CH6 (M22), CU_CH7 (M38), CU_CH8 (M43), and CU_CH9 (M44), where each construct was generated using cross-over technologies to create a chimera derived from peptide fragments of two or more different Casl2a nucleases. In certain methods, off- targeting efficiency rates were evaluated for each chimera Casl2a compared to a control Casl2a to demonstrate improved off-targeting rates. Constructs disclosed and claimed herein include, but are not limited to, CU_CHl : 1 to 927 bp from PC_CASl2A, 928 to 3876 bp from a positive control derived from a Casl2a of Eubacterium recuile CU_CH2 : 1 to 912 bp from SC_CASl2A, 913 to 3861 bp from a positive control derived from a Casl2a of Eubacterium rectale CU_CH3 : 1 to 86lbp from FB_CASl2A, 862 to 3810 bp from a positive control derived from a Casl2a of Eubacterium rectal; CU_CH4 :l to 504 bp from TX_CASl2A, 505 to 3819 bp from a positive control derived from a Casl2a of Eubacterium rectale·, CU_CH5 : 1 to 900 bp from TX_CASl2A with mutation G218A, 901 to 3849 bp

49 from a positive control derived from a Casl2a of Eubacterium rectale; CU_CH6 : 1 to 900 bp from TX_CASl2A, 901 to 3174 bp from a positive control derived from a Casl2a of Eubacterium rectale ;, CU_CH7 : 1 to 840 bp from, 841 to 3789 bp from a positive control derived from a Casl2a of Eubacterium rectale·, CU_CH8 (M43) :l to 846 bp from a Casl2a, 847 to 3795 bp from a positive control derived from a Casl2a of Eubacterium rectale·, and CU_CH9: 1 to 900 bp from TX_CASl2A, 901 to 3849 bp from a positive control derived from a Casl2a of Eubacterium rectale and combinations thereof.

[00206] In certain exemplary methods, constructs contemplate of use herein are exemplified by at least a construct having 80% homology with CU-CH2: SEQ ID NO:l : CU-CH1 : SEQ ID NO:2: CU-CH5: SEQ ID NO:3: CU-CH5 : SEQ ID NO:4: CU-CH3 : SEQ ID NO:5 :; CU- CH6: SEQ ID NO:6:; CU-CH7: SEQ ID NO:7;; CU-CH8: SEQ ID NO:8; CU-CH9: SEQ ID NO:9: : or a combination or derivative or mutant thereof.

[00207] In accordance with the methods described above, intergenic regions of Casl2a molecules were targeted to create chimeric constructs disclosed herein. In some methods, a 5 to 35 intergenic amino acid region of Casl2a was targeted for crossover creations of chimeric constructs. In one example, amino acid sequence FATSFKDYFKNRAN SEQ ID NO: 149 (corresponding nucleic acid sequence

TTTGCGACTAGCTTTAAAGATTACTTCAAGAACCGTGCAAAT) SEQ ID NO: 150 was used to create a chimeric construct containing fragments derived from 2 Casl2a nucleases. In another example, amino acid sequence LHKQILCIADTSYE SEQ ID NO: 151

(corresponding nucleic acid sequence

CTTCACAAACAGATTCTATGCATTGCGGACACTAGCTATGAG) SEQ ID NO: 152 was used to create a chimeric construct containing fragments derived from 2 Casl2a nucleases or 3 Casl2a nucleases (CU_CH6 (M22),. In yet another example, amino acid sequence VELQGYKIDWTYI SEQ ID NO: 153 (corresponding nucleic acid sequence GT AG AGTT AC AAGGTT AC A AG ATTG ATTGGAC AT AC ATT) SEQ ID NO: 154 was used to create a chimeric construct containing fragments derived from 3 Casl2a nucleases (CU_CH6 (M22).

