WO2020097360A1 - Methods and compositions for genome-wide analysis and use of genome cutting and repair - Google Patents

Abstract

Embodiments of the present disclosure relate to compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing. In certain embodiments, engineered nucleic acid guided nucleases can include a mutated or modified gRNA spacer region to increase editing efficiency and reduce off-targeting of the engineered nuclease. In some embodiments, gRNA spacer regions can be mutated by one or more of a nucleic acid insertion, deletion, point mutation, substitution or other modification to the spacer region to alter recognition and/or binding of a target DNA. In certain embodiments, target DNA can include genomic DNA. In some embodiments, modified spacers of gRNAs can be used in high-throughput editing systems to create specific gRNAs for improved editing efficiency and targeted control over gene editing of use in experimental and therapeutic situations.

Description

METHODS AND COMPOSITIONS FOR GENOME-WIDE ANALYSIS AND USE OF GENOME CUTTING AND REPAIR

PRIORITY

[001] This PCT application claims priority to U.S. Provisional Application No. 62/757,106 filed November 07, 2018. This application is incorporated herein by reference in its 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 November 05, 2019 referred to as‘l0l877639908SequenceListing.txt’ and is 29 kilobytes having 103 sequences. Further, the provisional application as filed contained sequence listings in Appendices A-E and are hereby incorporated by reference in their entirety for all purposes.

FIELD

[004] Embodiments of the present disclosure relate to compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing. In certain embodiments, engineered nucleic acid guided nucleases can include a mutated or modified gRNA spacer region to increase editing efficiency and reduce off-targeting of the engineered nuclease. In some embodiments, gRNA spacer regions can be mutated by one or more of a nucleic acid insertion, deletion, point mutation, substitution or other means to alter recognition and/or binding of a target DNA by the modified gRNA. In certain embodiments, target DNA can include genomic DNA. In some embodiments, modified spacers of gRNAs can be used in high-throughput screening methods to quickly identify specific editing systems to extort specificities of modified gRNAs for improved editing efficiency and targeted control over gene editing of use in experimental, diagnostic and therapeutic situations. In some embodiments, compositions, methods and systems disclosed herein include a Cas nuclease and a gRNA having a modified spacer for targeted gene editing with increased efficiency compared to a reference gRNA. In other embodiments, the gRNAs include, but are not limited to, the gRNAs represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 20, SEQ ID NO: 32 to SEQ ID NO: 40, SEQ ID NO: 50 to SEQ ID NO:

60, SEQ ID NO: 72 to SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91 to SEQ ID NO:94. In other embodiments, the gRNAs include, but are not limited to, the gRNAs and/or homology regions represented by Table 1.

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] 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 guide RNA (gRNA) into a cell, the cell's genome can be cut at a predetermined location, allowing existing genes to be removed and/or new ones added with certain efficiency. These systems are useful but have some important limitations regarding efficiency of targeted editing, imprecise editing complications, as well as, impediments when used for commercially relevant uses. Therefore, a need exists for creating improved accuracy and efficiency of genome editing with commercial relevance.

SUMMARY

[007] Embodiments of the present disclosure relate to compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing. In certain embodiments, engineered nucleic acid guided nucleases can include a mutated or modified gRNA spacer region to increase editing efficiency and reduce off-targeting of the engineered nuclease. In some embodiments, gRNA spacer regions can be mutated by nucleic acid insertion, deletion, point mutations, substitution or other means to alter recognition and/or binding of a target DNA. In certain embodiments, targeted DNA can include genomic DNA. In some embodiments, modified spacers of gRNAs can be used in high-throughput screening systems to identify improved editing systems having selected gRNAs for improved editing efficiency and improved targeted control over gene editing of use in experimental and therapeutic situations.

[008] In certain embodiments, a nucleic acid guided nuclease system disclosed herein can include a nucleic acid guided nuclease; and a modified gRNA capable of improved editing efficiency of a target DNA, wherein the modified gRNA hybridizes with the target DNA to increase gene editing efficiency compared to a reference gRNA not having the modification. In other embodiments, a modified gRNA comprises a mutated spacer region where the modification is capable of altering editing efficiency. In certain embodiments, the modified spacer region comprises an insertion, deletion, substitution or point mutation. In some embodiments, a modified spacer region of the gRNA modifies binding to the target DNA by the gRNA spacer. In some embodiments, these alterations can modify recognition of by the gRNA of the targeted DNA to facilitate repair by repair enzymes. In accordance with these embodiments, a modified spacer region can reduce binding affinity to the DNA, allowing recognition for cleavage by the nuclease while increasing repair of the targeted DNA to increase recombination events.

[009] In some embodiments, targeted DNA can be genomic DNA. In other embodiments, targeted DNA can include prokaryotic or eukaryotic DNA. In certain embodiments, targeted DNA can include mammalian DNA such as human or other mammals such as pets or livestock or other animals. In yet other embodiments, targeted DNA can include fish, bird or plant DNA.

[0010] Certain embodiments disclosed herein include kits of use for gene editing having a mutated gRNA spacer region and other components needed for gene editing.

[0011] In some embodiments, methods disclosed herein include methods for modifying genome editing in order to improve editing efficiency and reduce off-targeting. In

accordance with these embodiments, methods disclosed herein can include contacting a target DNA molecule having a target sequence with a nuclease complex including: (a) a nuclease protein; and (b) a novel nucleotide gRNA having: (i) a modified spacer sequence creating modified target recognition sequence hybridization compared to a reference gRNA that hybridizes with the same target sequence, and (ii) a scaffold tracrRNA for guiding binding of the nuclease to the target sequence wherein the complex forms a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the modified gRNA has increased editing efficiency and/or reduced off-targeting compared to a reference nuclease complex having a control gRNA. In some embodiments, modification of the gRNA spacer sequence increases targeted gene replacement compared to a reference gRNA. In other embodiments, modification of the gRNA spacer sequence increases gene editing by enhancing gene recombination in the target DNA molecule having a defective gene. In certain embodiments, a modified spacer of the gRNA has at least one mutation of a deletion, insertion, point mutation or a substitution in the spacer. In some embodiments, these spacer alterations create a gRNA having reduced binding and increased recombination allowing the repair enzymes of the target organism to repair cut DNA. In accordance with these embodiments, improved gene editing provided by the modified spacer of the gRNA can be used to increase editing efficiency in a given target DNA ( e.g . prokaryotic or eukaryotic DNA). In other

embodiments, the gRNAs include but are not limited to the gRNAs represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 20, SEQ ID NO: 32 to SEQ ID NO: 40, SEQ ID NO: 50 to SEQ ID NO: 60,

SEQ ID NO: 72 to SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91 to SEQ ID NO:94. In some embodiments, gRNAs disclosed herein can be used in CREATE methods to arrive at all possible changes in the spacer regions of the gRNAs of interest to assess changes in the spacer region compared to a control gRNA in genome editing technologies. The nucleic acid guided nuclease system according to any one of claims 1-3, wherein the modified spacer region of the gRNA modifies binding to a non target or off-target DNA. In some embodiments, gRNA spacer region can be modified in order to manipulate on target editing and off target editing, for optimization of specificity to the targeted genome. In accordance with these embodiments, the specificity to the targeted genome is the ratio of on-target to all off target editing (e.g. Specificity = On Target Editing / SUM of all Off target editing).

Brief Description of the Drawings

[0012] 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. [0013] Figure 1 illustrates a graph depicting the correlation between Cas9:gRNA-induced cell death and editing efficiency using unique gRNAs of some embodiments disclosed herein.

[0014] Figure 2 illustrates a graph depicting the variation in editing across 37 cassette designs of some embodiments disclosed herein.

[0015] Figure 3 illustrates graphs depicting variations in editing efficiency with target genomic position having editing cassettes 1-8 of some embodiments disclosed herein.

[0016] Figure 4 illustrates graphs depicting variations in editing efficiency with a target Cas9 promoter having editing cassettes 1-8 of some embodiments disclosed herein.

[0017] Figures 5A and 5B illustrate graphs depicting variations in editing efficiency for Group 1 cassettes with target genomic positions and comparisons of gRNAs according to some embodiments disclosed herein.

[0018] Figures 6A-6D illustrate escape from Cas9:gRNA induced cell death with Group 1 gRNAs due to non-targeted mutations of some embodiments disclosed herein.

[0019] Figures 7A-7C illustrate variations in editing efficiency for Group 1 cassettes with target Cas9 promoters and gRNA comparisons (8B) to wild type gRNAs of some

embodiments disclosed herein.

[0020] Figures 8A-8D illustrate editing efficiency compared between the wild-type E coli genome and another genome with significant off-target site ( e.g . dgoK gene) deleted with Group 1 cassettes of some embodiments disclosed herein.

[0021] Figures 9A-9D illustrate histogram plots depicting variations in editing efficiency for Group 2 cassettes with various target Cas9 promoters of some embodiments disclosed herein.

[0022] Figure 10 illustrates graphs of wild type gRNAs depicting unintended mutations with Group 2 cassettes and frequency thereof relative to cas9 promoter strength of some embodiments disclosed herein.

