Inducible crispr / CAS systems with high dynamic range and methods of use

Abstract

Systems, methods, and compositions used for the control of the timing of gene expression and activation involving sequence targeting, such as genome perturbation or gene-editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas and components thereof are provided. Inducible CRISPR-Cas systems having high dynamic range and reduced background or "leaky" expression in the off state are provided.

Description

Attorney Ref.59868.00075WO01 (GNE-0006-WO) INDUCIBLE CRISPR / CAS SYSTEMS WITH HIGH DYNAMIC RANGE AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 687,278 filed August 26, 2024, entitled “Inducible Crispr / CAS Systems With High Dynamic Range And Methods of Use,” and U.S. Provisional Patent Application No.63 / 737,446 filed December 20, 2024, entitled “Inducible Crispr / CAS Systems With High Dynamic Range And Methods of Use,” each of which are incorporated entirely by reference for all purposes. FIELD OF THE INVENTION

[0002] The present invention generally relates to systems, methods and compositions used for the control of the timing of gene expression and activation involving sequence targeting, such as genome perturbation or gene-editing, that use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas and components thereof. In particular, the present disclosure provides inducible CRISPR-Cas systems having high dynamic range and reduced background or “leaky” expression in the off state. BACKGROUND OF THE INVENTION

[0003] Gene expression is a dynamic process with precisely orchestrated temporal and spatial components that are necessary for normal development, homeostasis, and response to environmental perturbations. As such, the dysregulation of gene expression, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. Technologies capable of modulating gene expression in a spatiotemporally precise fashion have enabled the elucidation of the genetic cues responsible for normal biological processes and disease mechanisms.

[0004] For example, CRISPR / Cas is a powerful and versatile programmable genome editing tool that has ushered in a new era of biological discovery with applications in disease modeling, the study of gene regulation, as well as gene and cell therapies. Inducible molecular elements that can alter gene expression in response to external stimuli, such as a chemical or biochemical ligand, are known in the art, and inducible control of CRISPR / Cas activity using such systems enables precise timing of gene editing, offering several advantages over continuous Cas expression, including reduced off-target effects, target perturbation at specific developmental stages, and study of essential genes.

[0005] However, the utility of inducible expression systems is hampered by unwanted background or “leaky” expression in the OFF state. In the case of CRISPR / Cas systems, this leads to unwantedAttorney Ref.59868.00075WO01 (GNE-0006-WO) leaky expression and subsequent gene editing in the absence of induction. Although a number of alternative inducible strategies have been attempted, these approaches are limited by the tradeoff between leakiness and potency. As such, there is an evident need for efficient and precise spatial and temporal control over activity of CRISPR / Cas systems that not only provide inducible expression and potent activation of the CRISPR / Cas, but that also achieve a suitable reduction in unwanted leaky expression and / or a dampening of background activity from leaky expression, thereby providing a high dynamic range for the system. SUMMARY OF THE INVENTION

[0006] To address these issues, the systems and methods provided herein make use of molecular elements that involve conditionally destabilizing and inhibiting Cas proteins to significantly reduce the effects of leaky expression, without compromising maximum CRISPR activity upon induction. These “ultra-tight” inducible CRISPR / Cas systems demonstrate an up to 50-fold increase in dynamic range and maintain similar performance across many cell lines and target genes.

[0007] An aspect of the present disclosure is the provision of an inducible CRISPR-Cas system comprising a polynucleotide encoding a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element; and an anti-CRISPR protein.

[0008] In some embodiments, at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein.

[0009] In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. In some embodiments, the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the SF3B3-on switch is activated by branaplam (LMI070), and wherein activation induces expression of the encoded Cas protein.

[0010] In some embodiments, the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. In some embodiments, the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). In some embodiments, the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein.

[0011] In some embodiments, at least one inducible expression element comprises an inducible promoter element. In some embodiments, the inducible promoter element comprises a TET- responsive promoter element (TRE). In some embodiments, the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). In some embodiments, the inducible promoter element is induced by a tetracycline molecule.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0012] In some embodiments, the inducible promoter element is induced by a derivative of a tetracycline molecule. In some embodiments, the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. In some embodiments, the derivative of the tetracycline molecule is doxycycline.

[0013] In some embodiments, the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. In some embodiments, the anti-CRISPR protein comprises AcrIIA2. In some embodiments, the anti-CRISPR protein comprises AcrIIA4. In some embodiments, the anti- CRISPR protein comprises a fusion protein further comprising a degron tag. In some embodiments, the degron tag is activated by IKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. In some embodiments, the degron tag comprises an FK506 binding protein (FKBP). In some embodiments, the FKBP is a modified or mutant FKBP12. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). In some embodiments, FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the degron tag is activated by a ligand that targets a von Hippel-Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. In some embodiments, the ligand is dTag- V1. In some embodiments, the degron tag comprises a LID domain.

[0014] In some embodiments, the LID domain comprises a FKBP12(F36V) fused to a C-terminal cryptic degron sequence of SEQ ID NO: 44. In some embodiments, the LID domain comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the degron tag is activated by Shield1.

[0015] In some embodiments, the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a homolog or modified version of the Cas9 protein. In some embodiments, the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9. In some embodiments, the Cas protein is dCas9.

[0016] In some embodiments, the Cas protein further comprises a Cas fusion protein domain. In some embodiments, the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. In some embodiments, the KRAB domain is selected from: Zim3KRAB, KRAB-MeCP2, and Kox1KRAB. In some embodiments, the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0017] In some embodiments, the polynucleotide encodes one or more guide RNA molecules. In some embodiments, further comprising a separate polynucleotide encoding one or more guide RNA molecules. In some embodiments, one or more of the guide RNA molecules comprises an sgRNA molecule. In some embodiments, the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. In some embodiments, the constitutively expressed promoter comprises a U6 promoter. In some embodiments, comprising two or more sgRNA molecules. In some embodiments, each sgRNA molecule is driven by the same Pol III promoter. In some embodiments, each sgRNA molecule is driven by different Pol III promoter. In some embodiments, the different Pol III promoters are U6 and H1.

[0018] In some embodiments, In some embodiments, the polynucleotide encodes an sgRNA molecule library. In some embodiments, the sgRNA molecule library comprises a Pol III promotor. In some embodiments, the sgRNA molecule library comprises a plurality of Pol III promoters.

[0019] In some embodiments, at least one sgRNA molecules targets a gene of interest. In some embodiments, the polynucleotide further encodes a selectable marker.

[0020] In some embodiments, the anti-CRISPR is driven by a weak regulatory element. In some embodiments, the weak regulatory element comprises an IRES. In some embodiments, the weak regulatory element is selected from the group consisting of CMV-A, UbC, PGK, PGK100, and SV40.

[0021] In some embodiments, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by a single inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element. In some embodiments, the inducible expression element comprises the ligand inducible alternative splicing switch.

[0022] In some embodiments, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. In some embodiments, the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43), or LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the inducible expression element comprises the inducible promoter element. In some embodiments, the inducible promoterAttorney Ref.59868.00075WO01 (GNE-0006-WO) element comprises pTET (SEQ ID NO: 14). In some embodiments, the anti-CRISPR protein is a fusion protein comprising ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43) or LID (SEQ ID NO: 45). In some embodiments, the degron tag comprises LID (SEQ ID NO: 45). In some embodiments, the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising: (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element. In some embodiments, the inducible promoter element comprises pTET (SEQ ID NO: 14). In some embodiments, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. In some embodiments, the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43), or LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43).

[0023] In another aspect of the present disclosure, an inducible CRISPR-Cas system is provided comprising a polynucleotide encoding a destabilizing domain fused to a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element.

[0024] In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain. In some embodiments, the ligand controllable destabilizing domain is selected from the group consisting of: FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof.

[0025] In some embodiments, the ligand controllable destabilizing domain comprises a modified or mutant FKBP12. In some embodiments, the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*). In some embodiments, the modified or mutant FKBP12 comprises FKBPF36VL106P (SEQ ID NO: 40). In some embodiments, the ligand that controls the destabilizing domain is Shield1.

[0026] In some embodiments, the ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand that controls the destabilizing domain is trimethoprim (TMP).Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0027] In some embodiments, at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein.

[0028] In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. In some embodiments, the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the SF3B3-on switch is activated by branaplam (LMI070), and wherein activation induces expression of the encoded Cas protein.

[0029] In some embodiments, the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. In some embodiments, the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). In some embodiments, the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein.

[0030] In some embodiments, at least one inducible expression element comprises an inducible promoter element. In some embodiments, the inducible promoter element comprises a TET- responsive promoter element (TRE). In some embodiments, the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). In some embodiments, the inducible promoter element is induced by a tetracycline molecule.

[0031] In some embodiments, the inducible promoter element is induced by a derivative of a tetracycline molecule. In some embodiments, the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline.

[0032] In some embodiments, the derivative of the tetracycline molecule is doxycycline.

[0033] In some embodiments, the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. In some embodiments, the anti-CRISPR protein comprises AcrIIA4. In some embodiments, in the anti-CRISPR protein comprises a fusion protein further comprising a degron tag. In some embodiments, the degron tag is activated byIKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. In some embodiments, the degron tag comprises an FK506 binding protein (FKBP). In some embodiments, the FKBP is a modified or mutant FKBP12. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). In some embodiments, FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the degron tag is activated by a ligand that targets a von Hippel- Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. In some embodiments, the ligand comprises dTagV1. In some embodiments, the degron tag comprises a LID domain. In some embodiments, the LID domain comprises a C-terminus cryptic degron sequence. In some embodiments, the cryptic degron sequence comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the degron tag is activated by Shield1.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0034] In some embodiments, the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a homolog or modified version of the Cas9 protein. In some embodiments, the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9.

[0035] In some embodiments, the Cas protein is dCas9. In some embodiments, the Cas protein further comprises a Cas fusion protein domain. In some embodiments, the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. In some embodiments, the Cas fusion protein domain is selected from a ZIM3KRA, KRAB-MeCP2, and a Kox1KRAB domain. In some embodiments, the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB.

[0036] In some embodiments, the polynucleotide encodes one or more guide RNA molecules. In some embodiments, the system further comprises a separate polynucleotide encoding one or more guide RNA molecules. In some embodiments, one or more of the guide RNA molecules comprises an sgRNA molecule. In some embodiments, further comprising an sgRNA molecule library. In some embodiments, the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. In some embodiments, the constitutively expressed promoter comprises a U6 promoter.

[0037] In some embodiments, system comprises two or more sgRNA molecules. In some embodiments, each sgRNA molecule is driven by the same Pol III promoter. In some embodiments, each sgRNA molecule is driven by different Pol III promoter. In some embodiments, the different Pol III promoters are U6 and H1.

[0038] In some embodiments, the system further comprises an sgRNA molecule library. In some embodiments, the sgRNA molecule library comprises a Pol III promotor. In some embodiments, the sgRNA molecule library comprises a plurality of Pol III promoters.

[0039] In some embodiments, at least one sgRNA molecules targets a gene of interest. In some embodiments, the polynucleotide further encodes a selectable marker.

[0040] In some embodiments, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by a single inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element. In some embodiments, the inducible expression element comprises the ligand inducible alternative splicing switch. In some embodiments, the ligand inducible alternative splicing switch is selectedAttorney Ref.59868.00075WO01 (GNE-0006-WO) from an Xon splicing switch modulator or a SMN2 splicing switch modulator. In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain selected from FKBP12(F36V) (SEQ ID NO: 43), ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the inducible expression element comprises the inducible promoter element. In some embodiments, the inducible promoter element comprises pTET (SEQ ID NO: 14). In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain selected from FKBP12(F36V) (SEQ ID NO: 43), or ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand controllable destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element. In some embodiments, the wherein the inducible promoter element comprises pTET (SEQ ID NO: 14). In some embodiments, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain selected from FKBP12(F36V) (SEQ ID NO: 43), or ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42).Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0041] In another aspect of the present disclosure, an inducible CRISPR-Cas system is provided comprising a polynucleotide encoding: a destabilizing domain fused to a Cas protein, operably linked to at least one inducible expression element; and an anti-CRISPR.

[0042] In some embodiments, at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein. In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. In some embodiments, the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the SF3B3-on switch is activated by branaplam (LMI070), risdiplam, or RG7800, wherein activation induces expression of the encoded Cas protein.

[0043] In some embodiments, the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. In some embodiments, the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). In some embodiments, the SMN2-ON switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein.