Chimeric Casl2a-type nuclease library construction

[00208] Using Casl2a-type nuclease sequences available from the NCBI database, alignments were performed (FIG. 22) to determine homologous domains to design chimera library sequences (FIG 1C). A ~40bp homology arm with a control nuclease was used and its plasmid as shown in FIGS 11A-11B. All the library sequences were obtained as gBlocks from a company. The library plasmid construction for 1 and 2 crossovers used a Gibson

50 assembly method. The assembly used NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA), and the assembly products were desalted using dialysis by spotting the reaction on a filter with 0.025 pm pores floating in ddfUO. Following desalting, the assembly products were electroporated into E. cloni 10G ELITE Electrocompetent Cells (Lu eigen Corporation, Middleton, WI, USA). Libraries were spot plated onto LB with 34 pg/mL chloramphenicol to estimate transformation efficiency. The library plasmids were purified using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA). All PCR steps were performed with the high-fidelity Phusion enzyme (New England Biolabs, Ipswich, MA, USA) to ensure production of a high-quality library.

Nuclease-mediated cell killing assay

[00209] A two plasmid system was constructed for genome editing, which expresses a Casl2a like protein and a single crRNA (with J23119 promoter) targeting the galK or lacZ gene. For each experiment, equal amounts were transformed of non-targeting and on- targeting (e.g. galKl) gRNA plasmids. The cutting efficiency was calculated as following:

[00210] The same amount of culture was plated in two LB agar plates with chloramphenicol and carbenicillin.‘a’ denotes the number of colonies that can grow on the plate with on-targeting gRNA plasmid, and‘b’ is the number of colonies that can grow on the plate with non-targeting gRNA plasmid.

Generation of Heterologous Plasmids

[00211] To generate the Casl2a locus for heterologous expression, the Casl2a-type DNA sequences after codon optimization was PCR amplified and cloned into pSClOl, pX2, pMINR, and pY094 using Gibson cloning kit (New England Biolabs). Sequences of all the chimera and gRNA design have been identified.

The isolation of functional Casl2a-type mutants

[00212] The host strain carried the plasmid expressing lambda red proteins and chimeric Casl2a like proteins library. The strain were cultured in 30°C and supplemented with 0.2% arabinose for inducing lambda red proteins. When OD_s¥ reached 0.5-0.6, the cells were induced for 15 min at 42°C to induce chimeric Casl2a like proteins. After chilling on ice for 15 - 30 min, the cells were washed twice with 20% of the initial culture volume of ddH20. Then, the gRNA plasmid was mixed with the cells, followed by chilling on ice for 5 min. Following electroporation, the cells were recovered in SOB medium for 3 h. Then, 1 pL of cells was plated in the M9 agar media supplemented with 2-deoxy-galactose (DOG).

51 [00213] The isolation of functional Casl2a-type mutants directly in vivo potentially enabled the identification of Casl2a-type variants with higher editing efficiency. The galK gene product, galactokinase, catalyzes the first step in the galactose degradation pathway, phosphorylating galactose to galactose- 1 -phosphate. Galactokinase also efficiently catalyzes the phosphorylation of a galactose analog, 2-deoxy-galactose (DOG). The product of this reaction cannot be further metabolized, leading to a toxic build-up of 2-deoxy-galactose- 1- phosphate. Strains with galK inactivation can grow in the media supplementary with 2-DOG and background following negative selection is reduced and no colony screening is necessary.

[00214] The selected Casl2a-type mutants were verified using the above competent cell preparation and transformation method. After 3 h recovery, 1 pL of cells was plated in the MacConkey agar. The color screening method based on the galK inactivation to evaluate the editing efficiency of CRISPR-Cas9 was same as the previous studies.

Casl2a PAM Screen

[00215] PAM plasmid libraries were constructed using synthesized oligonucleotides (IDT) containing the designed NNNN PAM library. The dsDNA product was assembled into a linearized plasmid (containing kanR gene) using Gibson cloning (New England Biolabs). The

PAM library was transformed into MG1655 with the plasmid expressing chimeric Casl2a like proteins using the electroporation method. We then transformed two equivalent gRNA plasmids individually into the E. coli MG1655. One gRNA design is targeted on the library sites, and another gRNA plasmid is non-targeting control. We collected the cells grown on kanamycin media using different gRNA plasmids, and amplified the region of the PAM library from the reported plasmid for the high throughput sequencing. The enrichment score of PAM and accompanying sequence logo for one of two library replicates were demonstrated in PAM screening revealed the PAM specificity were different between different chimeric Casl2a like proteins. The prepared cDNA libraries were sequenced on a

MiSeq with a single-end 300 cycle kit (Illumina). Indels were mapped using a Python implementation of the Geneious 6.0.3 Read Mapper.