[0023] Figures 11A and 11B represent graphs depicting base-change frequencies at the target site for Group 2 gRNAs in the absence of a homology repair template and in the (A) absence and (B) presence of heat induction of recombinant proteins of some embodiments disclosed herein.

[0024] Figures 12A-12D illustrate variations in editing efficiency for Group 3 cassettes with a target Cas9 promoters of some embodiments disclosed herein.

[0025] Figure 13 illustrate the effects of mutations in the gRNA on editing behavior of some embodiments disclosed herein. [0026] Figure 14 illustrates a graph depicting editing efficiencies for a Group 1 gRNA mutated into a Group 3 gRNA compared to wild type gRNA using 2 different cas9 promoters of some embodiments disclosed herein.

[0027] Figure 15 illustrates graphs depicting non-targeted mutations caused by mutated gRNAs of some embodiments disclosed herein.

[0028] Figure 16 is a schematic illustrating differences in editing with different gRNAs of some embodiments disclosed herein.

[0029] Figure 17 represents graphs of CFUs per transformation depicting correlations of Cas9:gRNA induced cell death to gRNA scores of some embodiments disclosed herein.

[0030] Figure 18 represents graphs depicting editing with changes in cell growth for Group 1- 3 cassettes of some embodiments disclosed herein.

[0031] Figure 19 represents a graph depicting editing efficiency of four cassettes mixed together and applied at once of some embodiments disclosed herein.

[0032] Figure 20 illustrates galK across different engineered strains to check for consistency of galactokinase activity at different positions of some embodiments disclosed herein.

[0033] Figure 21 represents photographic images depicting MacConkey screen data to measure apparent editing efficiency for galk 4 and galk 5 cassettes of some embodiments disclosed herein.

[0034] Figure 22 represents histogram plots depicting gRNA toxicity for group 1 cassettes with change in target position of some embodiments disclosed herein.

[0035] Figure 23 represents graphs depicting base change frequencies for Group 1 cassettes of some embodiments disclosed herein.

[0036] Figure 24 represents graphic images depicting gRNA toxicity for group 1 cassettes with change in promoter concentrations with respect to transformation efficiency of some embodiments disclosed herein.

[0037] Figure 25 represents histogram plots depicting gRNA toxicity for group 4 cassette 5 with change in target position and Cas9 promoter of some embodiments disclosed herein.

[0038] Figure 26 represents histogram plots depicting editing efficiency with group 2 cassette 6 using different promoters of some embodiments disclosed herein.

[0039] Figure 27 represents graphs depicting gRNA toxicity for group 3 cassettes 6, 7, and 8 with change in target position and Cas9 promoter of some embodiments disclosed herein. [0040] Figure 28 represents graphs depicting transformation efficiency and number of recombinants for group 3 Cassette 7 and 8 with different target positions and Cas9 promoters of some embodiments disclosed herein.

[0041] Figure 29 represents photographic images depicting MacConkey screen data for restreaks for different group 3 colonies of some embodiments disclosed herein.

[0042] Figure 30 represents photographic images depicting MacConkey screen data and a restreak of group 3 cassettes with and without the temperature curable cassettes at 2 different temperatures for an experimental gene of some embodiments disclosed herein.

[0043] Figure 31 represents photographic images depicting MacConkey screen data to measure apparent editing efficiency for group 3 cassettes of an experimental gene at different genomic loci of some embodiments disclosed herein.

[0044] Figure 32 represents graphs depicting changes in transformation efficiency and number of recombinants for each condition with cassettes with increasing toxicity of some embodiments disclosed herein.

DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] 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.

[0048] ln 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.

[0049] In certain embodiments, designer spacer gRNA sequences can be used to alter genetic editing efficiencies of a Cas-based nuclease system.

[0050] Embodiments of the present disclosure relate to compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing. In certain embodiments, engineered nucleic acid guided nucleases can include a mutated or modified gRNA spacer region to increase editing efficiency and reduce off-targeting of the engineered nuclease. In some embodiments, gRNA spacer regions can be mutated by one or more of nucleic acid insertions, deletions, point mutations, substitutions or other means to alter recognition and/or binding of a target DNA.

In certain embodiments, target DNA can include genomic DNA. In some embodiments, modified spacers of gRNAs can be used in high-throughput screening systems to identify improved editing systems having selective gRNAs for improved editing efficiency and targeted control over gene editing of use in experimental and therapeutic situations.

[0051] In certain embodiments, a nucleic acid guided nuclease system disclosed herein can include a nucleic acid guided nuclease; and a modified gRNA capable of improved editing efficiency of a target DNA, wherein the modified gRNA hybridizes with the target DNA to increase gene editing efficiency compared to a reference gRNA not having the modification. In other embodiments, a modified gRNA comprises a mutated spacer region where the modification is capable of altering editing efficiency. In certain embodiments, the modified spacer region can include one or more of an insertion, deletion, substitution or point mutation. In some embodiments, a modified spacer region of the gRNA modifies binding to the target DNA by the gRNA spacer. In some embodiments, these alterations can modify recognition by the gRNA of the targeted DNA to improve repair by repair enzymes or improve off-targeting and editing efficiency of a targeted genome. In accordance with these embodiments, a modified spacer region can reduce binding affinity to the DNA, allowing recognition for cleavage by the nuclease while increasing repair of the targeted DNA to increase recombination events.

[0052] In some embodiments, targeted DNA can be genomic DNA. In other embodiments, targeted DNA can include prokaryotic or eukaryotic DNA. Other embodiments disclosed herein can include kits of use for gene editing having a mutated gRNA spacer region and other components needed for gene editing. In some embodiments, kits can include at least one container and one or more novel gRNAs disclosed herein and optionally, a nuclease. [0053] In certain embodiments, methods disclosed herein can include methods for modifying genome editing in order to improve editing efficiency and reduce off-targeting. In accordance with these embodiments, methods disclosed herein can include contacting a target DNA molecule having a target sequence with a nuclease complex comprising: (a) a nuclease protein ( e.g . Cas nuclease or other nuclease); and (b) a DNA-targeting gRNA including: (i) a modified gRNA having a modified spacer sequence creating modified target recognition sequence hybridization compared to a reference gRNA that hybridizes with the same target sequence, and (ii) a scaffold tracrRNA for guiding binding of the Cas nuclease to the target sequence wherein the complex forms a double-stranded RNA (dsRNA) duplex of a protein binding segment, wherein the modified gRNA has increased editing efficiency and/or reduced off-targeting compared to a reference nuclease complex having a control gRNA. In certain methods, the targeted genome can be a eukaryotic or prokaryotic genome. In other embodiments, the targeted genome can be a mammalian genome. In some embodiments, the targeted genome is a human genome. In yet other embodiments, the targeted genome can be a human or other mammal such as a pet or livestock genome. In other embodiments, the gRNAs include but are not limited to the gRNAs represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 20, SEQ ID NO: 32 to SEQ ID NO: 40, SEQ ID NO: 50 to SEQ ID NO: 60, SEQ ID NO: 72 to SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91 to SEQ ID NO:94. In some embodiments, gRNAs disclosed herein can be used in CREATE methods to arrive at all possible changes in the spacer regions of the gRNAs of interest to assess changes in the spacer region compared to a control gRNA in genome editing technologies. In other embodiments, the gRNAs include, but are not limited to, the gRNAs and/or homology regions represented by Table 1.

[0054] In some embodiments, modification of the gRNA spacer sequence increases targeted gene replacement compared to a reference gRNA (e.g. native or control gRNA). In other embodiments, modification of the gRNA spacer sequence increases gene editing by enhancing gene recombination in the target DNA molecule having a defective gene. In certain embodiments, a modified spacer of the gRNA has at least one mutation of a deletion, insertion, point mutation or a substitution in the spacer. In some embodiments, these spacer alterations create a gRNA having reduced binding and increased recombination allowing the repair enzymes of the target organism to repair cut DNA. In accordance with these embodiments, improved gene editing provided by the modified spacer of the gRNA can be used to increase editing efficiency in a given target DNA (e.g. prokaryotic or eukaryotic DNA). [0055] In certain embodiments, multiplex mutational libraries can be created through the use of CRISPR Cas9 and synthetic oligomer libraries where tens to hundreds of thousands or more gRNA spacer mutants can be screened and assayed for altered targeting and improved editing efficiency. In accordance with these embodiments, modified gRNA spacer regions can be selected for decreased binding that correlates with increased editing efficiency and/or recombination. In other embodiments, Cas9 expression can be modulated to improve editing efficiency using the modified gRNA. In accordance with these embodiments, improved high- throughput editing can be achieved.