[0044] In some embodiments, at least one inducible expression element comprises an inducible promoter element. In some embodiments, the inducible promoter element comprises a TET- responsive promoter element (TRE). In some embodiments, the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). In some embodiments, the inducible promoter element is induced by a tetracycline molecule. In some embodiments, the inducible promoter element is induced by a derivative of a tetracycline molecule. The In some embodiments, the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. In some embodiments, the derivative of the tetracycline molecule is doxycycline.

[0045] In some embodiments, the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. In some embodiments, the anti-CRISPR protein comprises AcrIIA4. In some embodiments, the anti-CRISPR protein comprises a fusion protein further comprising a degron tag. In some embodiments, the degron tag is activated by IKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. In some embodiments, the degron tag comprises an FK506 binding protein (FKBP). In some embodiments, the FKBP is a modified or mutant FKBP12. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). In some embodiments, FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the degron tag is activated by a ligand that targets a von Hippel-Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. T In some embodiments, the ligand comprises dTagV1. In some embodiments, the degron tag comprises a LID domain. In some embodiments, the LID domain comprises a C-terminal cryptic degron sequence. In someAttorney Ref.59868.00075WO01 (GNE-0006-WO) embodiments, the cryptic degron sequence comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the degron tag is activated by Shield1.

[0046] In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain. In some embodiments, the ligand controllable destabilizing domain is selected from the group consisting of: FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof.

[0047] In some embodiments, the ligand controllable destabilizing domain comprises a modified or mutant FKBP12. In some embodiments, the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*). In some embodiments, the modified or mutant FKBP12 comprises FKBP12(F36V) (SEQ ID NO: 43). In some embodiments, the ligand that controls the destabilizing domain is Shield1. In some embodiments, ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand that controls the destabilizing domain is trimethoprim (TMP).

[0048] In some embodiments, the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a homolog or modified version of the Cas9 protein. In some embodiments, the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9. In some embodiments, the Cas protein is dCas9. In some embodiments, the Cas protein further comprises a Cas fusion protein domain. In some embodiments, the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. In some embodiments, the KRAB domain is selected from: Zim3KRAB, KRAB-MeCP2, and Kox1KRAB. In some embodiments, the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. In some embodiments, the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB.

[0049] In some embodiments, the polynucleotide encodes one or more guide RNA molecules. In some embodiments, further comprising a separate polynucleotide encoding one or more guide RNA molecules. In some embodiments, one or more of the guide RNA molecules comprises an sgRNA molecule. In some embodiments, the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. In some embodiments, the constitutively expressed promoter comprises a U6 promoter. In some embodiments, comprising two or more sgRNA molecules. In some embodiments, each sgRNA molecule is driven by the same Pol III promoter.Attorney Ref.59868.00075WO01 (GNE-0006-WO) In some embodiments, each sgRNA molecule is driven by different Pol III promoter. In some embodiments, wherein the different Pol III promoters are U6 and H1.

[0050] In some embodiments, the system further comprises an sgRNA molecule library. In some embodiments, the sgRNA molecule library comprises a Pol III promotor. In some embodiments, the sgRNA molecule library comprises a plurality of Pol III promoters. In some embodiments, at one or more sgRNA molecules targets a gene of interest. In some embodiments, the polynucleotide further encodes a selectable marker.

[0051] In some embodiments, the anti-CRISPR is driven by a weak regulatory element. In some embodiments, the weak regulatory element comprises an IRES. In some embodiments, the weak regulatory element is selected from the group consisting of CMV-A, UbC, PGK, PGK100, and SV40.

[0052] In some embodiments, the anti-CRISPR is a fusion protein comprising ACRIIA4 and a ligand inducible domain (LID), wherein the LID is activated by Shield1; the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40), and the ligand that controls the destabilizing domain is Shield1; the Cas protein is a Cas9 protein; the inducible promoter element is pTET; and the one or more guide RNA molecules comprises an sgRNA molecule library. In some embodiments, the anti-CRISPR fusion protein comprises the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. In some embodiments, upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to a target sequence and, optionally, edits the genomic locus to alter gene expression. In some embodiments, a first sgRNA molecule comprising a first guide sequence capable of hybridizing to a first target sequence in a genomic locus of interest in the cell; a second sgRNA molecule comprising a second guide sequence capable of hybridizing to a second target sequence in the genomic locus of interest in the cell; and wherein upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to the first and second target sequences and edits the genomic locus of the cell by removing a DNA segment located between the first and second target sequences from the genome of the cell.

[0053] In some embodiments, the dynamic range of the system is enhanced upon introduction of one or more of: a.) a ligand that controls the destabilizing domain; b.) an agent that activates the inducible degradation domain; and c.) an agent that activates the ligand inducible alternative splicing switch.

[0054] In another aspect of the provided disclosure, an isolated, non-naturally occurring polynucleotide is provided encoding: a Cas protein optionally fused to a destabilizing domain and operably linked to an inducible promoter element; one or more sgRNA molecules comprising one or more guide sequences capable of hybridizing to a target sequence in a genomic locus of interest in a cell; and optionally an anti-CRISPR.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0055] In some embodiments, the destabilizing domain comprises a ligand controllable destabilizing domain. In some embodiments, the ligand controllable destabilizing domain comprises a FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof. In some embodiments, ligand controllable destabilizing domain comprises a modified or mutant cytosolic signaling protein FKBP12.

[0056] In some embodiments, the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. In some embodiments, the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*). In some embodiments, the modified or mutant FKBP12 comprises FKBPF36VL106P (SEQ ID NO: 40). In some embodiments, the ligand that controls the destabilizing domain comprises Shield1.

[0057] In some embodiments, ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). In some embodiments, the ligand that controls the destabilizing domain comprises TMP.

[0058] In some embodiments, the Cas protein is selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. In some embodiments, the Cas protein is a homolog or modified versions of the Cas protein. In some embodiments, the homolog or modified version of the Cas protein is nCas9 or dCas9. In some embodiments, the Cas protein is Cas9.

[0059] In some embodiments, the inducible promoter element comprises a TET-responsive promoter element (TRE). In some embodiments, the TRE comprises pTET. In some embodiments, the inducible promoter element is induced by a tetracycline molecule. In some embodiments, the inducible promoter element is induced by a derivative of the tetracycline molecule. In some embodiments, the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. In some embodiments, the inducible promoter element is induced by doxycycline.

[0060] In some embodiments, one or more of the guide RNA molecules comprises an sgRNA molecule. In some embodiments, the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. In some embodiments, the constitutively expressed promoter comprises a U6 promoter. In some embodiments, comprising two or more sgRNA molecules. In some embodiments, each sgRNA molecule is driven by different Pol III promoter. In some embodiments, the different Pol III promoters are U6 and H1.

[0061] In some embodiments, further comprising an sgRNA molecule library. In some embodiments, the sgRNA molecule library comprises a Pol III promotor. In some embodiments, the sgRNAAttorney Ref.59868.00075WO01 (GNE-0006-WO) molecule library comprises a plurality of Pol III promoters. In some embodiments, at least one of the one or more sgRNA molecules targets a gene of interest.

[0062] In some embodiments, the polynucleotide further encodes a selectable marker. In some embodiments, further comprising a ligand inducible alternative splicing switch upstream from the encoded Cas protein.

[0063] In some embodiments, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. In some embodiments, activation of the Xon splicing switch modulator by branaplam (LMI070) activates expression of the encoded Cas protein. In some embodiments, the ligand inducible alternative splicing switch comprises SF3B3.

[0064] In some embodiments, the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. In some embodiments, the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). In some embodiments, the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein.

[0065] In some embodiments, the anti-CRISPR is a fusion protein comprising ACRIIA4 and a ligand inducible domain (LID), wherein the LID is induced by Shield1; the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40); and the ligand that controls the destabilizing domain is Shield1; the Cas protein is a Cas9 protein; the inducible promoter element comprises pTET; and the one or more guide RNA molecules comprises an sgRNA molecule library. In another aspect of the present disclosure, a vector is provided comprising the embodied polynucleotide of present disclosure, for delivery of an inducible CRISPR-Cas system to a cell.

[0066] In another aspect of the present disclosure, a cell is provided comprising the any one of the embodied inducible CRISPR-Cas systems provided herein. In some embodiments, the cell is an eukaryotic host cell.

[0067] In another aspect of the present disclosure, a cell line is provided comprising a plurality of clonal cells stably transfected with the any one of the embodied vectors provided herein.

[0068] In another aspect of the present disclosure, a transgenic organism is provided stably transfected with any one of the embodied vectors of the present disclosure or expressing any one of the embodied inducible CRISPR-Cas system as provided herein.

[0069] In another aspect of the present disclosure, a model organism is provided that constitutively expresses any one of the embodied inducible CRISPR-Cas systems as provided herein.

[0070] In another aspect of the present disclosure, a method of modifying a target nucleic acid in a cell is provided, the method comprising: introducing into the cell the inducible CRISPR-Cas system according to any one the provided embodiments; contacting the cell with a ligand that activates the inducible expression element; and contacting the cell with a ligand that deactivatesAttorney Ref.59868.00075WO01 (GNE-0006-WO) the anti-CRISPR and / or the destabilizing domain, thereby causing the Cas protein to modify the target nucleic acid.

[0071] In some embodiments, the inducible CRISPR-Cas system comprises both an anti-CRISPR and a destabilizing domain, and wherein the method comprises contacting the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain.

[0072] In some embodiments, modifying the target nucleic acid in the cell alters expression of a target gene in the cell. In some embodiments, altering expression of the target gene in the cell comprises decreasing expression of the target gene. In some embodiments, altering expression of the target gene in the cell comprises increasing expression of the target gene. In some embodiments, modifying the target nucleic acid in the cell comprises cutting the target nucleic acid at two different locations. In some embodiments, cutting the target nucleic acid at the two different locations removes a target gene or gene segment from the cell. In some embodiments, modifying the target nucleic acid in the cell further comprises editing the target nucleic acid.

[0073] In another aspect of the present disclosure, a method of increasing a dynamic range of an inducible expression system in a cell is provided, the method comprising: introducing into the cell the inducible CRISPR-Cas system according to any one of the provided embodiments; culturing the cell under a first set of conditions in which the inducible CRISPR-Cas system exhibits a first expression level; contacting the cell with a ligand that activates the inducible expression element and a ligand that deactivates the anti-CRISPR and / or the destabilizing domain to cause the inducible CRISPR-Cas system to exhibit a second expression level that is higher than the first expression level, thereby increasing the dynamic range of the inducible expression system.

[0074] In some embodiments, the inducible CRISPR-Cas system comprises both an anti-CRISPR and a destabilizing domain, and wherein the method comprises contacting the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain.

[0075] In some embodiments, inducible Cas9 formats across cell lines, including induced pluripotent stem cell (iPSC) lines, are provided, where targets show consistently high efficacy, low background, and superior overall performance with pTET:Ultra-tight.

[0076] These and further aspects will be further explained in the rest of the disclosure, including the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIGS.1A-1D illustrate the limitations of the TET-inducible Cas9 system across cell lines. FIG.1A shows a schematic representation of the pTET PiggyBac transposon plasmid (pB), and workflow. FIG.1B illustrates the treatment of selected stable lines with Cas9 inducing drug(s) or left untreated as a control, followed by flow cytometry analysis to measure CD81 levels as an indication of knockout efficiency. FIG.1C shows the percentage CD81 knockout in the untreatedAttorney Ref.59868.00075WO01 (GNE-0006-WO) (gray bars) and treated (striped bars) cell populations. FIG.1D are flow cytometry plots of HEK293T pTET and pTET-NTC stable cells with and without Dox treatment.

[0078] FIGS.2A-2K illustrate the features of an “ultra-tight” pTET system that exhibits minimal basal editing and has a high knockout efficiency across cell lines. FIG.2A is a schematic representation of the control modules added to the basic pTET system that significantly reduced uninduced Cas9 activity. FIG.2B is a cartoon depicting conditional degradation of DD or DHFR fused Cas9 in untreated cells. FIG.2C is a cartoon illustrating Cas9 inhibition with AcrIIA4 anti- CRISPR protein. FIG.2D is a schematic illustrating a conditional splicing switch mechanism where alternative splicing is activated in the presence of the drug. FIGS.2E-2G illustrate the CD81 knockout efficiency in the untreated (gray bars) and treated (striped bars) cell populations and the dynamic range of various pTET modified PiggyBac constructs that were stably transfected into 293T (FIG.2E) and A549 (FIG.2F) cells and K562 (FIG.2G). FIG.2H is a comparison of the knockout efficiency across cell lines for the pTET and pTET-Ultra-tight (pTET-DD-Cas9-AcrIIA4-LID) constructs (untreated gray, treated striped). FIG.2I illustrates the significantly enhanced dynamic range for pTET-Ultra-tight in each cell line. FIG.2J illustrates the comparison in SW480 cells of pTET:Ultra-tight to an expanded set of genetic circuits (pTET- NTC; pTET; pTET-DHFR-Cas9; pTET-DD-Cas9; pTET-AcrIIA4; pTET-AcrIIA4-FKBP; pTET- AcrIIA4-LID). FIG.2K is a box plot displaying the dynamic range of Cas9 activity revealing that pTET-Ultra-tight remained the superior option for balancing on versus off-state editing in the SW480 cell line.