[00216] Ei denotes the enrichment score. X; is the frequency of PAM i using on-targeting gRNA plasmid in the deep sequencing measurements. Y; is the frequency of PAM i using non-targeting gRNA plasmid in the deep sequencing measurements.

Yeast Transformation

52 [00217] High-efficiency yeast transformation was conducted using the LiAc/SS carrier DNA/PEG method.

PEI transfection

[00218] HEK293T were cultured in 6-well dish with 60% confluency. After cells attached on the surface of the dish, for each well, two l.5mL centrifuge tubes were loaded with 250mE serum-free and phenol red-free DMEM. One of the tubes was loaded with 3uL of polyehtyleimine (PEI, concentration: lmg/mL), and the other one tube was loaded with lpg of plasmid. After addition, tubes were mixed and placed for 4 min. After placing, tubes loaded with PEI were mixed to tubes with specific plasmid drop-wisely. Tubes were placed for 20 minutes after mixing and mixtures were added into wells drop-wisely.

Fluorescence- activated cell sorting (FACS)

[00219] HEK293T was incubated with lmL (0.5%) trypsin at 37°C for 5 minutes followed by pelleting and resuspension in DMEM with 5% fetal bovine serum (FBS). Resuspended cells were filtered with CellTrics® 50pm filter to discard debris. Cell sorting was performed using BD FACSAria™ Fusion equipped with OBIS 488 nm laser (SN: 177745) at 98.3mW of power. Forward scatter area (FSC-A), side scatter area (SSC-A) and side scatter width (SSC-W) were collected through a filter. The GFP signal was collected in the 488 nm channel through a 530/30-A band pass filter. The first gate was drawn in the SSC-A/FSC-A plot to include cells with universal size, and the second gate was drawn in the SSC-A/SSC-W plot to include single cells. The third gate was drawn in the FSC-A/488 B 530/30-A channel to sort cells with GFP signal.

T7E1 assay

[00220] Genomic DNA was extracted using the QuickExtract DNA Extraction Solution (Epicenter) following the manufacturer’ s protocol. The genomic region flanking the CRISPR target site for each gene was PCR amplified, and products were purified using QiaQuick Spin Column (QIAGEN) following the manufacturer’s protocol. 200-500 ng total of the purified PCR products were mixed with 1 pl 10 x Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 10 mΐ and were subjected to a re-annealing process to enable heteroduplex formation: 95°C for 10 min, 95°C to 85°C ramping at -2°C/s, 85°C to 25 °C at -0.25 °C/s, and 25 °C hold for 1 min. After re-annealing, products were treated with SURVEYOR nuclease and SURVEYOR enhancer S (Integrated DNA Technologies) following the manufacturer’s recommended protocol and analyzed on 4%-20% Novex TBE polyacrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 10 min and imaged with a Gel Doc gel imaging system (Bio-rad).

53 Quantification was based on relative band intensities. Indel percentage was determined by the formula, 100 x (1 - sqrt(l - (b + c)/(a + b + c))), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each cleavage product.

The foregoing discussion of the disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. Although the description of the disclosure has included description of one or more embodiments and certain variations and modifications, other variations and

modifications are within the scope of the disclosure, e.g., as can be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

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Claims

WHAT IS CLAIMED IS:

1. A method for creating a non-naturally occurring gene editing Casl2a nuclease library comprising,

combining two or more naturally-occurring Casl2a-type nucleases;

allowing crossover of the two or more Casl2a-type nucleases for one or more crossovers; and creating a mixed chimera Casl2a-like nuclease library of non-naturally occurring chimera Casl2a-like nucleases.