[0056] In some embodiments, modified spacers in a gRNA homologous to a targeted DNA controls activation, binding and cleavage of DNA by Cas9 having reduced binding and increased recombination. In certain embodiments, editing plasmids can be constructed with cassettes containing for example a 150 bp homology arm (HA) in cis with a contemplated modified or reference gRNA disclosed herein, expressed under a constitutive promoter to assess how gRNA activity could vary EE. In this example, the homology arm (HA) can have targeted non-synonymous point mutations, and optionally, a synonymous mutation in the PAM or gRNA SEED sequence (5 nucleotides next to the PAM, deemed crucial for binding for immunity to Cas9 mediated double stranded binding (DSB)). Editing efficiency (EE) can be measured by sequencing the edited locus from a represented number of colonies. gRNA activity ( e.g . toxicity) can be measured by transforming plasmids with only the gRNA (no repair template) in cells with active Cas9 to assess gRNA toxicity. gRNA activity-toxicity can be calculated as the logarithmic fold reduction in the transformation efficiency for the gRNA with respect to a non-targeting gRNA. Editing and toxicity can be measured for each cassette to assess the targeted location (exemplary Table disclosed herein). Editing across all constructs can be assessed and selected for increased editing efficiency (EE). In these examples and in practice, editing efficiency increased with an increase in gRNA toxicity. In certain embodiments, cassettes can be created and assessed for targeting ideal gRNA spacers with increased editing efficiency and assessed toxicity. A sigmoid-shaped trend can be created based on the data between the 3 groups accounting for editing versus gRNA toxicity (Figure attached) in order to select optimal gRNA modified spacers.

[0057] In some embodiments, compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing disclosed herein can include Cas9:gRNA-mediated recombineering. In some aspects, Cas9:gRNA-mediated recombineering can be achieved by following CRISPR EnAbled Trackable genome Engineering (CREATE) design. CREATE can be performed as described in at least Liang et al., Metab Eng. (2017) 41 : 1-10 and Garst et al., Nat Biotechnol. (2017) 35(l):48-55, the disclosures of which are incorporated herein in its entirety. In other aspects, CREATE can be applied to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. In some other aspects, CREATE can link each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. In some embodiments, gRNAs can be generated that include spacer region manipulations and can include all possible substitutions in the spacer region of the gRNA. In other examples disclosed herein, gRNAs that can be used in CREATE methods disclosed herein are represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 20, SEQ ID NO: 32 to SEQ ID NO: 40, SEQ ID NO: 50 to SEQ ID NO: 60, SEQ ID NO: 72 to SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91 to SEQ ID NO:94. In some examples disclosed herein, homologous repair cassettes that can be used in CREATE methods disclosed herein are represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 21 to SEQ ID NO: 31, SEQ ID NO: 41 to SEQ ID NO: 49, SEQ ID NO: 61 to SEQ ID NO: 71, SEQ ID NO: 76 to SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, and SEQ ID NO: 95 to SEQ ID NO: 99. In other aspects, homologous repair cassettes can include a homology repair template (HRT). In some examples disclosed herein, homology repair templates can be, but are not limited to, representation by SEQ ID NO: 80 to SEQ ID NO: 84.

[0058] In some embodiments, compositions and methods for creating and using engineered nucleic acid guided nucleases having a modified gRNA spacer region to modulate targeted gene editing 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 in human or other mammals.

[0059] 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 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

[0060] 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.

[0061] Genetic Disorders contemplated herein can include, but are not limited to:

[0062] Neoplasia: Genes linked to this disorder: PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notchl; Notch2; Notch3; Notch4; ART; AKT2; AKT3; HIF; HIFI a;

HIF3a; Met; HRG; Bcl2; 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: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Ape;

[0063] Age-related Macular Degeneration: Genes linked to these disorders Abcr; Ccl2; Cc2; cp (cemloplasmin); Timp3; cathepsinD; VIdlr; Ccr2;

[0064] 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;

[0065] 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;

[0066] Fragile X Syndrome: Genes linked to this disorder: FMR2; FXR1; FXR2;

mGLURS;

[0067] Secretase Related Disorders: Genes linked to this disorder: APH-l (alpha and beta); Presenil n (Psenl); nicastrin (Ncstn); PEN-2;

[0068] Others: Genes linked to this disorder: Nosl ; Paipl; Nati; Nat2; [0069] Prion - related disorders: Gene linked to this disorder: Prp;

[0070] ALS: Genes linked to this disorder: SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c);

[0071] Drug addiction: Genes linked to this disorder: Prkce (alcohol); Drd2; Drd4;

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

[0072] Autism: Genes linked to this disorder: Mecp2; BZRAP1; MDGA2; SemaSA; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; MglurS);

[0073] Alzheimer's Disease Genes linked to this disorder: El; CHIP; ETCH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1; CR1; VIdlr; Ubal; Uba3; CHIP28 (Aqpl,

Aquaporin 1); Uchll; Uchl3; APP;

[0074] Inflammation and Immune-related disorders Genes linked to this disorder: IL- 10; IL-l (IL-la; IL-lb); IL-13; IL-17 (IL-l7a (CTLA8); IL-l7b; IL-l7c; IL-l7d; IL-l7f); 11- 23; Cx3crl; ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-l2a; IL-l2b); CTLA4; Cx3cl 1, AAT deficiency/mutations, AIDS (KIR3DL1, NKAT3, NKB1, ANIB11, KIR3DS1, IFNG, CXCL12, SDF1); Autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A); Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-l (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, ATTD XPID, PIDX, TNFRSF14B, TACI); Inflammation (IL- 10, IL-l (IL-la, IL-lb), IL-13, IL-l 7 (IL-l 7a (CTLA8), IL-l 7b, IL-l7c, IL-l7d, IL-l7f), 11- 23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-l 2 (IL-l 2a, IL-l 2b), CTLA4, Cx3cl l); Severe combined immunodeficiencies (SCåDs)(JAK3, JAKL, DCLRE1C,

ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4);

[0075] Parkinson's, Genes linked to this disorder: x-Synuclein; DJ-l; LRRK2; Parkin; PINK1;

[0076] Blood and coagulation disorders: Genes linked to these disorders: Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH I, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, 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 XTTTR deficiency (F13B); Fanconi anemia

(FANCA, FAC A, FA1, FA, FA A, 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, UNC I 3D, 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);

[0077] 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, D 1, 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);

[0078] 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, PKHD1, ARPKD, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63);

[0079] 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

(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, BMNDl, 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);

[0080] 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, AEG); Autism (Mecp2, BZRAP I, MDGA2, Sema5A, Neurex 1, GLOl, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLER5); 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, RGNK1, 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);

[0081] Occular-related disorders: Genes linked to these disorders: Age-related macular degeneration (Aber, Cel 2, Cc2, cp (ceruloplasmin), Timp3, cathepsinD, Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, 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, M1 S1, 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);

[0082] P13K/AKT Cellular Signaling disorders: Genes linked to these disorders:

PRKCE; IT GAM; 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;

[0083] ERK/MAPK Cellular Signaling disorders: Genes linked to these disorders: PRKCE; IT GAM; 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;

[0084] 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; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7;

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

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

[0085] Axonal Guidance Cellular Signaling disorders: Genes linked to these disorders: PRKCE; IT GAM; 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;

[0086] Ephrin Recptor Cellular Signaling disorders: Genes linked to these disorders: PRKCE; IT GAM; 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;

[0087] 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;

[0088] 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; HD AC 5; CREB1; PRKC1; HS PA5 ;

REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HD AC 11 ; MAPK9; HD AC 9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK; HD AC 6; CASP3;

[0089] Apoptosis Cellular Signaling disorders: Genes linked to these disorders:

PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14;

MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3 : BTRC3 : PARPI;

[0090] B Cell Receptor Cellular Signaling disorders: Genes linked to these disorders: RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1;

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

MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1;

[0091] Leukocyte Extravasation Cellular Signaling disorders: Genes linked to these disorders: ACTN4; CD44; PRKCE; IT GAM; 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;

[0092] Integrin Cellular Signaling disorders: Genes linked to these disorders: ACTN4; IT GAM; 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;

[0093] 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; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB 1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6;

[0094] 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;

[0095] 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; HD AC 9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RAM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3;

[0096] 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;

[0097] 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; NFKB 1; KEAP1; PRKCA; EIF2AK3; IL6;

CYP1B1; HSP90AA1;

[0098] 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; [0099] 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; RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1;

IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1;

TGFBA1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOO;

[00100] NF-KB Cellular Signaling disorders: Genes linked to these disorders: IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB;

PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1;

[00101] 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; ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HS P90AA1; RPS6KB1;

[00102] Wnt and Beta catenin Cellular Signaling disorders: Genes linked to these disorders: CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; 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; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6; [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; RELA; PRKCD; MAPK9;

ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN; IL1R1; PRKCA; IL6;

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

PRKCZ; ELK1; 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; FTL; NFE2L2; 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; FLT1; SMAD2; FGFR1; MET; PGF; SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9;

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

NFKB1; JUN; IL1R1; HSP90AA1;

[00110] Fc Epsilon RI Signaling disorders: Genes linked to these disorders: PRKCE; RAC1; PRKCZ; LYN; 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; RELA; SRC; PIK3C2A; 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 D 3 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; VEGF A; 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; NFKB1; 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; 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; FRAPl; 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; [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; SMURF 1; 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; MAPK 14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF;

STAT1; and

[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- 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.