[0079] FIG.3A-3F illustrate the low background editing and high dynamic range provided by the “Ultra-tight” pTET constructs expressing sgRNA targeting B2M (FIGS.3A-3B), CD298 (FIGS. 3C-3D), or CD9 (FIGS.3E-3F) stably integrated into HEK293T cells. FIGS.3A, 3C, and 3E show the percentage of target protein negative cells as measured by flow cytometry. FIGS.3B, 3D, and 3F show box plots displaying the dynamic range of Cas9 activity for each system at each target loci.

[0080] FIGS.4A-4I illustrate the development of Dox independent inducible Cas9 system having low background editing in the absence of induction and high dynamic range. FIG.4A is a schematic diagram of engineered all-in-one piggyBac cassettes, where Cas9 expression is driven by a CMV constitutive promoter and activity is controlled by DD, DHFR, or Xon elements. FIG. 4B illustrates the CD81 knockout percentage in HEK293T cells for the various constructs. FIG. 4C illustrates a schematic diagram of an Xon based ultra-tight inducible Cas9 system. FIG.4D illustrates the unexpectedly high leaky cutting of CD81 in the HEK293T cells of the Xon based cassettes in combination with DD or DHFR elements fused to the N-terminus of Cas9. FIG.4E illustrates where an internal start codon within the DD or DHFR domain would cause the bypass of the splicing switch element. FIG.4F illustrates CD81 knockout efficiency in the HEK293T cells with the various versions of Xon based inducible Cas9 system where the DD or DHFRAttorney Ref.59868.00075WO01 (GNE-0006-WO) elements are fused to the C-terminus of Cas9. FIG.4G provides box plots showing the dynamic range for each of the identified Xon based inducible Cas9 systems in K562 cells. FIG.4H illustrates the CD81 knockout efficiency in the untreated and treated K562 cells with the different versions of Xon based inducible Cas9. FIG.4I provides box plots display the dynamic range for each of the identified Xon based inducible Cas9 systems in K562 cells. FIG.4J is a schematic diagram of the promoter variants engineered for the anti-CRISPR module in the Xon system.

[0081] FIGS.5A-5F illustrate a pTET Ultra-tight system that exhibits tight control over essential gene knockout and deletion of a ~2.9kb genomic region. FIG.5A is a schematic overview of the cell growth assay workflow. FIG.5B (sgRNA1) and FIG.5D (sgRNA2) illustrate the growth curves shown for each line in the presence and absence of drug treatment. FIGS.5C (sgRNA1) and 5E (sgRNA2) are box plots that display dynamic range as fold change in % confluence for each line. FIG.5F shows that the pTET-Ultra-tight system can be combined with dual gRNAs to inducibly delete a ~2.5kb genomic locus by introducing dual sgRNA into the pTET and pTET- Ultra-tight constructs to eliminate exons 2 and 3 within the H3-3A gene.

[0082] FIGS.6A – 6D illustrate an ultra-tight inducible CRISPRi system. FIG.6A illustrates the workflow for examining the inducible CRISPRi system in DLD1 cells. FIG.6B, on the left provides the CD81 silencing showed in percentage of cells and on the right CRISPRi dynamic range shown as the ratio of CD81 silencing in induced vs uninduced cells. FIG.6C are representative flow cytometry results of the CD81 stained cells with inducible CRISPRi systems. FIG.6D illustrates silencing of PLK1 gene in DLD1 cell line by the pTET inducible CRISPRi system and the Ultra-tight inducible CRISPRi system. On the left, workflow examining PLK1 silencing is displayed. On the right, PLK1 silencing is shown as normalized cell death.

[0083] FIG.7 illustrates a schematic diagram of an exemplary promoter hijacking strategy for CRISPR knock-in for stable integration of a splice switch based inducible Cas9 into a genome.

[0084] FIG. 8 illustrates representative flow cytometry results of HEK293T wild-type and CD81 knockout cells stained with APC-conjugated CD81 antibody or an IgG isotype control antibody, with unstained samples included as negative controls. The knockout line was generated by stably integrating a plasmid in which Cas9 and CD81 sgRNA are constitutively expressed from the CAG and U6 promoters, respectively (schematic shown).

[0085] FIGS. 9A-9C illustrate results of western immunoblotting and CD81 knockout by flow cytometry, and targeted amplicon sequencing in the constitutive pCAG-Cas9 line, respectively, for evaluation of Cas9 expression levels upon module induction and constitutive Cas9 disruption of CD81.

[0086] FIGS. 10A-10B illustrate data from pTET pTET:Ultra-tight (pTET-DD-Cas9;AcrIIA4-LID) HEK293T cells were either left untreated or drug-treated for the indicated times followed by measurement of CD81 knockout by flow cytometry, and targeted amplicon sequencing, respectively.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0087] FIGS. 11A-11C illustrate data assessing pTET:Ultra-tight performance in transformed cell lines and assessment of basal iPSC phenotypes engineered with pTET or pTET:Ultra-tight.

[0088] FIG.11D illustrates data assessing indel generation at the B2M locus in stable HEK293T cells before and after induction Cas9 from pTET vs. pTET:Ultra-tight.

[0089] FIGS. 12A-12B illustrate a time-course analyses of CD81 knockout in Xon-Cas9 vs. Xon- Cas9;AcrIIA4-FKBP HEK293T cells, that were left untreated or treated with either 20nM Branaplam (Xon-Cas9) or 20nM Branaplam + 1μM dTagV-1 (Xon-Cas9;AcrIIA4-FKBP). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0091] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0092] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0093] All references cited throughout the present disclosure, including patent applications and publications, are incorporated by reference herein in their entirety.Attorney Ref.59868.00075WO01 (GNE-0006-WO) Definitions

[0094] As used herein, the term “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)” refers to sequences based on regularly spaced clustered short repeat palindromic sequences that, unlike RNA interference, control gene expression at the transcriptional level.

[0095] In general, “CRISPR system” refers collectively to elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas protein, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system, and / or other sequences and transcripts from a CRISPR locus.

[0096] CRISPR / Cas systems or CRISPR-Cas systems can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., can be derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. CRISPR systems in accordance with embodiments of the invention are generally characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence”. In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.

[0097] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into aAttorney Ref.59868.00075WO01 (GNE-0006-WO) host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide RNA sequences. In some embodiments, the CRISPR enzyme and guide RNA sequence are operably linked to and expressed from the same promoter.

[0098] The term “CRISPR-associated protein” or “Cas protein” refers to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The term “Cas mutant” or “Cas variant” refers to a wild-type Cas protein, e.g., a protein or polypeptide derivative of a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. In certain embodiments, a “Cas mutant” or “Cas variant” substantially retains the nuclease activity of the Cas protein. In certain embodiments, a “Cas mutant” or “Cas variant” is mutated such that one or both nuclease domains are inactive. In certain embodiments, a “Cas mutant” or “Cas mutant” has nuclease activity. In certain embodiments, a “Cas mutant” or “Cas variant” lacks some or all of the nuclease activity of its wild-type counterpart.

[0099] A “Cas9” molecule or protein, as used herein, refers to a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A “Cas9 polypeptide” is a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site comprising a target domain and, in certain embodiments, a PAM sequence. Cas9 molecules include both naturally occurring Cas9 molecules and Cas9 molecules and engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule. The terms “altered”, “engineered” or “modified”, as used in this context, refer merely to a difference from a reference or naturally occurring sequence, and impose no specific process or origin limitations. A Cas9 molecule may be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or an enzymatically inactive (or dead) Cas9 molecule.

[0100] The term “fusion protein”, as used herein, refers to a single protein created by joining two or more proteins, protein domains, or protein fragments that are not naturally found together in aAttorney Ref.59868.00075WO01 (GNE-0006-WO) single protein. For example, a fusion protein may contain a first domain from one Cas protein and a second domain from a different Cas protein or may contain a first domain from a degradation protein and a second domain from a Cas protein. Modification to include such domains in fusion proteins may confer additional activity on the modified polypeptides. Such activities may include nuclease activity, methyltransferase activity, demethylase activity, DNA or RNA repair activity, DNA or RNA damage activity, deamination activity, dismutase activity, alkylation activity, scavenging activity, oxidation, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, desSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, or any combination thereof, that modifies a polypeptide associated with the target sequence nucleic acid. A fusion protein may also comprise epitope tags (e.g., histidine tags, FLAG® tags (Sigma Aldrich, St. Louis, MO), Myc tags), reporter protein sequences (e.g., glutathione-S-transferase, beta-galactosidase, luciferase, green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein) and / or nucleic acid binding domains (e.g., a DNA binding domain, an RNA binding domain). A fusion protein may also comprise activator domains (e.g., heat shock transcription factors, NFKB activators) or repressor domains (e.g., a KRAB domain). As described by Lupo, A., et al., Current Genomics 14(4): 268-278 (2013), the KRAB domain is a potent transcriptional repression module, and is located at the amino-terminal sequence of most the C2H2 zinc finger proteins (see, e.g., Margolin, J., et al., Proceedings of the National Academy of Sciences of the United States of America 91:4509-4513 (1994); Witzgall, R., et al. al., Proceedings of the National Academy of Sciences of the United States of America 91:4514-4518 (1994)). The KRAB domain generally binds corepressor proteins and / or transcription factors through protein-protein interactions, resulting in transcriptional repression of genes to which KRAB zinc finger proteins (KRAB-ZFPs) bind (see, e.g., Friedman JR, et al., Genes & Development 10:2067-2678 (1996)). In some embodiments, linker nucleic acid sequences are used to join the two or more proteins, protein domains, or protein fragments.

[0101] As used herein, the term “gRNA molecule” or “gRNA” refers to a guide RNA which is capable of targeting a Cas molecule to a target nucleic acid. In one embodiment, the term “gRNA molecule” refers to a guide ribonucleic acid. In another embodiment, the term “gRNA molecule” refers to a nucleic acid encoding a gRNA. In one embodiment, a gRNA molecule is non-naturally occurring. In one embodiment, a gRNA molecule is a synthetic gRNA molecule.

[0102] As used herein, the term “single guide RNA” or “sgRNA” refers to a single RNA molecule that combines a custom-designed short crRNA sequence with a scaffold tracrRNA sequence, as frequently used in connection with CRISPR-Cas systems.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0103] The term “polynucleotide” refers to a polymeric form of two or more nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides according to the present disclosure may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, selection markers, regulatory elements, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0104] The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0105] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to meanAttorney Ref.59868.00075WO01 (GNE-0006-WO) that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0106] The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more Pol III promoters (e.g., 1, 2, 3, 4, 5, or more Pol III promoters). Examples of Pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of other promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the CMV-A (truncation of CMV promoter), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the PGK100 promoter (the first 100bp of PGK promoter), the EF1α promoter, and the UbC promoter.

[0107] Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol.8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol.78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, RNAs, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, guide RNAs. sgRNAs, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). Advantageous vectors include lentiviruses and adeno- associated viruses, and types of such vectors can also be selected for targeting particular types of cells.

[0108] The term “inducible expression element” as used herein refers to a system or a set of components that act in a coordinated manner to affect a change to an expression cassette in response to an external stimulus, such as the introduction of a chemical or biochemical ligand,Attorney Ref.59868.00075WO01 (GNE-0006-WO) i.e., an “inducer” or “an activator”. In one aspect, an inducible expression element comprises an inducible promoter element having a regulatory element that can interact with an external stimulus, such as a chemical or biochemical ligand, to induce expression of the downstream elements in the expression cassette. In one aspect, an inducible expression element comprises a splicing switch modulator that splices transcript elements in a first manner in the absence of an external stimulus, such as a chemical or biochemical ligand, and splices transcript elements in a second manner in the presence of the external stimulus. In one non-limiting example, a splicing modulator splices out a transcription start codon in the absence of the external stimulus, but when the external stimulus is applied to the system, the splicing modulator splices transcript elements so as to include the start codon, thereby facilitating functional translation of the transcript into a protein product when the external stimulus is present.