2. The method according to claim 1, further comprising analyzing the library for genome editing efficiency compared to a naturally-occurring Casl2a-type nuclease.

3. The method according to claim 1 or claim 2, wherein the mixed chimera Casl2a- like nuclease library comprises a crossover between REC1 and REC2.

4. The method according to claim 1, 2 or 3, wherein the mixed chimera Casl2a-like nuclease library comprises a crossover between REC2 and WEDI.

5. The method according to any one of claims 1 to 4, wherein the mixed chimera Casl2a-like nuclease library comprises a first crossover between PI and WEDIII.

6. The method according to any one of claims 1 to 5, wherein the mixed chimera Casl2a-like nuclease library comprises a second crossover between PI and WEDIII different than the first crossover between PI and WEDIII or in addition to a first crossover between PI and WEDIII.

7. The method according to any one of claims 1 to 6, wherein the mixed chimera Casl2a-like nuclease library comprises a crossover between WEDIII and RuvC-l.

8. The method according to any one of claims 1 to 7, wherein the mixed chimera Casl2a-like nuclease library comprises a crossover between RuvC-II and Nuc.

9. The method according to claim 1, wherein there are no more than 6 crossovers for chimera Casl2a-like nuclease of the library.

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10. The method according to claim 1, wherein the chimera Casl2a-like nuclease library comprises chimeras having intact modules of WED-I and REC1; REC2; WEDII and PI; WED III RuvC- 1 , BH and RuvC-II, Nuc or combinations thereof from the two or more Casl2a-type nucleases.

11. The method according to any of the preceding claims, wherein a chimera Casl2a- like nuclease of the chimera Casl2a-like nuclease library is functional in a prokaryote having improved editing efficiency.

12. The method according to any of the preceding claims, wherein a chimera Casl2a- like nuclease of the chimera Casl2a-like nuclease library is functional in a eukaryote having improved editing efficiency.

13. The method according to any of the preceding claims, wherein a chimera Casl2a- like nuclease of the chimera Casl2a-like nuclease library is further selected for reduced off targeting of a targeted genome.

14. The method according to any of the preceding claims, wherein two or more Casl2a-type nucleases are from bacteria, yeast or a combination thereof.

15. A method for creating a non-naturally occurring gene editing nuclease library comprising, combining two or more naturally-occurring Casl2a-type nucleases to allow crossover of the two or more Casl2a-type nucleases having one crossover to create a mixed chimera Casl2a-like nuclease library of non-naturally occurring chimera Casl2a-like nucleases, wherein the crossover occurs between REC1 and REC2.

16. The method according to claim 15, wherein the chimera Casl2a-like nuclease library comprises chimeras having a recombined intact module of REC1 from at least a second naturally-occurring Casl2a-type nuclease.

17. A method for creating a non-naturally occurring gene editing nuclease library comprising, combining two or more naturally-occurring Casl2a-type nucleases to allow crossover of the two or more Casl2a-type nucleases having two and optionally, one crossovers to create a mixed chimera Casl2a-like nuclease library of non-naturally occurring

56 chimera Casl2a-like nucleases, wherein the crossovers occur between REC1 and REC2; and between RuvC-II and Nuc.

18. The method according to claim 17, wherein the chimera Casl2a-like nuclease library comprises chimeras having a recombined intact module of REC1 from at least a second naturally-occurring Casl2a-type nuclease.

19. The method according to claim 17 or 18, wherein the chimera Casl2a-like nuclease library comprises chimeras having a recombined intact module of Nuc from at least a second naturally-occurring Casl2a-type nuclease.

20. A kit comprising, the mixed chimera Casl2a-like nuclease library of any one of claims 1 to 19 and a container.

21. The kit according to claim 20, wherein the kit is of use to edit a prokaryote genome, a plant genome, a eukaryotic genome or yeast genome.

22. The kit according to claim 21, wherein the kit is of use to edit a eukaryotic genome of a human or other mammal.

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