EXAMPLES

[00135] 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.

[00136] Some challenges with inconsistent editing efficiency (EE) can be overcome by predicting gRNAs that more efficiently lead to double-stranded breaks (DSBs). However, even using prescreened gRNAs that lead to high Cas9:gRNA-induced cell death, editing efficiency can vary the spectrum from 0-100%. Therefore, an improved understanding of the system with respect to gRNA predictability is needed to improve editing in single and high-throughput applications of Cas9-mediated recombineering.

Example 1

[00137] Efficient gRNA-mediated cell death does not ensure high editing efficiency. In one exemplary experiment, studies were performed following CRISPR EnAbled Trackable genome Engineering (CREATE) design for Cas9:gRNA-mediated recombineering as described in Liang et al., Metab Eng. (2017) 41 : 1-10 and Garst et al., Nat Biotechnol. (2017) 35(l):48-55_j_the disclosures of which are incorporated herein in its entirety. As an initial evaluation, variation in Cas9:gRNA-induced cell death was evaluated using 35 gRNAs (Table 1). Of these, 16 targeted galK , which encodes galactokinase, and 19 targeted other genes across the genome.

Table 1: List of editing cassettes with gRNA and repair HA (homology arm) Sequences.

[00138] In some exemplary methods, to quantify Cas9:gRNA-induced cell death, fold reduction was calculated in colony-forming units (CFUs) after transforming a plasmid encoding either a targeting gRNA or a non-targeting gRNA into cells expressing Cas9. The fold-reduction in CFUs varied over four orders of magnitude between gRNAs (Figure 1).

[00139] In another exemplary method, the level of Cas9:gRNA-induced cell death required to enable high EE was evaluated by selecting against cells without targeted genomic mutations. Plasmids were constructed with an editing cassette encoding each gRNA in cis with a 150 bp HRT containing a synonymous PAM/seed mutation and a targeted point mutation(s) using previously optimized design rules. Plasmids with the editing cassettes were transformed into cells with active Cas9 and lambda Red recombination proteins. EE was measured by Sanger sequencing the target genomic locus from 20 colonies. EE increased with an increase in the level of Cas9:gRNA-induced cell death measured for the gRNA of each cassette in the absence of a HRT (See Figure 1). gRNAs that resulted in efficient killing of unedited cells ( e.g . those that demonstrated greater than a 1000-fold reduction in CFETs after transformation when compared to the CFETs upon transforming a non-targeting gRNA in the absence of a HRT) enabled nearly 100% EE (Figure 1).

[00140] In one method, a selection based model was applied to further describe editing. In this exemplary model, cell depletion due to gRNA toxicity and cell growth were first order reactions with death constant ( k_death ) and growth rate (growth) respectively. Because recombineering is temporarily induced, it was assumed that modeling would start with a fixed population of recombined cells (Nrecombined,o) and wild-type cells(N_Wiid-type„o ). Nrecombined,o was fit as a linear function of death constant ( kdeath ) to explain induction of recombination with gRNA toxicity. Change was calculated over a fixed recovery time (t), of 180 minutes. Variation of editing efficiency with toxicity could be explained with the following sigmoidal function:

[00141] In another exemplary method, editing cassettes were classified into 3 groups based on the model. Group 1 gRNA had kdeath greater than p_growth. The 15 gRNAs above this threshold were categorized into Group 1. Cassettes with gRNA toxicity, kdeath, comparable to a non-targeting gRNA (p>0.1) were clustered into Group 3. Nine gRNAs that caused negligible Cas9:gRNA-induced cell death in the absence of a HRT (with a p-value>0.05 compared to a non-targeting gRNA) were categorized into Group 3. The remaining gRNAs were categorized into Group 2. [00142] Cas9:gRNA-induced cell death in E. coli in the absence of a HRT and lambda Red recombination was used as a proxy for Cas9:gRNA-mediated DNA DSB activity. Therefore, it was inferred that the Cas9:gRNA-mediated DNA DSB activity increases from Group 3 to Group 1. Cas9:gRNA DSB activity was controlled primarily by interactions between the 20 bp spacer, the target sequence, and Cas9. The variation in binding affinity between the spacer and the target sequence was likely responsible for the large variation observed in Cas9:gRNA- induced death even using gRNAs targeting different positions within the same gene (Figure 1).

[00143] In another exemplary method, genome editing was evaluated to see if it would occur by recombination between the genome and the HRT either before or after Cas9:gRNA-induced DSB formation. A low level of edited CPUs (-10⁴ edited out of 10⁶ total cells) was observed in the presence of a HRT even with a non-targeting gRNA, which cannot cause DNA DSBs, suggesting that recombination catalyzed by the lambda Red recombination enzymes occurs in the absence of Cas9:gRNA-induced DSBs (Figure 2). If recombination were entirely independent of DNA DSBs, the number of recombinants would have remained the same across all gRNA groups, but editing efficiency (EE) would have increased due to elimination of non- edited cells as Cas9:gRNA-induced cell death increases across Groups 3 to 1. It was observed that the number of edited CFUs significantly increased from Group 3 to Group 1 (P < 0.0001, Figure 2), suggesting that recombination can occur after DSB formation. Therefore, both DNA DSB-independent and DSB-dependent recombination could occur. The fold-reduction in total CFEis across groups due to Cas9:gRNA-induced cell death exceeded the fold-increase in the number of edited CFUs by nearly two orders of magnitude (Figure 2), suggesting that DSB- independent recombination was the dominant mechanism for genome editing. Consequently, high EE results primarily from selection against non-edited cells, but there was a small contribution from ITRT -mediated recombination after DSB formation.

Example 2

[00144] Apparent EE for the same cassette changes with target genome position. In another study, other parameters that could impact EE were assessed using the MacConkey agar screen to measure EE in galK. On MacConkey agar, colonies with inactive GalK were white, and colonies with active GalK were red. Nine editing cassettes were used to introduce a premature stop codon into galK. Of these, four had Group 1, two had Group 2, and three had Group 3 gRNAs.

[00145] To test if EE varies with the target position, galK was first deleted from its native operon, then constructed strains with galK were integrated at seven different genomic positions (Figure 2 and Figure 20). For editing cassettes with Group 1 gRNAs, the apparent EE remained high for all positions (Figure 3). For editing cassettes with Group 2 gRNAs, the apparent EE increased with increasing distance from the terminus (Figure 3). Group 3 did not have a clear position-dependent trend, but the apparent EE was highest furthest from the terminus of replication (Figure 3). Therefore, it was observed that the genomic position of the target was an important determinant of EE; for example, for gRNAs that cause low Cas9:gRNA-induced cell death.

Example 3

[00146] Apparent EE for the same cassette depends on cas9 promoter strength. In other exemplary experiments, a araC promoter that controlled cas9 in the initial experiments was exchanged with three previously characterized constitutive promoters: prol (weak), proC (medium), and proD (strong) were used in its place. gRNA was expressed under the strong J23119 promoter in all cases. There was a significant increase in apparent EE for the two Group 2 cassettes and two of the three Group 3 cassettes with stronger cas9 promoters (Figure 4). In Group 2, it was also observed that the unedited colonies had significantly smaller colony size with stronger Cas9 promoters (data not shown). Surprisingly, for cassettes with Group 1 gRNAs (which already confer high Cas9:gRNA-induced cell death and EE), apparent EE decreased with increasing promoter strength (Figure 4). Therefore, it was observed that the effect on EE of cas9 promoter strength varies by gRNA and in these examples, by the gRNA group.

Example 4

[00147] Introduction of targeted mutations using Group 1 gRNAs is reduced due to incomplete editing and deletions in the editing plasmid. In another exemplary method, genomic targets were deep sequenced after editing with eight galK editing cassettes at three different positions (0.30, 0.82 and 2.28 Mb from ter ) using four cas9 promoters (araC, prol, proC, and proD) to measure“actual” EE. For Group 1 gRNAs, actual EE for galK integrated at three different sites in the genome was high (80.5 +/- 1.5 % and 90.5 +/- 2.2% cells had all the desired mutations with cassettes 1 and 2, respectively) (Figure 5A). The number of CFETs after transforming the plasmids encoding the editing cassettes (Figure 5A) was low (10⁴ - 10⁵ CFETs per transformation) across all genomic positions compared to the number after transformation with plasmids encoding a non-targeting gRNA (10⁶-10⁷ CFETs per transformation) (Figure 2). Group 1 gRNAs was projected to have had high and consistent DNA DSB activity across all genomic positions, allowing efficient elimination of non-edited cells and therefore high EE. [00148] In one observation, due to the high Cas9:gRNA-induced cell death observed in Group 1, escape from Cas9:gRNA activity would be necessary for a cell to survive. In another method, the escape mechanism in cells was evaluated without all the desired mutations with Group 1 cassettes (-20% and -10% of all cells for cassettes 1 and 2, respectively). In Cassette 1, the point mutations in the PAM and target site were separated by 18 bases. After editing with Cassette 1, the deep-sequencing data demonstrated that the base-change frequency at the target site was about 10% lower than at the PAM site (Figure 5B). Cassette 2 had several target mutations in the seed region but no PAM site mutation. Although the actual EE with Cassette 2 was 10% higher than with Cassette 1, the base-change frequency of the mutations within the seed region varied by over 8% (Figure 5B). Therefore, editing was incomplete. However, all partially edited cells for both cassettes harbored either a PAM site mutation or at least 1 mutation in the seed region permitting escape from Cas9:gRNA activity.