[0109] In general, an inducible expression element may be “off” when the inducer or activator ligand is not present but can be switched on when the system is contacted with the inducer. The manner by which this happens is dependent on the control mechanisms as well as differences in cell type. The term “inducible” as used herein may encompass all aspects of a switch irrespective of the molecular mechanism involved. Accordingly, an “inducible expression element” as provided by the disclosure herein may include, but is not limited to, antibiotic based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In preferred embodiments, an “inducible expression element” may be a tetracycline (Tet) / DOX inducible system, an Xon splicing switch modulator, or an SMN2 splicing switch modulator. These and other elements are described further herein.

[0110] An “anti-CRISPR” as used herein refers to a protein that inhibits the normal activity of a CRISPR-Cas system through one or more mechanisms, such as, e.g., crRNA loading interference, DNA binding blockage, DNA cleavage prevention, or any combination thereof. See, e.g., Nakamura et al. Nat Comm.2019 Jan 12; 10(194); Arake de Tacca et al. bioRxiv.2023 Mar 29; and Meacham et al. PLOS Biol.2023 Dec 8; 21(12), each of which is incorporated by reference.

[0111] A “degron”, “degron domain,” or “degron tag” as used herein refer to a class of protein tag which can be added to either the N or C terminal of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing domains which will be identified by various enzymes and sent to the proteasome for degradation. The tags discussed here are all conditional, meaning that they only facilitate degradation of their fused protein under certain conditions (typically the addition of an exogenous small molecule). Degron tags can range in size from several kDas up to ~35 kDa and are typically knocked-in via gene targeting or cloned in- frame to the protein of interest for plasmid-based expression systems. In some embodiments, the degron is FKBP12(F36V) abbreviated as “dTAG” herein. The FKBP12(F36V) degron is activated by small molecules that to bind VHL and that target CRBN complex. In some embodiments, the degron is a LID domain comprised FKBP and a 19-amino-acid cryptic degron sequence (e.g.,Attorney Ref.59868.00075WO01 (GNE-0006-WO) fused to the C terminus of FKBP). In some embodiments, the degron tag comprises a cryptic degron sequence TRGVEEVAEGVVLLRRRGN (SEQ ID NO: 44). In some embodiments, the degron domain is as described in any one of the following references, each of which are incorporated herein in their entirety: Nabet et al. Nat Chem Biol.2018 Mar 26;14(5); Nabet et al. Nat Comm.2020 Sept 18;11(1); Koduri et al. PNAS.2019 Jan 25;116(7); Buckley et al. ACS Chem Biol.2015 Jun 23;10(8); Chung et al. Nat Chem Biol.2015 Jul 27;11(9); Mercer et al. Science.2024 Mar 15;383(6688); Nowak et al. J Med Chem.2021 Aug 12;64(15); Bond et al. J Med Chem.2021 Oct 15;64(20); Bonger et al. ACS Chem Biol.2014 Jan 17;9(1); and Bonger et al. Nat Chem Biol.2011 July 3;7.

[0112] A “destabilizing domain” as used herein refers to a protein domain that is inherently unstable under default conditions, and when fused to a protein of interest, can result in rapid degradation of the fusion protein. But when contacted with a ligand, the destabilizing domain can be stabilized, abating the degradation of the fusion protein. See, e.g., Chu et al. Bioorg Med Chem Lett.2008 Nov 15;18(22), which is incorporated by reference, herein. In embodiments, herein, the destabilizing domain is encoded to be, or is fused to a Cas9 protein.

[0113] A “marker”, selection marker” or a “selectable marker” is a protein expressed by the system provided herein that allows for the isolation or continued culture of transfected cells expressing the marker from the majority of cells in the population. Such markers include, but are not limited to, Puro, G418r, Blastr, Hygror, and others known in the art.

[0114] By “comprising” it is meant that the recited elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the scope of the claim.

[0115] By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.

[0116] By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. Detailed Description Inducible CRISPR-Cas systems

[0117] The present disclosure provides inducible CRISPR-Cas systems comprising a polynucleotide encoding a Cas protein, configured to bind a guide RNA and operably linked to at least one inducible expression element, wherein the system further comprises at least one of: 1) a destabilizing domain fused to a Cas protein; and 2) an anti-CRISPR protein, as described in detail herein.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0118] The provided inducible CRISPR-Cas systems control CRISPR-Cas activity and enable precise timing of gene editing, offering several advantages over continuous Cas expression, including reduced off-target effects and enhanced target perturbation at specific developmental stages. The present disclosure provides inducible CRISPR-Cas systems that are characterized by a high dynamic range and reduced uninduced Cas activity (i.e., low leakiness).

[0119] Inducible control of CRISPR-Cas activity enables precise timing of gene editing, offering several advantages over continuous Cas expression, including reduced off-target effects, target perturbation at specific developmental stages, and study of essential genes. However, utility of the most commonly used inducible system, Tet-ON, is hampered by “leaky” Cas expression leading to gene knockout in the absence of induction. Although a number of alternative inducible strategies have been reported, these approaches are limited by the tradeoff between leakiness and inducibility (low transcription efficiency in the induced state) or are only confirmed viable in a limited number of cell types.

[0120] In the present disclosure, various strategies to reduce the activity of the Tet-inducible CRISPR system in the OFF state are employed. By combining methods to conditionally destabilize and inhibit Cas, the present disclosure establishes for the first time that Cas activity in the uninduced state is significantly reduced without compromising maximum CRISPR activity upon induction. These “ultra-tight” CRISPR-Cas systems demonstrate an up to 50-fold increase in dynamic range and maintain similar performance across many cell lines and target genes. Tet- independent “ultra-tight” inducible CRISPR-Cas systems that can be multiplexed with Tet-ON for orthogonal inducible control are also provided in the present disclosure.

[0121] Aspects of the disclosure relate to inducible CRISPR-Cas systems that enable precise timing of gene editing. Such CRISPR-Cas systems suitable for use with the provided disclosure are well known in the art. For instance, relevant examples are described in Pacesa et al. Cell.2024 Feb 29;187(5), the disclosure of which is incorporated by reference herein for all purposes. CRISPR- Cas systems in accordance with embodiments of the disclosure can induce double stranded breaks (DSBs) or single stranded breaks at the target site, followed by disruptions, as discussed herein. In some embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5′ overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor (e.g., KRAB) or activator, to affect gene expression.

[0122] Cas proteins contemplated for use with the provided disclosure include, but are not limited to, Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6,Attorney Ref.59868.00075WO01 (GNE-0006-WO) CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, Csf4, Cas12a, Cas12a2, Cas12g, Cas13, spCas9, nCas9, dCas9 and include functional variants and derivatives thereof.

[0123] Cas fusion proteins are also contemplated for use with the present disclosure. In particular, suitable Cas fusion proteins include, but are not limited to, those that are base editors (fusion to cytosine / adenosine deaminases), prime editors (fusion to reverse transcriptases and DNA polymerases), transcriptional effectors (CRISPRa or CRISPRi, for example, those which utilize activating domains such as VP64 and VPR), and epigenome editors (fusions to DNA methyltransferases).

[0124] In some embodiments, a Cas fusion protein comprises a Krüppel associated box (KRAB) domain including, but not limited to, those described in Yeo et al. Nat Meth.2018 July 16;15; Alerasool et al. Nat Meth.2020 Oct 5;17; and Tycko J et al. Cell.2020 Dec 23;183(7):2020- 2035.e16 each of which are incorporated by reference herein for all purposes. Other KRAB domains are described in Tycko J. et al., High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell.2020 Dec 23;183(7):2020-2035.e16. doi: 10.1016 / j.cell.2020.11.024. Epub 2020 Dec 15. PMID: 33326746; PMCID: PMC8178797, which is incorporated by reference herein for all purposes. In some embodiments, the KRAB domain is selected from Zim3KRAB, Kox1KRAB, and KRAB-MeCP2 domains. In some embodiments, the dCas9-Zim3KRAB comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the dCas9-Kox1KRAB fusion domain comprises the amino acid sequence of SEQ ID NO: 50.

[0125] In embodiments of the present disclosure, inducible CRISPR-Cas systems as provided herein comprise one or more guide RNA molecules that are suitable for directing the editing of DNA or RNA. In some embodiments, the guide RNA comprises single guide RNA (sgRNA). In some embodiments, the guide RNA is located on the same vector as the encoded Cas protein. In some embodiments, the guide RNA is located on a different vector from the vector encoding the Cas protein. In some embodiments, there are two sgRNAs, which are located on the same or a different vector from the vector encoding Cas protein. In some embodiments, there are two or more sgRNAs located on the vector encoding Cas protein. In some embodiments, there are two or more sgRNAs, wherein one or more sgRNA is located on a different vector from the vector encoding Cas protein.

[0126] In some embodiments, the guide RNA comprises a guide RNA library, (e.g., an sgRNA library) comprising a plurality of guide RNA molecules, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more guide RNA molecules, including, but not limited, to a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequence for genome-wide knockout of human and mouse genes, and CRISPR Synergistic Activating Mediator (SAM) library for transcriptional activation of every gene in the human and mouse genome.Attorney Ref.59868.00075WO01 (GNE-0006-WO) Inducible Expression Elements

[0127] Aspects of the disclosure include inducible expression elements that conditionally facilitate expression of one or more components of an expression cassette or vector in response to the presence of an inducer. Inducible expression elements include inducible promoter elements as well as inducible splicing switch modulators.

[0128] Inducible promoter elements generally contain a regulatory element that can interact with an external stimulus, such as a chemical or biochemical ligand, to induce expression of the downstream elements in the expression cassette. Splicing switch modulators (also referred to herein as “ligand inducible alternative splicing switches”) generally function by splicing transcript elements in a first manner in the absence of an external stimulus, such as a chemical or biochemical ligand, and splicing transcript elements in a second manner in the presence of the external stimulus. In one non-limiting example, a splicing modulator splices out a transcription start codon in the absence of the external stimulus, but when the external stimulus is applied to the system, the splicing modulator splices transcript elements so as to include the start codon, thereby facilitating functional translation of the transcript into a protein product when the external stimulus is present. In general, an inducible expression element may be “off” when the inducer is not present but is switched “on” when the system is contacted with the inducer. The manner by which this happens is dependent on the control mechanisms as well as differences in the host cell type.

[0129] One non-limiting example on an inducible promoter element is the Tet-On system, which regulates expression of a target gene that is under transcriptional control of a tetracycline- responsive promoter element (TRE, e.g., TRE3G). The TRE is an artificial promoter responsive to rtTA. It consists of 7 serial tet operons (tetO7) and a strong minimal CMV promoter (mCMV), which itself is not active and only recruits the transcriptional machinery upon binding of rtTA to the seven tet operons. Doxycycline, a tetracycline derivative, activates binding of the mutant TetR to the TRE, leading to expression of the transgene.

[0130] In this system, the transcriptional activator protein is a tetracycline-responsive transcriptional activator protein (rtTa), or a derivative thereof. The rtTA protein is able to bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE).

[0131] The system further comprises a reverse tetracycline-controlled transactivator, rtTA. The rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the targetAttorney Ref.59868.00075WO01 (GNE-0006-WO) transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

[0132] One non-limiting example of a splicing switch modulator is the Xon splicing switch modulator, which can be activated by Branaplam (LMI070). The Xon system is generally described, for example, in Monteys et al. Nature.2021 Aug;596(7871):291-295, and is based on a splicing cassette from SF3B3. In use, the Xon system splices out a translation start codon (ATG) in the absence of the Branaplam, but when Branaplam is applied to the system, the Xon system splices transcript elements so as to include the ATG start codon, thereby facilitating functional translation of the transcript into a protein product. In one embodiment, the Xon system comprises a SF3B3 switch comprising a nucleic acid sequence of SEQ ID NO: 8. In one embodiment, the Xon system comprises a mini SF3B3 switch comprising a nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the Xon system comprises a SF3B3 switch comprising a nucleic acid sequence of SEQ ID NO: 10.

[0133] Another non-limiting example of a splicing switch modulator is the SMN2 splicing switch modulator, which can be activated by Risdiplam or RG7800. The SMN2 system is generally described, for example, in PCT / EP2024 / 065689, the disclosure of which is incorporated by reference herein for all purposes. In use, the SMN2 system skips a middle exon and causes frameshift or translation start codon loss for the target in the absence of the Risdiplam or RG7800, but when Risdiplam or RG7800 is applied to the system, the SMN2 system splices transcript elements so as to include in-frame coding sequence of the target, thereby facilitating functional translation of the transcript into a protein product. In one embodiment, the SMN2 system comprises a pMM596 ON-switch comprising a nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the SMN2 system comprises a pMM569 ON-switch comprising a nucleic acid sequence of SEQ ID NO: 12. In one embodiment, the SMN2 system comprises a pLS41ON- switch comprising a nucleic acid sequence of SEQ ID NO: 13.

[0134] Aspects of the disclosure include anti-CRISPR proteins that interfere with a function of a CRISPR-Cas system, and which are described, for example, in U.S. Patent No.11,530,405, the disclosure of which is incorporated by reference herein for all purposes. By “interfere with a function of a CRISPR-Cas system”, it is meant that the anti-CRISPR protein as defined herein significantly downmodulates the activity of the CRISPR-Cas system, i.e., decreases, from partial to complete inhibition, the activity of the CRISPR-Cas system. Anti-CRISPR proteins are also described, for example, in U.S. Patent Publication No. US2023 / 016742, the disclosure of which is incorporated by reference herein for all purposes.

[0135] Non-limiting examples of anti-CRISPR proteins in accordance with embodiments of the disclosure generally include members of the AcrE family (E1, E2, E3, E4); the AcrF family (F1, F2, F3, F4, F5, F6, F7, F8, F9, F10); the AcrIIA family (A1, A2, A3, A4); and the AcrCII family (C1, C2, C3).Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0136] In some embodiments, the strength of the regulatory element placed upstream of and operably linked to the anti-CRISPR is tuned to achieve a diminished or attenuated expression level of the anti-CRISPR to ensure that the anti-CRISPR has the desired activity level in the “off” or uninduced state. For example, in some embodiments, a relatively weak promoter, or another relatively weak regulatory element, such as an internal ribosome entry site (IRES), is operably coupled to the anti-CRISPR to achieve a desired diminished expression level of the anti-CRISPR when the system is in the “off” state. This allows the system to achieve the desired dynamic range of CRISPR-Cas activity between the “off” and “on” states. When the system is induced, for example, by introducing Dox to turn on expression of the CRISPR-Cas, and introducing a ligand that binds to the degron tag on the anti-CRISPR to degrade and thereby inactivate the anti- CRISPR, the starting low level expression of the anti-CRISPR allows that system to achieve a high dynamic range, achieving negligible background CRISPR-Cas activity in the uninduced “off” state, but then achieving highly potent CRISPR-Cas activity in the induced “on” state. As shown in the accompanying examples, utilizing a strong regulatory element to drive expression of the anti-CRISPR can lead to anti-CRISPR levels that are too high, and that impact the upper end of the dynamic range of the system by interfering with Cas activity, even when the deactivating ligand is introduced to inactivate the anti-CRISPR via the degron tag.

[0137] Degrons, or degron tags, are a class of protein tag that can be fused to either the N- or C- terminal end of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing domains that are identified by various enzymes and sent to the proteasome for degradation. Degron tags are conditionally active, meaning that they only facilitate degradation of their fused protein under certain conditions, such as following contact with an exogenous ligand, such as a small molecule. Degron tags can range in size from several kDa up to roughly 35 kDa. Degron tags are known in the art and are described, for example, in Bondeson, D.P. et al. Nat Commun.13, 5495 (2022).

[0138] Non-limiting examples of degron tags in accordance with embodiments of the invention include SMASh, dTag (FKBP12(F36V)), ligand-inducible degradation (LID) domains, HaloTag, and auxin-inducible degron (AID). Degron tags are generally described in Bondeson, D.P. et al. Nat Commun.13, 5495 (2022), which is incorporated by reference herein for all purposes. The dTag system is described in Nabet B. et al. Nat Chem Biol.2018 May;14(5):431-441, which is incorporated by reference herein for all purposes.

[0139] Destabilizing domains are protein domains that are inherently unstable under default conditions, and when fused to a protein of interest, can result in rapid degradation of the fusion protein. When contacted with a stabilizing ligand, the destabilizing domain is stabilized, thereby blocking or abating the degradation of the fusion protein. Destabilizing domains are known in the art and include, for example, FKBPF36VL106P and ecDHFR, which are described in Banaszynski et al. Cell.2006 Sep 8;126(5):995-1004. , the disclosure of which is incorporated byAttorney Ref.59868.00075WO01 (GNE-0006-WO) reference herein for all purposes, and Iwamoto et al. Chem Biol.2010 Sep 24;17(9):981-8., the disclosure of which is incorporated by reference herein for all purposes. FKBPF36VL106P (also referred to herein as “DD”) is an engineered variant of the FKBP12 protein that is continuously degraded until treated with the ligand SHIELD1, which stabilizes the tag. ecDHFR is an E.coli dihydrofolate reductase mutant (DHFR) that renders tagged proteins unstable and is stabilized upon exposure to Trimethoprim (TMP). This system is compatible with mammalian cells as endogenous mammalian DHFR protein is impervious to TMP treatment. In various embodiments, the inducible CRISPR-Cas systems described herein incorporate a DD or an ecDHFR destabilizing domain. In some embodiments, a DD or an ecDHFR destabilizing domain is fused to a Cas protein. In some embodiments, a DD or an ecDHFR destabilizing domain is fused to the N- terminal end of a Cas protein (or, alternatively, is encoded 5’ to, or upstream of a polynucleotide sequence encoding the Cas protein). In some embodiments, a DD or an ecDHFR destabilizing domain is fused to the C-terminal end of a Cas protein (or, alternatively, is encoded 3’ to, or downstream of a polynucleotide sequence encoding the Cas protein). SHIELD1 is a specific, cell- permeant and high-affinity ligand of FK506-binding protein-12 (FKBP), and reverses the instability by binding to mutated FKBP (mtFKBP), allowing conditional expression of mtFKBP- fused proteins. SHIELD1 can stabilize proteins tagged with a mutated FKBP12-derived destabilizing domain (DD). The chemical structure of SHIELD1 is shown below, and SHIELD1 is assigned CAS No.914805-33-7. Trimethoprim (TMP) can stabilize proteins tagged with an ecDHFR destabilizing domain (DD). TMP is assigned CAS No.738-70-5.

[0140] In some embodiments, a common ligand, such as SHIELD1, is used to deactivate both the anti-CRISPR and the destabilizing domain (DD) of an inducible CRISPR-Cas system. For example, in some embodiments, an inducible CRISPR-Cas system comprises a destabilizing domain (DD) that comprises FKBPF36VL106P, and also comprises an anti-CRISPR, such as AcrIIA4, fused to LID. In such embodiments, both the DD and the anti-CRISPR can be deactivated by SHIELD1. This allows a single ligand to be introduced to the system to inactivate both the DD and the anti-CRISPR simultaneously. In other embodiments, separate ligands can be used to inactivate the DD and to inactivate the anti-CRISPR. “Ultra-tight” Dox independent inducible CRISPR / Cas9 systems and Xon switch systems

[0141] Also provided herein are “ultra-tight” dox independent (or non-Dox-inducible) inducible CRISPR / Cas9 systems. In some embodiments, Cas9-DD and DD-Cas9 systems are provided, where the DD domain is fused to Cas9 at the C- or N- terminus, respectively. In some embodiments of the DHFR-Cas9 system, the DHFR domain is fused to the Cas9 at the N-terminus. In some embodiments of the Xon-Cas9 system, ATG is deleted from the Cas9 coding sequence and provided in the alternatively spliced exon.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0142] Also provided herein are Xon switch systems. In some embodiments, Xon switch systems are provided where the destabilizing domain DD or DHFR is fused to the C- terminus of Cas9.

[0143] Also provided herein are pTET-Ultra-tight (also referred to herein as “pTET:Ultra-tight”) systems. In some embodiments, the pTET-:Ultra-tight systems are combined with dual gRNAs to inducibly delete a ~2.5kb genomic locus by introducing dual sgRNA into the pTET and pTET- Ultra-tight constructs to eliminate exons 2 and 3 within the H3-3A gene. Ultra-tight inducible CRISPRi systems

[0144] In some embodiments, CRISPRi systems are provided, having an ultra-tight and highly inducible tool for gene silencing, wherein the system comprises a combination of a DD destabilizing domain with the ZIM3 based CRISPRi system. In some embodiments, a leaky effect is suppressed by the fusion of a DD domain to the ZIM3-dCas9. Promoter hijacking

[0145] Also provided herein are methods for promoter hijacking strategies. In some embodiments, the promoter hijacking strategy results in stable integration of the splice switch based inducible Cas9 into a genome. In some embodiments, the splice switch based inducible Cas9 cassette is inserted downstream of a promoter of a housekeeping gene, and the inducible Cas9 is driven by the endogenous promoter, wherein the promoter is less susceptible to silencing. In some embodiments, only one allele is hijacked to maintain the housekeeping gene’s function. In some embodiments, the insertion is achieved by CRISPR medicated homology directed repair or homology independent target integration. Preferred embodiments:

[0146] The various elements described herein can be combined in suitable expression vectors to facilitate inducible control over the gene editing activity of a CRISPR-Cas system. For example, in some embodiments, an inducible CRISPR-Cas system comprises a polynucleotide encoding: a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element, and an anti-CRISPR protein.

[0147] In one preferred embodiment, an inducible CRISPR-Cas system comprises a polynucleotide encoding a Cas9 protein, or a homolog or modified version thereof, which is controlled by a single inducible expression element comprising a ligand inducible alternative splicing switch.

[0148] In one preferred embodiment, an inducible CRISPR-Cas system comprises a polynucleotide encoding a Cas9 protein, or a homolog or modified version thereof, which is controlled by aAttorney Ref.59868.00075WO01 (GNE-0006-WO) single inducible expression element comprising a ligand inducible alternative splicing switch selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator.

[0149] In one preferred embodiment, the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBPF36VL106P (SEQ ID NO: 40), or LID (SEQ ID NO: 45).

[0150] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45).

[0151] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBPF36VL106P (SEQ ID NO: 40).

[0152] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45).

[0153] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBPF36VL106P (SEQ ID NO: 40).

[0154] In one preferred embodiment, the inducible expression element comprises the inducible promoter element comprising pTET (SEQ ID NO: 14).

[0155] In one preferred embodiment, the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBPF36VL106P (SEQ ID NO: 40), or LID (SEQ ID NO: 45).

[0156] In one preferred embodiment, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising: (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element.

[0157] In one preferred embodiment, the inducible promoter element comprises pTET (SEQ ID NO: 14).

[0158] In one preferred embodiment, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator.

[0159] In one preferred embodiment, the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBPF36VL106P (SEQ ID NO: 40), or LID (SEQ ID NO: 45).

[0160] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45).

[0161] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBPF36VL106P (SEQ ID NO: 40).

[0162] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45).Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0163] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBPF36VL106P (SEQ ID NO: 40).

[0164] In one preferred embodiment, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by a single inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element.

[0165] In one preferred embodiment, the inducible expression element comprises the ligand inducible alternative splicing switch.

[0166] In one preferred embodiment, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator.

[0167] In one preferred embodiment, the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBPF36VL106P (SEQ ID NO: 40), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42).

[0168] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40).

[0169] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42).

[0170] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40).

[0171] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42).

[0172] In one preferred embodiment, the inducible expression element comprises the inducible promoter element.

[0173] In one preferred embodiment, the inducible promoter element comprises pTET (SEQ ID NO: 14).

[0174] In one preferred embodiment, the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBPF36VL106P (SEQ ID NO: 40), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42).

[0175] In one preferred embodiment, the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40).

[0176] In one preferred embodiment, the ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42).Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0177] In one preferred embodiment, the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising: (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element.

[0178] In one preferred embodiment, the wherein the inducible promoter element comprises pTET (SEQ ID NO: 14).

[0179] In one preferred embodiment, the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator.

[0180] In one preferred embodiment, the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBPF36VL106P (SEQ ID NO: 40), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42).

[0181] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40).

[0182] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR FKBPF36VL106P (SEQ ID NO: 40).

[0183] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40).

[0184] In one preferred embodiment, the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42).