[00149] In another method, it was observed that about 9% and 4% of all the deep-sequencing reads did not have any PAM or seed mutation after editing with Cassettes 1 and 2, respectively (Figure 5B). In these colonies, it was observed that neither deletion of galK or mutations in galK could have allowed escape from high Cas9:gRNA-induced cell death (Figures 6A-6B). In some of these cases, escape could be attributed to random deletions in for example, the editing plasmid that included the gRNA sequence (Figures 6C-6D).

Example 5

[00150] Off-target DSB activity reduces EE for Group 1 gRNAs. In another exemplary method, the number of CFETs after transformation with plasmids carrying cassettes using a Group 1 gRNA decreased with an increase in cas9 promoter strength (Figure 7A), suggesting an increase in Cas9:gRNA-induced cell death. It is projected that an increase in Cas9:gRNA- induced cell death should increase EE due to the elimination of unedited cells. Contrary to this projected result, actual EE and the fraction of cells with incomplete editing decreased with an increase in cas9 promoter strength (Figures 7A-7B). However, the number of non-edited CFETs remained unchanged (Figure 7C).

[00151] In another method, to test the impact of off-target DSBs on EE, the actual EE in the wild-type strain was compared to a strain lacking dgoK , which contains a possible off-target site for Group 1 Cassette 1 (Figure 8A). With the strongest cas9 promoter (proD), actual EE decreased by 48+/-3% in the wild-type strain but remained unchanged for the AdgoK strain (Figure 8B-8C) There was also a significant increase in the number of CFETs after transformation with plasmid carrying the editing cassette in the AdgoK strain compared to the wild-type strain (P<0.05) (Figure 8D), suggesting a decrease in Cas9:gRNA-induced cell death due to lack of the off-target site. Data suggested that off-target sites for Group 1 gRNAs reduce EE because they confer both high on-target DSB activity and substantial off-target activity.

Example 6

[00152] Actual EE with Group 2 gRNAs changes with target position and cas9 promoter strength. In another exemplary method, it was observed that Group 2 gRNAs caused lower Cas9:gRNA-induced cell death in the absence of a HRT than Group 1 gRNAs. Therefore, less- efficient selection against non-edited cells after editing using a Group 2 gRNA leads to lower EE. For Group 2 Cassette 4, a 34.0 +/- 1.2-fold increase in actual EE which is furthest from ter was observed compared to actual EE at a position closer to ter and 5.4±0.05-fold increase in actual EE with the strongest cas9 promoter was also observed (Figure 9A). These increases in actual EE could be explained by an increase in Cas9:gRNA-induced cell death that was observed with increasing distance from ter and with increasing cas9 promoter strength (Figure 9B). A significant increase in actual EE with increasing distance from ter and the strength of the cas9 promoter was also observed for Group 2 Cassette 5 (Figure 9C). Interestingly, the number of CFETs remained unchanged after transformation with plasmids encoding Cassette 5 with or without the HRT across positions and cas9 promoters (p-value > 0.1) (Figures 9C- 9D).

Example 7

[00153] Unintended mutations increased significantly with Group 2 gRNAs. In another exemplary method, a significant number of mutations was observed within the DNA spacer region that were not targeted for editing with both Group 2 cassettes at all levels of Cas9 expression (Figure 10). To evaluate the role of error-prone repair of DSBs, cells expressing Cas9 were transformed with plasmids encoding gRNAs without a HRT. A high mutation frequency was observed in the spacer region of the DNA target (Figure 11A), suggesting mutations introduced by error-prone DSB repair of the Cas9:gRNA target can prevent subsequent Cas9:gRNA-mediated DSBs. Further evidence that error-prone polymerases induced by the SOS response are involved in the unintended mutations observed with Group 2 cassettes was provided by the decreased frequency of mutations when lambda Red enzymes are induced prior to the introduction of Group 2 gRNA plasmids without a HRT (Figure 11B).

[00154] In one observation of these exemplary methods, frequency of unintended mutations with Cassette 2 gRNAs increased slightly with an increase in cas9 promoter strength (Figure 10). An increase in cas9 promoter strength increased the frequency of DSBs, which were positively correlated with the SOS response. Surprisingly, the frequency of unintended mutations was negligible with Group 1 gRNAs, despite their greater ability to cause Cas9:gRNA-induced cell death (Figure 7B). The difference in the frequency of unintended mutations with Group 2 gRNAs versus Group 1 gRNAs could be due to differences in the binding affinity of the Cas9:gRNA complexes. These results suggest that Cas9:gRNA complexes with Group 2 gRNAs dissociate from the target site faster than Cas9:gRNA complexes with Group 1 gRNAs, which could then permit error-prone repair proteins to access the DSB. In contrast, Cas9:gRNA complexes with tightly binding Group 1 gRNAs may not dissociate from the DSB, reducing the incidence of error-prone repair and leading to cell death. Example 8

[00155] Group 3 gRNAs lead to binding of Cas9 but not DSB formation, dependent upon target position and cas9 promoter. In another exemplary method, apparent EE approached 100% at positions farther from ter for all Group 3 cassettes and approached 100% with a change in the cas9 promoter for Group 3 Cassettes 6 and 7 (Figure 12A). Surprisingly, actual EE as determined by sequencing galK in white colonies was significantly lower than apparent EE for all Group 3 cassettes at most positions as well as with stronger cas9 promoters (Figure 12A). It was observed that Group 3 gRNAs lead to negligible Cas9:gRNA-induced cell death in the absence of a HRT (Figure 12B), that could be due to low DNA DSB activity. Data suggest that Group 3 gRNAs promote binding of Cas9, but not the conformational change that is required to induce a DNA DSB; therefore, the high level of apparent EE with Group 3 gRNAs may be due to repression of galK transcription by bound Cas9. Further, loss of GalK function without any genomic mutations was observed when Cas9-expressing cells were transformed with plasmids containing the Group 3 gRNA but no HRT (Figure 12B). The loss of GalK function was also observed when cells expressing dead Cas9 (dCas9), which is capable of repressing gene expression but not of creating DSBs, were transformed with Group 3 gRNAs (Figure 12C). In addition, two Group 3 gRNAs were cloned into plasmids with a temperature- sensitive origin of replication that are maintained at 30°C but can be cured at 37°C. As such, colonies containing GalK inactivated by editing or unintended genomic mutations should remain white regardless of growth temperature; for example, whether or not the gRNA is still present. After transforming cells with plasmids carrying Group 3 gRNAs, the cells formed white colonies at 30°C, but red colonies upon re-plating at 37°C, conditions under which the plasmids expressing the gRNA were cured (Figure 12D). These data demonstrate that the observed loss of function of GalK was due to transcriptional repression rather than actual editing.

Example 9 [00156] Editing behavior can be controlled by the gRNA spacer sequence. In one exemplary method, to determine if limited DSB activity of Cas9:gRNA complexes explain the cas9 promoter strength- and target position-dependent EE observed for Group 2 gRNAs (Figures 9A-9D) and the repression of transcription observed for Group 3 gRNAs (Figure 12C), the 4th, 8th and l2th nucleotides of the gRNA spacer proximal to the PAM site of two Group 1 gRNAs was mutated, for example, in effort to decrease DSB activity and performed editing using the same HRT (Figure 13). When cas9 was expressed using the araC promoter, actual EE decreased dramatically for both Cassette 1 and Cassette 2 variants (Figure 13). It was observed that expressing cas9 using a proC promoter increased actual EE when in the presence of a Mut-4 and Mut-l2 variants of Cassette 1 by 6.1 ±1.0- and 15.0 ±l.0-fold, respectively, compared to the actual EE when cas9 was expressed using the araC promoter (Figure 13). However, the actual EE for variants of Cassette 2 demonstrated a different trend. Actual EE with the Mut-4 variant of Cassette 2 was similar to that with the wild-type gRNA, but actual EE for the Mut-8 and Mut-l2 variants was negligible when cas9 was expressed using either the araC promoter or proC promoter (Figure 13). Interestingly, apparent EE for with the Mut-8 and Mut-l2 variants of Cassette 2 improved with the proC cas9 promoter (Figure 14). It was observed that the mutations in Cassette 1 gRNA changed its behavior making the system promoter-dependent similar to Group 2, and the mutations in the Cassette 2 gRNA transformed it to the transcriptional repression phenotype observed in Group 3 gRNAs. These data reiterate that DSB activity limitations play an important role in the outcome of editing with Cas9:gRNA-mediated recombineering.