[0185] In one preferred embodiment, the inducible promoter is pTET, the Cas protein comprises dCas9, the destabilizing domain comprises ecDHFR, and the KRAB domain is Zim3KRAB.

[0186] In one preferred embodiment, the inducible promoter is pTET, the Cas protein comprises dCas9, the destabilizing domain comprises ecDHFR, and the KRAB domain comprises Kox1KRAB.

[0187] In one preferred embodiment, the inducible promoter is pTET, the Cas protein comprises dCas9, the destabilizing domain comprises FKBPF36VL106P, and the KRAB domain is Zim3KRAB.

[0188] In one preferred embodiment, the inducible promoter is pTET, the Cas protein comprises dCas9, the destabilizing domain comprises FKBPF36VL106P, and the KRAB domain comprises Kox1KRAB. Polynucleotides, Vectors, Host Cells, Model Organisms and Transgenic Animals:

[0189] Aspects of the disclosure include polynucleotides encoding various combinations of the elements of the inducible expression systems described herein. Aspects of the disclosure includeAttorney Ref.59868.00075WO01 (GNE-0006-WO) vectors that are suitable for delivery of one or more of such polynucleotides into a host system, such as, for example, into a host cell or model organism to generate, for example, a stable cell line or a transgenic animal that inducibly expresses one or more of the components described herein, or various combinations thereof. Non-limiting examples of host cell lines that can be used with the systems described herein include SW480 cells, HAP-1 cells, HeLa cells, HCT116 cells, 293T cells, A549 cells, K562 cells. Aspects of the disclosure include model organisms that constitutively express one or more of the components described herein, or various combinations thereof. Sequence Tables: Table 1: Nucleotide Sequences Description Nucleic Acid Sequence SEQ ID NOAttorney Ref.59868.00075WO01 (GNE-0006-WO) dTag(FKBP12( GGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCT 4 F36V)) TCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGAT GCTTGAAGATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAttorney Ref.59868.00075WO01 (GNE-0006-WO) GCGACAGGAGGATTTTTACCCTTTCCTCAAAGATAATAGAGAA AAGATCGAAAAGATTCTCACTTTCCGGATTCCCTACTATGTTGG GCCCCTCGCACGAGGCAATTCTCGGTTTGCTTGGATGACAAGAAAttorney Ref.59868.00075WO01 (GNE-0006-WO) AAAAAGGAAACGAGTTGGCACTCCCCAGTAAGTATGTAAACTT TCTTTACCTGGCAAGCCACTACGAGAAGTTGAAGGGAAGTCCT GAGGATAACGAGCAGAAACAACTGTTCGTGGAGCAACATAAACAttorney Ref.59868.00075WO01 (GNE-0006-WO) SF3B3 full TTTCTGTACAACTTAACCTTGCAGAGAGCCACTGGCATCAGCTT 10 length Xon TGCCATTCTTGGAAACTTTTCTGGTAAGTTCTCTCGTTACCATCT TTTGAAATTTTAAGTGAATTAATACATATCTTGCTTAGTCTCTTGAttorney Ref.59868.00075WO01 (GNE-0006-WO) pMM596 ON- GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCA 11 switch GGTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGT TATGTGACTTTGTTTTGTAAATTTATAAAATACTACTTGCTTCTC 2 3Attorney Ref.59868.00075WO01 (GNE-0006-WO) GGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAGGAAATGCTGGCATAGAGCAGCAC 4 8Attorney Ref.59868.00075WO01 (GNE-0006-WO) ATGAGGTGTTAAGTTCTGGTGCATTATCTGTTACCTATTTCAGAT GCATTTCCTAGTTCACAAATTGTGTAATGATTCTTGTCAGGGCA CACTTTTCTTGGCTGCTTACCTAGTGCCAAGTCGTGTGCCTATTGAttorney Ref.59868.00075WO01 (GNE-0006-WO) TGATGAGGTAGCCTACCATGAGAAATACCCAACTATCTACCATC TTCGGAAAAAACTTGTGGACTCTACAGATAAGGCTGACTTGAG GCTCATTTATCTGGCACTGGCTCACATGATAAAATTCAGAGGTCAttorney Ref.59868.00075WO01 (GNE-0006-WO) GAAACTAGGCAAATCACAAAGCACGTTGCCCAGATCCTCGATA GTCGAATGAATACCAAGTATGATGAGAATGACAAACTCATTAG AGAGGTTAAGGTTATTACTCTTAAGAGTAAGCTCGTTTCTGATTAttorney Ref.59868.00075WO01 (GNE-0006-WO) CATCCTGAGCCCTTGCGGCAGATGCAGACAGGTGCTGCTGGATC TGCACCCCGGCATCAAGGCCATCGTGAAGGATAGCGACGGCCA GCCTACAGCCGTGGGCATCAGAGAGCTGCTGCCTAGCGGCTATAttorney Ref.59868.00075WO01 (GNE-0006-WO) GAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGA GGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCC CTCTTGAACGACCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCAttorney Ref.59868.00075WO01 (GNE-0006-WO) TGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGG 9Attorney Ref.59868.00075WO01 (GNE-0006-WO) CACCCACCTTAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGC TACTGTGCCCTGCTGAAAGCACATTTTTAATACTAATTTTATCTT TCAAATTCCTTTTCCAATTCAGTCTTCCTTTTTATCTAAAATGATAttorney Ref.59868.00075WO01 (GNE-0006-WO) AGAAAGTCCTTCCAAAACACTCACTCTTGTACGAATACTTTACT GTATATAACGAACTGACAAAGGTTAAATATGTGACCGAGGGCA TGCGAAAACCAGCCTTCCTTTCCGGTGAGCAAAAAAAGGCTATTAttorney Ref.59868.00075WO01 (GNE-0006-WO) ATCCGAGAACAAGCTGAAAACATCATACATTTGTTTACCCTCAC AAACTTGGGTGCACCCGCAGCATTTAAGTACTTTGACACAACCA TTGACAGGAAGAGATATACCAGTACCAAAGAAGTACTGGATGC 0Attorney Ref.59868.00075WO01 (GNE-0006-WO) GCAAACTATAACCCTCAAGTCCGAGCTGAACGGCGGAAGCGGA GTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCC CCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCT 1 2Attorney Ref.59868.00075WO01 (GNE-0006-WO) AGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCTACTGTGCCC TGCTGAAAGCACATTTTTAATACTAATTTTATCTTTCAAATTCCT TTTCCAATTCAGTCTTCCTTTTTATCTAAAATGATGGAGAAGTTTAttorney Ref.59868.00075WO01 (GNE-0006-WO) CAAAACACTCACTCTTGTACGAATACTTTACTGTATATAACGAA CTGACAAAGGTTAAATATGTGACCGAGGGCATGCGAAAACCAG CCTTCCTTTCCGGTGAGCAAAAAAAGGCTATTGTCGATCTGTTGAttorney Ref.59868.00075WO01 (GNE-0006-WO) AACAAGCTGAAAACATCATACATTTGTTTACCCTCACAAACTTG GGTGCACCCGCAGCATTTAAGTACTTTGACACAACCATTGACAG GAAGAGATATACCAGTACCAAAGAAGTACTGGATGCCACCTTG 3 4 5 6Attorney Ref.59868.00075WO01 (GNE-0006-WO) CCAGTGAATTGGGCGCGCCTGCTCGACACGCTGCAGAACACGC AGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTT AAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTAttorney Ref.59868.00075WO01 (GNE-0006-WO) TTTTTCCTTGAAACAGAGTCCTGCTCTGTCACCCAGGCTGGAGC GCAGTGGTGTGATCTTGGCTCACTGCCACCTCCGCCTCCTGGGT TCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAttorney Ref.59868.00075WO01 (GNE-0006-WO) TACCCTTTCCTCAAAGATAATAGAGAAAAGATCGAAAAGATTC TCACTTTCCGGATTCCCTACTATGTTGGGCCCCTCGCACGAGGC AATTCTCGGTTTGCTTGGATGACAAGAAAGTCTGAAGAGACCATAttorney Ref.59868.00075WO01 (GNE-0006-WO) GGCACTCCCCAGTAAGTATGTAAACTTTCTTTACCTGGCAAGCC ACTACGAGAAGTTGAAGGGAAGTCCTGAGGATAACGAGCAGAA ACAACTGTTCGTGGAGCAACATAAACATTACCTTGACGAGATAAttorney Ref.59868.00075WO01 (GNE-0006-WO) GGAACGAGTATGTCATATCCGAATCTGAGAATGAATCTATAGTC GAAAAGTTCATATCCGCGTTTAAGAATGGTTGGAATCAAGAAT ACGAGGATGAAGAGGAATTTTATAATGACATGCAAACTATAACAttorney Ref.59868.00075WO01 (GNE-0006-WO) GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG 7Attorney Ref.59868.00075WO01 (GNE-0006-WO) GGAGTGCTGTCTGCATAGAAACAGATAATTTGCTTACGTTTACC ATGTGGGGAATATTTTTGTAAAGATGGATTAAGGCTAGGTTTGA ATTGTGTGAAATTTCAAATATTGGATTAGGAAATACAAAGTTACAttorney Ref.59868.00075WO01 (GNE-0006-WO) AAGAAAAACGGACTGTTCGGTAATCTCATAGCTCTTTCACTCGG TCTGACTCCTAATTTTAAGAGCAATTTTGACCTTGCCGAAGACG CTAAACTTCAGCTCTCAAAGGACACTTATGACGATGATTTGGACAttorney Ref.59868.00075WO01 (GNE-0006-WO) TTGGCAAGGCTACTGCAAAGTATTTCTTCTACTCAAACATAATG AATTTCTTTAAAACAGAGATTACTCTCGCCAACGGAGAAATCCG AAAACGGCCTCTCATCGAAACAAACGGCGAGACTGGAGAAATCAttorney Ref.59868.00075WO01 (GNE-0006-WO) GTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAA GACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCC CCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTA 0Attorney Ref.59868.00075WO01 (GNE-0006-WO) CAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTT TTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAA AGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGA 1 2 3Attorney Ref.59868.00075WO01 (GNE-0006-WO) AAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCA CCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGG AGCTTCTAAAACCGGAAGGAAGTGCTGGGTCCGCCGCAGGAAGAttorney Ref.59868.00075WO01 (GNE-0006-WO) GAGAATATCGTTATTGAAATGGCACGAGAAAATCAAACAACTC AAAAAGGTCAGAAGAACTCACGAGAACGGATGAAGCGCATTG AAGAAGGTATCAAGGAACTCGGTAGCCAAATATTGAAGGAACAAttorney Ref.59868.00075WO01 (GNE-0006-WO) CTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATT AGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA CGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAttorney Ref.59868.00075WO01 (GNE-0006-WO) AATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAC CCTAGCGCTACCGGATCCCAACCATGACCGAGTACAAGCCCACAttorney Ref.59868.00075WO01 (GNE-0006-WO) GTCAATAATGACGGGAACGAGTATGTCATATCCGAATCTGAGA ATGAATCTATAGTCGAAAAGTTCATATCCGCGTTTAAGAATGGT TGGAATCAAGAATACGAGGATGAAGAGGAATTTTATAATGACAAttorney Ref.59868.00075WO01 (GNE-0006-WO) CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT 4Attorney Ref.59868.00075WO01 (GNE-0006-WO) AAATACCCAACTATCTACCATCTTCGGAAAAAACTTGTGGACTC TACAGATAAGGCTGACTTGAGGCTCATTTATCTGGCACTGGCTC ACATGATAAAATTCAGAGGTCATTTTTTGATCGAGGGTGACCTGAttorney Ref.59868.00075WO01 (GNE-0006-WO) GCACGTTGCCCAGATCCTCGATAGTCGAATGAATACCAAGTATG ATGAGAATGACAAACTCATTAGAGAGGTTAAGGTTATTACTCTT AAGAGTAAGCTCGTTTCTGATTTCAGGAAAGATTTTCAATTTTA 5Attorney Ref.59868.00075WO01 (GNE-0006-WO) ACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGG CCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAA GCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATAttorney Ref.59868.00075WO01 (GNE-0006-WO) hPGKp- TTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGG 36 AcrIIA4-LID- CTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCT GGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGAT 7Attorney Ref.59868.00075WO01 (GNE-0006-WO) GGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTG GAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTT GGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCC 8Description Amino Acid Sequence SEQ ID NO 0 1 2 3 4Attorney Ref.59868.00075WO01 (GNE-0006-WO) LID GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK 45 PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHP GIIPPHATLVFDVELLKLETRGVEEVAEGVVLLRRRGN 6 7 8 9Attorney Ref.59868.00075WO01 (GNE-0006-WO) NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE 0Description Nucleic Acid Sequence SEQ ID NOAttorney Ref.59868.00075WO01 (GNE-0006-WO) B2M AGTAGCGCGAGCACAGCTA 54Table 4: CRISPRi guides. Description Nucleic Acid Sequence SEQ ID NOTabe 5: Prmers used to generate amp cons or next-generaton sequencng Primer name Primer sequence 5’ to 3’ SEQ IDAttorney Ref.59868.00075WO01 (GNE-0006-WO) Methods of Use:

[0190] The inducible CRISPR-Cas systems described herein can generally be used in connection with methods that involve modifying a target nucleic acid in a cell. In some embodiments, the inducible CRISPR-Cas systems can be used to modify a target nucleic acid in a cell by first introducing the inducible CRISPR-Cas system into the cell, contacting the cell with a ligand that activates the inducible expression element, and contacting the cell with a ligand that deactivates the anti-CRISPR and / or the destabilizing domain, thereby causing the Cas protein to modify the target nucleic acid.