[00157] Other previously discussed trends with Group 2 and Group 3 cassettes were observed with the mutant gRNAs in these experiments. With the Cassette 1 Mut-l2 variant, despite the increase in actual EE with a proA cas9 promoter, the CFETs after transformation remained unchanged (data not shown). Therefore, it appears that EE increased due in part to an increase in frequency of DSB-dependent recombination with the HRT, rather than an increase in selection against non-edited cells. Further, the frequency of non-targeted mutations increased for the Cassette 1 Mut-4 and Mut-l2 mutants after editing with proC-cas9 (Figure 15); such mutations were not observed with the wild-type Group 1 gRNA (Figure 5B).

[00158] These observations suggest that faster dissociation of Cas9:gRNA complexes with Group 2 gRNAs after DSB formation compared to slow dissociation of complexes with Group 1 gRNAs provided for an increase in access to error-prone polymerases and recombination proteins to the DSB.

Example 10 [00159] Cas9:gRNA DNA DSB activity and the rate of dissociation following DSB formation may explain editing trends across groups. In another exemplary method, properties of the N20 spacer, which controls DNA DSB activity and rate of dissociation of the Cas9:gRNA complex after DSB formation, likely provide for the observed trend for recombination with the HRT, off-target activity, alternate escape mechanisms, and non- targeted mutations across gRNA groups as illustrated in Figure 17.

[00160] Group 1 : Group 1 gRNAs likely had the highest DNA DSB activity, which permitted high and consistent EE across genomic loci due to strong selection against unedited cells (Figure 6A-6B). Strong persistent Cas9:gRNA binding blocked access of repair proteins, precluding repair and causing cell death. Deletions within the gRNA-encoded region of the editing plasmid were the dominant escape mechanism (Figures 6A-6B and Figures 7A-7D). The strong on-target activity of these gRNAs also increased the probability of off-target activity, which reduced EE by killing properly edited cells, particularly when cas9 was expressed at high levels (Figures 8A-8C and Figures 9A-9D).

[00161] For Group 1 cassettes, the k_death exceeds m^^_nϋi. No significant change in gRNA toxicity was observed with change in position (p-value = 0.258; Figure 22). Actual editing efficiency remained high at all positions but varied between cassettes. Editing with cassette 2 was -10% higher than cassette 1. The homology arm design for the 2 cassettes was different. Cassette 1 had single point PAM and targeted mutations separated by a distance, and cassette 2 had several simultaneous mutations adjacent to PAM/SEED mutation. For Cassette 1, base change for the PAM mutation was 12% higher than the targeted mutation. The incomplete recombinants could be due to corrections made by the Mismatch repair (MMR) machinery. Since the repair of the PAM mutation is lethal, base correction was higher for the targeted mutation. Six percent of sequences for design 2 also had inconsistent base change frequencies, suggesting that even several simultaneous bases possibly cannot completely evade mismatch repair (Figure 23). Accounting for the MMR, actual editing efficiency approached 100%. The deleted region included the gRNA sequence (Figures 6C-6D). The primary mechanism of escape for group 1 gRNA was by deletions in the editing plasmid.

[00162] With change in Cas9 promoter, there was increased gRNA toxicity, corresponding to the expected increase in expression (Figure 24). The observed behavior could be due to Cas9’s off-target activity. Off-target damage could continue to stay toxic even after mutating the on-target. The escape mechanisms like gRNA deletion are favored as they provide immunity from both on and off-target toxicity. [00163] Grow 2: Cas9:gRNA complexed with Group 2 gRNAs likely had weaker DSB activity, but dissociated faster after DSB formation than complexes with Group 1 gRNAs. Consequently, cassettes with Group 2 gRNAs demonstrated a different dependence on position and Cas9 promoter compared to cassettes with Group 1 gRNAs (Figures 9A-9D). Faster dissociation of Cas9:gRNA complexed with Group 2 gRNAs may have increased access of both lambda Red recombination proteins and error-prone polymerases, leading to increasing the frequency of DSB-dependent recombination with the HRT (Figures 9A-9D) and the frequency of unintended mutations (Figure 10 and Figures 11A-11B).

[00164] Upon transforming plasmids with only the gRNA for cassettes 4 and 5, high mutation frequency was observed at several bases at their target (Figures 11A-11B). The mutation frequencies and the transformation efficiency were significantly higher with un-induced Lambda red recombination (p<0.005, Figures 11A-11B). Cassette 4’s gRNA toxicity was comparable to group 1 gRNA with proC and proD Cas9 promoters, whereas increased non- targeted mutations was never observed in any Group 1 gRNA. Also, the transformation efficiency for cassette 5 was comparable to a non-targeting RNA even for high editing conditions (p-value for t-test > 0.1) (Figure 25).

[00165] Grow 3 : Cassettes with a weak Group 3 gRNA caused repression of gene expression rather than editing under some conditions, suggesting that the Cas9:gRNA complex was capable of binding to the target site but has very low DNA cleaving activity. Presumably bound Cas9 eventually dissociated without inducing a DSB to enable cell survival (Figures 12A- 12D).

[00166] In another method, it was observed that Group 3 Cassette 6 also permitted a 20-60% increase in editing with altered Cas9 expression but the toxicity remained comparable to nt- gRNA (Figure 26). For some cassettes, editing increased primarily by increase in recombination and not selection against non-recombinants, deviating the sigmoidal editing behavior.

[00167] In another observation, it is noted that Group 3 cassette gRNA had k_death comparable to non-targeting guide RNA. The toxicity remained consistently low regardless of position or Cas9 promoter (Figure 27). Although Group 3 cassette gRNA had a significant increase in apparent editing efficiency with position and Cas9 promoter, to our surprise, the actual editing was negligible. It was suspected that the colonies could be unsegregated, but the number of recombinants did not change in any condition (Figure 28). Several colonies were re-streaked to assess a mixture of red and white colonies (Figure 29). The sequenced white colonies had no mutations or deletions in GalK. It was noted that Group 3 gRNA demonstrated loss of function without any observed genomic change.

[00168] Regarding Group 3, this observed loss of function could be due to repression. In order to assess repression phenotype, two Group 3 editing cassettes and one Group 1 editing cassette were cloned into a plasmid with a temperature sensitive origin which is maintained in the cell at 30°C but cured at 37°C. Cells transformed with Group 3 cassettes reverted to the wild-type red phenotype at 37°C, while cells edited with Group 1 guides did not. Colonies re- streaked at 30°C were mixed but not at 37°C (Figure 30). Loss of function was contingent upon presence of the gRNA confirming the repression phenotype. The same phenotype was replicated with catalytically dead Cas9 (dCas9) (Figure 12C). Further, an increase in repression was observed for all Group 3 gRNA as we moved away from the terminus (Figure 31). Similarly an increase in gRNA toxicity was seen with Cassette 4 (Figure 28). Because, both repression and toxicity, are dependent on Cas9 activity data suggest cas9 activity could be position dependent.

[00169] The varying behavior of experimental Groups 1-3 gRNAs indicates that custom designed gRNAs could allow better control over genome editing and outcomes such as off- target DSBs and non-intended mutations. However, there was no correlation between the number of CFUs obtained after transforming Cas9-expressing cells with plasmids encoding gRNAs and predicted Cas9:gRNA DSB activity using seven different algorithms optimized in different hosts (Figure 17).

Example 11

[00170] Editing behavior changes with growth phase. The impact of reduced cell growth rate on actual EE was evaluated by (i) recovering the cells in M9 minimal medium rather than in rich medium (LB); and (ii) editing cells in early stationary phase (defined as OD₆oo = 2.5) using Cassette 1 from Group 1, Cassette 5 from Group 2 and Cassette 6 from Group 3 and using the araC promoter for cas9 expression. Slowing cell growth substantially improved EE for Group 2 and 3 gRNAs (Figure 18). Editing in stationary phase increased EE by 15.6±1.7- fold and 5.4±0.2-fold with single Group 2 and Group 3 gRNAs, respectively (Figure 18). The increase in actual EE occurred without significant change in total CFUs per transformation for Groups 2 and Groups 3, suggesting an increase in DSB-dependent recombination. With the Group 1 gRNA, EE remained high, but the number of CFUs after transformation increased significantly when editing was carried out in the stationary phase.

Example 12 [00171] Editing behavior can be controlled by the gRNA sequence. Editing displayed gRNA group dependent trends: (1)) Group 3 was non-toxic, 2 had programmable toxicity and 1 had high on and off-target toxicity; (2) Group 3 had low editing, 2 had programmable editing, and 1 had high editing; (3) Number of recombinants were low in 3 and 1 but varied in 2; and (4) Non-template mutations were absent in groups 1 and 3 and high in Group 2. Non-template driven behavior like repression (Group 3) and plasmid mutations (Group 1) were group dependent as well. In Group 2 we observed increased editing with induction of recombination and no increase in cassette toxicity (Figure 32). Such improvement in recombination has been attributed to donor template type: linear versus circular. Therefore, editing across groups was compared by co-transforming the gRNA and linearized HA. The number of recombinants increased slightly with linear donor (p<0.005). Group dependent editing trends were similar between linear and plasmid HA donor. Actual editing was low for Group 3 and high for Group 1 at both positions.

Example 13

[00172] High EE with a mixture of editing cassettes requires complex control over several parameters. In other exemplary methods, the impact of variable editing behavior was evaluated across cassettes on editing in multiplex using an equimolar mixture of one cassette from each group of gRNAs (Cassettes 1, 5 and 6) and a non-targeting gRNA at the wild-type position of galK , with cas9 expressed using the araC promoter. The non-targeting gRNA represented non-functional gRNAs in high-throughput libraries introduced by errors in synthesis and cloning, which can comprise up to about 30% of a library. The total actual EE using the mixture of cassettes was only l . l±0.04% (Figure 19). ETsually, plasmids encoding cassettes with high EE yield orders of magnitude fewer CFETs after editing than plasmids encoding non-targeting gRNAs and poorly editing Group 2 and Group 3 cassettes (Figure 19). Improved EE was achieved with single cassettes by editing in stationary phase (Figure 18). ETpon editing with a mixture of cassettes from each of the three groups and a non-targeting gRNA, the total actual EE increased by l l .0±0.9-fold (Figure 19). Edited cells arising from the Group 2 and Group 3 editing cassettes, which demonstrated higher EE and high CFETs after transformation in stationary phase than Group 1 (Figure 19), constituted 95% of the edited population. Therefore, the high EE achieved with Group 1 gRNAs was apparently offset in multiplex applications due to low CFETs after transformation.

[00173] While gRNAs are useful for genome editing of single targets, gRNAs can be suboptimal in multiplex applications. The above examples suggest that multiplex applications can in certain cases be improved using gRNAs having Group 2 behavior promoting both DSB- dependent and DSB-independent mechanisms of recombination with the homology repair template (HRT) to enable high actual EE and CFETs after transformation. Alternatively, multiplex editing can be improved by engineering Cas9 nucleases with increased dissociation rates after DNA DSB formation. However, gRNA design appears to be crucial to ensure consistent EE across cassettes. Partial editing, off-target DSB activity, unintended mutations, and binding of Cas9 without DSB formation, also interfere with targeted editing. These factors pose problems for use of editing cassettes as barcodes to estimate the fitness of cells with targeted mutations because the plasmid barcode does not necessarily correspond to the targeted edit in the genome.

[00174] Based on the data in the examples herein, the following strategies can be used to minimize some of these issues: (1) improve HRT design to avoid incomplete editing; (2) use engineered high-fidelity nucleases to prevent elimination of edited cells due to off-target DSB activity; and (3) establish control over error-prone polymerases to prevent the introduction of unintended mutations.

Material and Methods

Design of editing constructs

[00175] All editing was performed using S. pyogenes Cas9-mediated lambda recombineering. In order to design gRNAs, the NGG PAM closest to the target site was identified. A 20 bp sequence homologous to the sequence upstream of the PAM was used as the gRNA spacer. In our editing cassettes for point mutations, 150 bp homology repair templates (HRTs) were designed centered around the NGG PAM. This template consisted of a synonymous mutation to mutate the G in the NGG PAM to provide immunity to subsequent cleavage by Cas9 after editing as well as targeted mutation(s) within 20 bp of the synonymous PAM mutation. This repair homology template was placed in cis with the gRNA, which was expressed under control of the J23119 promoter, in a 230 bp editing cassette that was ordered as a gblock from Eurofms.

[00176] For targeted deletions and insertions, the plasmid containing the designed gRNA was co-transformed with a linear HRT. For targeted deletion of dgoK, the HRT was ordered as a 200 bp g-block with 100 bp homology to sequences upstream and downstream of the targeted gene (data not shown)

[00177] In order to construct strains with galK integrated at different genomic loci, the galK gene was deleted from its operon in E. coli MG1655. Several bases towards the end of the galK gene are involved in the expression of subsequent genes in the galactokinase pathway. In order to keep this pathway functional for the MacConkey agar screen, a portion of the galK gene needed to be retained after deletion. Consequently, the HRT was amplified for deletion of galK from the SW105 strain using primers galk lOO for and galk lOO rev to include this functional region. For integration of galactokinase (galK) at different genomic loci, a previously disclosed method described by Bassalo et ah, ACS Syth. Biol. (2016) 5, 561-568 was used, the disclosure of which is incorporated herein in its entirety. Each integration plasmid contained a -1200 bp region homologous to the integration site centered around the PAM in a pBR322 backbone. The plasmid initially contained a uvGFP cassette with 600 bp sequences homologous to the upstream and downstream regions of the destination site. Primers were used to replace uvGFP with galK in the plasmids (data not shown). galK flanked by 600 bp homology regions was subsequently amplified for genome integration. Integration was performed by co-transforming the gRNA with the linear HRT as described above. Proper integration was verified using PCR and subsequent Sanger sequencing.

Cloning

[00178] NEB Q5 polymerase was used to amplify all gblocks, integration cassettes, and plasmid backbones. PCR was performed with 2.5 pL each of 10 pM forward and reverse primers, 1 pL of template (-1-10 ng/pL), 25 pL of 2X NEB Q5 2X Master Mix and 19 pL of nuclease-free double distilled water under standard PCR conditions for HF Phusion polymerase (98°C for 30 s, 34 cycles of 98°C for 15 s, T_m °C for 15 s and 72°C for 15 s*(length of amplicon), and one cycle of 72 °C for 5 m). Primer T_m values were calculated using the NEB T_m calculator. For all plasmid backbones, the amplification was followed by a Dpnl digestion reaction; 1 pL of NEB Dpnl was added to the PCR amplification reaction and the solution was incubated at 37°C for 1 hour. Amplicons were purified by gel extraction using the Qiagen gel extraction kit. The inserts were cloned into plasmids using Circular Polymerase Extension Cloning (CPEC) with 12.5 pL of an equimolar mixture of insert and backbone with at least 100 ng of backbone, and 12.5 pL of NEB 2X HF Phusion Master Mix. PCR reactions were carried out at 98°C for 30 seconds, followed by 10 cycles of 98°C for 10 seconds, 55°C for 10 seconds, 72°C for 90 seconds, and then 72°C for 120 seconds followed by a hold at l2°C). Ten pL of the CPEC reaction was transformed into competent cells by electroporation. The transformed cells were plated on LB and appropriate antibiotics as listed in the table.

Cas9-mediated Lambda red recombination and selection

[00179] All editing was performed in E. coli MG1655 (a K12 strain). The lambda Red recombineering protocol was followed using cells that had been previously transformed with either the pX2cas9 or proN-X2cas9 plasmid and the pSIM5 lambda recombination plasmid. For a description of the plasmid, see Sharan et ak, Nat. Protoc. (2009) 4, 206-223, the disclosure of which is incorporated herein in its entirety. Starter cultures of cells containing the pSim5 recombineering plasmid and the plasmid expressing cas9 were grown at 30°C overnight (- 15 hours). The starter cultures were diluted 1 : 100 fold into 50-250 mL cultures and grown to mid-exponential phase (OD₆oo of 0.35-0.4). The cells were incubated at 42°C for 15 minutes to induce lambda Red recombination proteins and then cooled on ice for 15-20 minutes. Aliquots (45 mL) were subjected to centrifugation at 7500 x g at 4°C for 3 minutes. The supernatant was discarded, and the pellets were washed in 25 mL ice-cold sterilized double distilled water by resuspending and centrifuging at 7500 x g at 4°C for 3 minutes. Cells were washed thrice in prechilled (4°C) water and once in prechilled (4°C) 10% glycerol. Finally, the cells were resuspended in a 100-fold lower volume of 10% glycerol than the culture volume. For editing, cells were transformed with either the editing plasmids or with the guide RNA and the linear HRT in 0.1 mm electroporation cuvettes. Transformed cells were allowed to recover for three hours in either LB or LB with 0.2% arabinose when the araC promoter was used to control the expression of cas9. The recovered cells were plated on MacConkey or LB agar plates containing the appropriate antibiotics. When the araC promoter was used to control Cas9 expression, the inducer (arabinose) was not added in the plates. For the experiment with M9 medium, the recovery was performed in M9 medium. For the stationary phase experiments, the cells were grown in LB until an OD₆oo of 2.8-3 was reached. Five mL of cells were incubated at 42°C to induce lambda Red recombination proteins followed by washing as described with the regular protocol. With the stationary phase cells, two extra wash steps with water to better remove excess salts were performed for preparation of competent cells.

Curing of plasmids with a temperature-sensitive origin of replication

[00180] gRNAs used for insertions, deletions and for tests of gene repression were cloned into editing plasmids with a temperature-sensitive pSClOl origin in a backbone with a carbenicillin resistance marker. For a description of the plasmid, see Phillips et ak, Plasmid (1999) 41, 78-81, the disclosure of which is incorporated herein in its entirety. For initial editing/repression, the cells with the temperature-sensitive plasmids were grown at 30°C. To initiate curing, these plasmids were grown at 37°C overnight, then diluted lOO-fold into fresh medium and grown until late-exponential phase. Apart from the test for repression of gene expression, we removed gRNA plasmids used for gene insertions and deletions to avoid potential off-target effects before carrying out subsequent gene editing with different cassettes. pSim5, which also has a temperature-sensitive origin of replication, was also cured from the strains by this procedure. Consequently, it was necessary to re-introduce the pSim5 plasmid into cells for subsequent genome editing. Calculation of transformation efficiency, apparent editing efficiency and number of recombinant

[00181] In order to measure CFUs per transformation, we plated several dilutions of cells onto LB plates after three hours of recovery at 30°C after transformation. Colonies were counted on plates with the most well-resolved colonies and CFUs per transformation were calculated as the number of colony forming units (CFUs) per pg DNA per transformation.

[00182] Apparent editing efficiencies were calculated based upon the numbers of red and white colonies on MacConkey agar plates. MacConkey agar plates were prepared as per the manufacturer’s directions with 2% galactose as the sugar. After 3 hours recovery post- recombineering, several dilutions of the recovery mixture were spread on MacConkey agar plates using glass beads. Colonies were counted on plates with 250-500 resolved colonies to enable calculations of statistical significance. The apparent editing efficiency was calculated as the ratio of white to total colonies.

[00183] The actual editing efficiencies were calculated by Sanger sequencing galK from single colonies and subsequently from deep sequencing data as described in the next section. The number of edited was calculated as a product of transformation efficiency and actual editing efficiency.

Genomic deep sequencing

[00184] Cells for genome sequencing were scraped from LB agar plates with IX PBS after overnight growth of the recovery mixture. Care was taken to collect at least 250-500 colonies for adequate representation of the population. Fifty pL of the scraped cell suspension was subjected to centrifugation in a 1.5 mL tube. The cells were washed twice with PBS and as much of the supernatant was removed as possible. Finally, the cells were suspended in 50 pL TE buffer (pH 8.0). The resuspended cells were boiled at l00°C for 10 minutes. The genomic region to be sequenced was amplified using primers with Illumina Nextera adapters (data not shown). Amplification was performed with 2X OneTaq™ polymerase with standard conditions using 25 pL of the 2X OneTaq™ mastermix, 2.5 pL of each primer, 1 pL of the boiled cell sample and 19 pL of double distilled water. PCR was performed with standard conditions (94°C for 10 minutes, 34 cycles of 94°C for 30 seconds, 54°C for 30 seconds, 68°C for 60 seconds and one cycle at 72°C for 5 minutes followed by a hold at l2°C). Each cassette- experiment PCR product was then amplified with a unique experimental Nextera barcode. All sequencing was performed using the Nextera Next-generation sequencing kit with Illumina.

[00185] An analysis pipeline was developed for estimating the number of reads with the targeted genomic edit. The initial assembly of forward and reverse reads was performed using the PandaSeq tool and the USEARCH algorithm. Experimental barcodes were split using our analysis code. Reads were mapped to their mutant genotypes using the EiSEARCH algorithm with correctly mapped reads having >98.3% identity to the target sequence, allowing an error of three mismatches over a -450 sequenced region. The actual editing efficiencies were calculated as the ratio of read counts mapped to the mutant phenotype to the total number of reads obtained for the sample. In order to map base change frequencies, the reads were compared to the wild-type sequence with a 95% identity threshold using the USEARCH algorithm. A code was developed to use the identity/mismatch mapping to calculate the frequency of mismatches at each position. The base change frequency was the ratio of the total number of reads with the base changed to the total number of reads with perfect (100%) identity to wild-type. Scipy and Numpy kits from python were used for all data analysis.

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.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid guided nuclease system comprising:

a nucleic acid guided nuclease; and

a modified gRNA capable of improved editing of a target DNA,

wherein the modified gRNA hybridizes with the target DNA to alter gene editing efficiency compared to a reference gRNA not having the modification.

2. The nucleic acid guided nuclease system according to claim 1, wherein the modified gRNA comprises a mutated spacer region.

3. The nucleic acid guided nuclease system according to claim 1, wherein the modified spacer region comprises at least one insertion, deletion, substitution or point mutation.

4. The nucleic acid guided nuclease system according to any one of claims 1-3, wherein the modified spacer region of the gRNA modifies binding to the target DNA.

5. The nucleic acid guided nuclease system according to any one of claims 1-3, wherein the modified spacer region of the gRNA modifies binding to a non-target or off-target DNA for optimization of specificity to the targeted genome.

6. The nucleic acid guided nuclease system according to any one of claims 1-4, wherein the modified spacer region of the gRNA decreases binding to the target DNA and non-target or off-target DNA.

7. The nucleic acid guided nuclease system according to claim 5, wherein the specificity to the target DNA comprises the ratio of on-target editing to the sum of all off target editing.

8. The nucleic acid guided nuclease system according to any one of claims 1-7, wherein the target DNA comprises genomic DNA.

9. The nucleic acid guided nuclease system according to any one of claims 1-8, wherein the modified gRNA comprises DSB-dependent and DSB-independent mechanisms of recombination with the homology repair template (HRT) to enable high editing efficiency (EE).

10. The nucleic acid guided nuclease system according to claim 1, wherein the modified spacer region comprises at least one point mutation.

11. The nucleic acid guided nuclease system according to any of the preceding claims, wherein the modified gRNA has decreased binding compared to a control gRNA binding the same target and has increased editing efficiency.

12. A method for modifying genome editing, the method comprising: contacting a target DNA molecule having a target sequence with a nuclease complex comprising:

(a) a Cas nuclease protein; and

(b) a DNA-targeting gRNA comprising: (i) a modified gRNA having a modified spacer sequence creating modified target recognition sequence hybridization compared to a positive control gRNA that hybridizes with the same target sequence, and (ii) a scaffold tracrRNA for guiding binding of the Cas nuclease to the target sequence wherein the complex forms a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the modified gRNA has increased editing efficiency and/or reduced off-targeting compared to a reference nuclease complex having the control gRNA.

13. The method according to claim 12, wherein the modification of the gRNA spacer sequence increases editing efficiency of the targeted gene replacement compared to the control gRNA.

14. The method according to claim 12, wherein the modification of the gRNA spacer sequence increases gene editing by enhancing gene recombination in the target DNA molecule having a defective gene.

15. The method according to claim 12, wherein the modification of the gRNA spacer sequence retains both DSB-dependent and DSB-independent mechanisms of recombination with the homology repair template.

16. The method according to claim 12, wherein the modified spacer of the gRNA comprises at least one mutation of a deletion, insertion, point mutation or substitution in the spacer.

17. The method according to any one of claims 12 to 16, wherein the target DNA molecule is genomic DNA in a mammalian organism.

18. The method according to any one of claims 12 to 17, wherein the modified spacer region of the gRNA modifies binding to a non-target or off-target DNA for optimization of specificity to the targeted genome.

19. The method according to claim 18, wherein the specificity to the target DNA comprises the ratio of on-target editing to the sum of all off target editing.

20. A kit comprising, the nucleic acid guided nuclease system according to any one of claims 1 to 11; and at least one container.

21. A multiplex mutational library comprising modified gRNAs wherein the spacer region of a target gRNA is mutated to include all possible nucleic acid substitutions for each position in the spacer region of the target gRNA to form a library of modified target gRNAs.

22. The multiplex mutational library according to claim 21, wherein the modified target gRNAs further comprise a nucleic acid guided nuclease.

23. The multiplex mutational library according to claim 2 lor 22, wherein the modified target gRNAs are further operably linked to a protospacer adjacent motif (PAM) sequence.

24. A nucleic acid-guided nuclease system comprising:

(a) a nucleic acid-guided nuclease; and

(b) a modified gRNA capable of improved editing efficiency of a target DNA, wherein the modified gRNA hybridizes with the target DNA to increase gene editing efficiency compared to a reference gRNA not having the modification and wherein the modified spacer region of the gRNA modifies binding to a non-target or off-target DNA for optimization of specificity to the targeted genome.

25. The system according to claim 24, wherein the specificity to the targeted genome comprises the ratio of on-target editing to the sum of all off target editing.

26. The system according to claim 24 or 25, wherein the engineered guide polynucleotide (gRNA) comprises a polynucleotide represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 20, SEQ ID NO: 32 to SEQ ID NO: 40, SEQ ID NO: 50 to SEQ ID NO: 60, SEQ ID NO: 72 to SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91 to SEQ ID NO:94.

27. The system according to any one of claims 24 to 26, further comprising an editing sequence having a change in sequence relative to the sequence of the targeted genomic region.

28. The system according to any one of claims 24 to 27, wherein the modified gRNA and the editing sequence are provided as a single nucleic acid.

29. The system according to any one of claims 24 to 28, wherein the target region is within a eukaryotic cell.

30. The system according to any one of claims 24 to 28, wherein the target region is within a bacterial cell.

31. The system according to any one of claims 24 to 28, wherein the target region is within a plant cell.

32. The system according to any one of claims 24 to 28, wherein the target region is within a mammalian cell.

33. The system according to any one of claims 24 to 28, wherein the target region is within a human cell.