[0191] Aspects of the disclosure provide methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, a method comprises sampling a cell or population of cells from a human or non-human animal and modifying the cell or cells. Culturing may occur at any stage ex vivo.

[0192] In some embodiments, a method comprises allowing a CRISPR complex to bind to the target nucleic acid to effect cleavage of the target nucleic acid, thereby modifying the target nucleic acid, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target nucleic acid.

[0193] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the methods comprise allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide.

[0194] In some embodiments, the inducible CRISPR-Cas system comprises an anti-CRISPR, and the method comprises contacting the cell with a ligand that deactivates the anti-CRISPR. This increases the dynamic range of the inducible expression system by ensuring that in the OFF state, the inducible expression system exhibits exceedingly low activity of the CRISPR-Cas, and any “leaky” expression has little to no impact due to the presence of the anti-CRISPR, which serves to impair the ability of any Cas molecules that are expressed to interact with and modify the target nucleic acid.

[0195] In some embodiments, the inducible CRISPR-Cas system comprises a destabilizing domain, and the method comprises contacting the cell with a ligand that deactivates the destabilizing domain. This increases the dynamic range of the inducible expression system by ensuring that in the OFF state, any Cas activity from the inducible expression system is reduced due to the presence of the destabilizing domain, which serves to degrade any Cas molecules that are expressed, thus preventing their ability interact with and modify the target nucleic acid.

[0196] In some embodiments, the inducible CRISPR-Cas system comprises both an anti-CRISPR fusion comprising a degron tag and a destabilizing domain, and the method comprises contactingAttorney Ref.59868.00075WO01 (GNE-0006-WO) the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain. This increases the dynamic range of the inducible expression system by ensuring that in the OFF state, the inducible expression system exhibits exceedingly low expression of the CRISPR-Cas, and any “leaky” expression has little to no impact due to the presence of the anti- CRISPR and the destabilizing domain, which serve to impair the ability of any Cas molecules that are expressed to interact with and modify the target nucleic acid, and degrade expressed Cas molecules, respectively.

[0197] In some embodiments, modifying the target nucleic acid in the cell alters expression of a target gene in the cell, for example, by introducing a double stranded break in the target nucleic acid, or by introducing a single stranded break in the target nucleic acid (i.e., a nick). In certain embodiments, altering expression of the target nucleic acid in the cell comprises decreasing expression of the target gene. In certain embodiments, altering expression of the target nucleic acid in the cell comprises increasing expression of the target gene.

[0198] In some embodiments, modifying the target nucleic acid in the cell comprises cutting the target nucleic acid at two different locations. Such a method can be used, for example, to remove or excise an entire gene segment or an entire gene from a genomic locus of a cell.

[0199] In some embodiments, modifying the target nucleic acid in the cell further comprises editing the target nucleic acid, for example, editing the target nucleic acid to introduce an edited sequence, in which one or more nucleotides have been changed in reference to a starting sequence. Such methods can be used to determine, for example, the functional impact of editing the target nucleic acid on one or more functional attributes of the cell.

[0200] Aspects of the disclosure include methods of increasing the dynamic range of an inducible expression system in a cell by first introducing an inducible CRISPR-Cas system into the cell, then culturing the cell under a first set of conditions in which the inducible CRISPR-Cas system exhibits a first expression level, then contacting the cell with a ligand that activates the inducible expression element and a ligand that deactivates the anti-CRISPR and / or the destabilizing domain to cause the inducible CRISPR-Cas system to exhibit a second expression level that is higher than the first expression level. This increases the dynamic range of the inducible expression system by ensuring that in the OFF state, the inducible expression system exhibits exceedingly low expression of the CRISPR-Cas, and any “leaky” expression has little to no impact due to the presence of the anti-CRISPR and / or the destabilizing domain, which serve to impair the ability of any Cas molecules that are expressed to interact with and modify the target nucleic acid.

[0201] In some embodiments, the inducible CRISPR-Cas system comprises both an anti-CRISPR and a destabilizing domain, and the method comprises contacting the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain. Accordingly, the inducer ligand activates expression of the Cas, and the one or more ligands that deactivate the anti-CRISPR and / or destabilizing domain eliminate the ability of these components to impair theAttorney Ref.59868.00075WO01 (GNE-0006-WO) ability of the Cas molecules to modify the target nucleic acid. Combining the various components of the systems described herein leads to a non-linear increase in the dynamic range of the inducible CRISPR-Cas systems. For example, and described in further detail in the examples section, combinations of the various components described herein can lead to a greater than 100- fold increase in the dynamic range of the system, in comparison to an inducible expression system that does not contain an anti-CRISPR and / or a destabilizing domain. Kits:

[0202] Also within the scope of the invention are kits containing any one or more of the elements disclosed in the above methods and compositions. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube.

[0203] In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilize form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more of the vectors and / or one or more of the polynucleotides described herein. Further embodiments:

[0204] Additional embodiments of the disclosure are provided in the following clauses: 1. An inducible CRISPR-Cas system comprising a polynucleotide encoding: a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element; and an anti-CRISPR protein. 2. The system of clause 1, wherein at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein. 3. The system of clause 2, wherein the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator.Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 3, wherein the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The system of clause 4, wherein the SF3B3-on switch is activated by branaplam (LMI070), and wherein activation induces expression of the encoded Cas protein. The system of clause 2, wherein the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. The system of clause 6, wherein the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). The system of clause 7, wherein the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein. The system of any one of clauses 1-8, wherein at least one inducible expression element comprises an inducible promoter element. The system of clause 9, wherein the inducible promoter element comprises a TET- responsive promoter element (TRE). The system of clause 10, wherein the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). The system of any one of clauses 9-11, wherein the inducible promoter element is induced by a tetracycline molecule. The system of any one of clauses 9-11, wherein the inducible promoter element is induced by a derivative of a tetracycline molecule. The system of clause 13, wherein the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. The system of clause 14, wherein the derivative of the tetracycline molecule is doxycycline. The system of any one of clauses 1-15, wherein the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. The system of clause 16, wherein the anti-CRISPR protein comprises AcrIIA4. The system of any one of clauses 1-17, wherein the anti-CRISPR protein comprises a fusion protein further comprising a degron tag. The system of clause 18, wherein the degron tag is activated by IKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. The system of clause 18, wherein the degron tag comprises an FK506 binding protein (FKBP).Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 20, wherein the FKBP is a modified or mutant FKBP12. The system of clause 21, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). The system of clause 22, wherein FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). The system of any one of clauses 18- 23, wherein the degron tag is activated by a ligand that targets a von Hippel-Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. The system of clause 24, wherein the ligand is dTag-V1. The system of clause 18, wherein the degron tag comprises a LID domain. The system of clause 26, wherein the LID domain comprises a FKBP12(F36V) fused to a C-terminal cryptic degron sequence of SEQ ID NO: 44. The system of 27, wherein the LID domain comprises the amino acid sequence of SEQ ID NO: 45. The system of any one of clauses 26- 28, wherein the degron tag is activated by Shield1. The system of any one of clauses 1-29, wherein the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. The system of clause 30, wherein the Cas protein is Cas9. The system of clause 30, wherein the Cas9 protein is a homolog or modified version of the Cas9 protein. The system of clause 31, wherein the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9. The system of clause 33, wherein the Cas protein is dCas9. The system of any one of clauses 1-34, wherein the Cas protein further comprises a Cas fusion protein domain. The system of clause 35, wherein the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. The system of clause 36, wherein the KRAB domain is selected from: Zim3KRAB, KRAB-MeCP2, and Kox1KRAB.Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 37, wherein the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. The system of clause 38, wherein the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. The system of clause 37, wherein the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB. The system of any one of clauses 1-40, wherein the polynucleotide encodes one or more guide RNA molecules. The system of any one of clauses 1-41, further comprising a separate polynucleotide encoding one or more guide RNA molecules. The system of clause 41 or 42, wherein one or more of the guide RNA molecules comprises an sgRNA molecule. The system of any one of clauses 41-43, wherein the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. The system of clause 44, wherein the constitutively expressed promoter comprises a U6 promoter. The system of any one of clauses 41-45, comprising two or more sgRNA molecules. The system of clause 46, wherein each sgRNA molecule is driven by the same Pol III promoter. The system of clause 46, wherein each sgRNA molecule is driven by different Pol III promoter. The system of clause 48, wherein the different Pol III promoters are U6 and H1. The system of any one of clauses 41-49, further comprising an sgRNA molecule library. The system of clause 50, wherein the sgRNA molecule library comprises a Pol III promotor. The system of clause 51, wherein the sgRNA molecule library comprises a plurality of Pol III promoters. The system of any one of clauses 41-45, wherein at least one sgRNA molecules targets a gene of interest. The system of any one of clauses 1-53, wherein the polynucleotide further encodes a selectable marker. The system of any one of clauses 1-54, wherein the anti-CRISPR is driven by a weak regulatory element.Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 55, wherein the weak regulatory element comprises an IRES. The system of clause 56, wherein the weak regulatory element is selected from the group consisting of CMV-A, UbC, PGK, PGK100, and SV40. The system of any one of clauses 1- 57, wherein: the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by a single inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element. The system of clause 58, wherein the inducible expression element comprises the ligand inducible alternative splicing switch. The system of clause 59, wherein the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. The system of any one of clauses 54-60, wherein the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43), or LID (SEQ ID NO: 45). The system of clause 61, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). The system of clause 61, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). The system of clause 61, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45).Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 61, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). The system of clause 58, wherein the inducible expression element comprises the inducible promoter element. The system of clause 66, wherein the inducible promoter element comprises pTET (SEQ ID NO: 14). The system of clause 66 or 67, wherein the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43), or LID (SEQ ID NO: 45). The system of clause 68, wherein the degron tag comprises LID (SEQ ID NO: 45). The system of clause 68, wherein the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). The system of any one of clauses 1-54, wherein: the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising: (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element. The system of clause 71, wherein the inducible promoter element comprises pTET (SEQ ID NO: 14). The system of clause 71 or 72, wherein the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. The system of any one of clauses 71-73, wherein the anti-CRISPR protein is a fusion protein comprising: ACRIIA4 (SEQ ID NO: 47), and a degron tag selected from: FKBP12(F36V) (SEQ ID NO: 43), orAttorney Ref.59868.00075WO01 (GNE-0006-WO) LID (SEQ ID NO: 45). The system of clause 74, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). The system of clause 74, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). The system of clause 74, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises LID (SEQ ID NO: 45). The system of clause 74, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the degron tag comprises FKBP12(F36V) (SEQ ID NO: 43). An inducible CRISPR-Cas system comprising a polynucleotide encoding: a destabilizing domain fused to a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element. The system of clause 79, wherein the destabilizing domain comprises a ligand controllable destabilizing domain. The system of clause 80, wherein the ligand controllable destabilizing domain is selected from the group consisting of: FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof. The system of clause 81, wherein the ligand controllable destabilizing domain comprises a modified or mutant FKBP12. The system of clause 82, wherein the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. The system of clause 83, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*).Attorney Ref.59868.00075WO01 (GNE-0006-WO) The system of clause 84, wherein the modified or mutant FKBP12 comprises FKBPF36VL106P (SEQ ID NO: 40). The system of clause 85, wherein the ligand that controls the destabilizing domain is Shield1. The system of clause 81, wherein ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). The system of clause 85, wherein the ligand that controls the destabilizing domain is trimethoprim (TMP). The system of any one of clauses 79-88, wherein at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein. The system of clause 89, wherein the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. The system of clause 90, wherein the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The system of clause 91, wherein the SF3B3-on switch is activated by branaplam (LMI070), and wherein activation induces expression of the encoded Cas protein. The system of clause 89, wherein the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. The system of clause 93, wherein the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). The system of clause 94, wherein the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein. The system of any one of clauses 79-95, wherein at least one inducible expression element comprises an inducible promoter element. The system of clause 96, wherein the inducible promoter element comprises a TET- responsive promoter element (TRE). The system of clause 97, wherein the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). The system of any one of clauses 96-98, wherein the inducible promoter element is induced by a tetracycline molecule. . The system of any one of clauses 96-99, wherein the inducible promoter element is induced by a derivative of a tetracycline molecule.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 100, wherein the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. . The system of clause 101, wherein the derivative of the tetracycline molecule is doxycycline. . The system of any one of clauses 79-102, wherein the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. . The system of clause 103, wherein the anti-CRISPR protein comprises AcrIIA4. . The system of clause 104, wherein the anti-CRISPR protein comprises a fusion protein further comprising a degron tag. . The system of clause 105, wherein the degron tag is activated byIKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. . The system of clause 106, wherein the degron tag comprises an FK506 binding protein (FKBP). . The system of clause 107, wherein the FKBP is a modified or mutant FKBP12. . The system of clause 108, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). . The system of clause 109, wherein FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of any one of clauses 105-110, wherein the degron tag is activated by a ligand that targets a von Hippel-Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. . The system of clause 111, wherein the ligand comprises dTagV1. . The system of clause 105, wherein the degron tag comprises a LID domain.. The system of clause 113, wherein the LID domain comprises a C-terminus cryptic degron sequence. . The system of 114, wherein the cryptic degron sequence comprises the amino acid sequence of SEQ ID NO: 44. . The system of any one of clauses 113-115, wherein the degron tag is activated by Shield1. . The system of any one of clauses 79-116, wherein the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel,Attorney Ref.59868.00075WO01 (GNE-0006-WO) Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. . The system of clause 117, wherein the Cas protein is Cas9. . The system of clause 117, wherein the Cas9 protein is a homolog or modified version of the Cas9 protein. . The system of clause 119, wherein the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9.. The system of clause 118, wherein the Cas protein is dCas9. . The system of any one of clauses 79-121, wherein the Cas protein further comprises a Cas fusion protein domain. . The system of clause 122, wherein the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. . The system of clause 123, wherein the Cas fusion protein domain is selected from a ZIM3KRA, KRAB-MeCP2, and a Kox1KRAB domain. . The system of clause 124, wherein the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. . The system of clause 125, wherein the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. . The system of clause 125, wherein the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB. . The system of any one of clauses 79-127, wherein the polynucleotide encodes one or more guide RNA molecules. . The system of any one of clauses 79-128, wherein the system further comprises a separate polynucleotide encoding one or more guide RNA molecules.. The system of clause 128 or 129, wherein one or more of the guide RNA molecules comprises an sgRNA molecule. . The system of clause 130, further comprising an sgRNA molecule library.. The system of any one of clauses 128-130, wherein the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. . The system of clause 132, wherein the constitutively expressed promoter comprises a U6 promoter. . The system of any one of clauses 128-133, comprising two or more sgRNA molecules.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 134, wherein each sgRNA molecule is driven by the same Pol III promoter. . The system of clause 135, wherein each sgRNA molecule is driven by different Pol III promoter. . The system of clause 136, wherein the different Pol III promoters are U6 and H1. . The system of any one of clauses 128- 137, further comprising an sgRNA molecule library. . The system of clause 138, wherein the sgRNA molecule library comprises a Pol III promotor. . The system of clause 139, wherein the sgRNA molecule library comprises a plurality of Pol III promoters. . The system of any one of clauses 128-133, wherein at least one sgRNA molecules targets a gene of interest. . The system of any one of clauses 79-141, wherein the polynucleotide further encodes a selectable marker. . The system of any one of clauses 79-142, wherein: the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by a single inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element. . The system of clause 143, wherein the inducible expression element comprises the ligand inducible alternative splicing switch. . The system of clause 144, wherein the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator. . The system of any one of clauses 79-145, wherein the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBP12(F36V) (SEQ ID NO: 43), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42). . The system of clause 146, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43).. The system of clause 146, wherein:Attorney Ref.59868.00075WO01 (GNE-0006-WO) the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The system of clause 146, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator. . The system of clause 146, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The system of clause 144, wherein the inducible expression element comprises the inducible promoter element. . The system of clause 151, wherein the inducible promoter element comprises pTET (SEQ ID NO: 14). . The system of clause 151 or 152, wherein the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBP12(F36V) (SEQ ID NO: 43), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42). . The system of clause 153, wherein the ligand controllable destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of clause 153, wherein the ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The system of any one of clauses 79-142, wherein: the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by two inducible expression elements comprising: (i) a ligand inducible alternative splicing switch; and (ii) an inducible promoter element. . The system of clause 156, wherein the inducible promoter element comprises pTET (SEQ ID NO: 14). . The system of clause 156 or 157, wherein the ligand inducible alternative splicing switch is selected from an Xon splicing switch modulator or a SMN2 splicing switch modulator.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of any one of clauses 156-158, wherein the destabilizing domain comprises a ligand controllable destabilizing domain selected from: FKBP12(F36V) (SEQ ID NO: 43), ecDHFR (SEQ ID NO: 41), or ecDHFR (SEQ ID NO: 42). . The system of clause 159, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of clause 160, wherein: the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The system of clause 160, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of clause 160, wherein: the ligand inducible alternative splicing switch comprises an SMN2 splicing switch modulator; and the destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . An inducible CRISPR-Cas system comprising a polynucleotide encoding: a destabilizing domain fused to a Cas protein, operably linked to at least one inducible expression element; and an anti-CRISPR. . The system of one of clause 164, wherein at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein. . The system of clause 165, wherein the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. . The system of clause 166, wherein the Xon splicing switch modulator comprises an SF3B3-on switch comprising a nucleic acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 167, wherein the SF3B3-on switch is activated by branaplam (LMI070), risdiplam, or RG7800, wherein activation induces expression of the encoded Cas protein. . The system of clause 165, wherein the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. . The system of clause 169, wherein the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NO: 12), and pLS41 (SEQ ID NO: 13). . The system of clause 170, wherein the SMN2-ON switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein. . The system of any one of clauses 164-171, wherein at least one inducible expression element comprises an inducible promoter element. . The system of clause 172, wherein the inducible promoter element comprises a TET-responsive promoter element (TRE). . The system of clause 173, wherein the TRE comprises pTET (also referred to as TRE3G) (SEQ ID NO: 14). . The system of any one of clauses 172-174, wherein the inducible promoter element is induced by a tetracycline molecule. . The system of any one of clauses 172-174, wherein the inducible promoter element is induced by a derivative of a tetracycline molecule. . The system of clause 176, wherein the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. . The system of clause 177, wherein the derivative of the tetracycline molecule is doxycycline. . The system of any one of clauses 164-178, wherein the anti-CRISPR protein is selected from the group consisting of: AcrIIA4 and AcrII2. . The system of clause 179, wherein the anti-CRISPR protein comprises AcrIIA4. . The system of clause 180, wherein the anti-CRISPR protein comprises a fusion protein further comprising a degron tag. . The system of clause 181, wherein the degron tag is activated by IKZF3, HaloTag, SMASh tag, SD40, BD1L94V, BD3L387A, or B-LID. . The system of clause 181, wherein the degron tag comprises an FK506 binding protein (FKBP).Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 183, wherein the FKBP is a modified or mutant FKBP12. . The system of clause 184, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein FKBP12*). . The system of clause 185, wherein FKBP12* comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of any one of clauses 171- 186, wherein the degron tag is activated by a ligand that targets a von Hippel-Lindau (VHL) E3 ligase complex or a ligand that targets CRBN complex. . The system of clause 187, wherein the ligand comprises dTagV1. . The system of clause 171, wherein the degron tag comprises a LID domain.. The system of clause 189, wherein the LID domain comprises a C-terminal cryptic degron sequence. . The system of 190, wherein the cryptic degron sequence comprises the amino acid sequence of SEQ ID NO: 44. . The system of any one of clauses 189- 191, wherein the degron tag is activated by Shield1. . The system of clause 192, wherein the destabilizing domain comprises a ligand controllable destabilizing domain. . The system of clause 193, wherein the ligand controllable destabilizing domain is selected from the group consisting of: FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof. . The system of clause 194, wherein the ligand controllable destabilizing domain comprises a modified or mutant FKBP12. . The system of clause 195, wherein the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. . The system of clause 196, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*). . The system of clause 197, wherein the modified or mutant FKBP12 comprises FKBP12(F36V) (SEQ ID NO: 43). . The system of clause 198, wherein the ligand that controls the destabilizing domain is Shield1.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 194, wherein ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The system of clause 200, wherein the ligand that controls the destabilizing domain is trimethoprim (TMP). . The system of any one of clauses 164-201, wherein the Cas protein is selected from the group consisting of: Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. . The system of clause 202, wherein the Cas protein is Cas9. . The system of clause 202, wherein the Cas9 protein is a homolog or modified version of the Cas9 protein. . The system of clause 204, wherein the homolog or modified version of the Cas9 protein is selected from the group consisting of spCas9, nCas9, and dCas9.. The system of clause 205, wherein the Cas protein is dCas9. . The system of any one of clauses 164-206, wherein the Cas protein further comprises a Cas fusion protein domain. . The system of clause 208, wherein the Cas fusion protein domain comprises a Krüppel associated box (KRAB) domain. . The system of clause 207, wherein the KRAB domain is selected from: Zim3KRAB, KRAB-MeCP2, and Kox1KRAB. . The system of clause 208, wherein the KRAB domain is selected from a Zim3KRAB or Kox1KRAB domain. . The system of clause 210, wherein the Cas protein comprises dCas9 and the KRAB domain is Zim3KRAB. . The system of clause 210, wherein the Cas protein comprises dCas9 and the KRAB domain comprises Kox1KRAB. . The system of any one of clauses 164-212, wherein the polynucleotide encodes one or more guide RNA molecules. . The system of any one of clauses 164-213, further comprising a separate polynucleotide encoding one or more guide RNA molecules. . The system of clause 213 or 214, wherein one or more of the guide RNA molecules comprises an sgRNA molecule. . The system of any one of clauses 213- 215, wherein the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 216, wherein the constitutively expressed promoter comprises a U6 promoter. . The system of any one of clauses 213-217, comprising two or more sgRNA molecules. . The system of clause 218, wherein each sgRNA molecule is driven by the same Pol III promoter. . The system of clause 218, wherein each sgRNA molecule is driven by different Pol III promoter. . The system of clause 220, wherein the different Pol III promoters are U6 and H1. . The system of any one of clauses 213-221, further comprising an sgRNA molecule library. . The system of clause 222, wherein the sgRNA molecule library comprises a Pol III promotor. . The system of clause 223, wherein the sgRNA molecule library comprises a plurality of Pol III promoters. . The system of any one of clauses 213-224, wherein at one or more sgRNA molecules targets a gene of interest. . The system of any one of clauses 164 -225, wherein the polynucleotide further encodes a selectable marker. . The system of any one of clauses 164-226, wherein the anti-CRISPR is driven by a weak regulatory element. . The system of clause 227, wherein the weak regulatory element comprises an IRES. . The system of clause 227, wherein the weak regulatory element is selected from the group consisting of CMV-A, UbC, PGK, PGK100, and SV40. . The system of any one of clauses 164 or 165, wherein: the anti-CRISPR is a fusion protein comprising ACRIIA4 and a ligand inducible domain (LID), wherein the LID is activated by Shield1; the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40), and the ligand that controls the destabilizing domain is Shield1; the Cas protein is a Cas9 protein; the inducible promoter element is pTET; and the one or more guide RNA molecules comprises an sgRNA molecule library.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The system of clause 230, wherein: the anti-CRISPR fusion protein comprises the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. . The system of any one of clauses 1-231, wherein, upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to a target sequence and, optionally, edits the genomic locus to alter gene expression.. The system of clause 232, wherein the system comprises: a first sgRNA molecule comprising a first guide sequence capable of hybridizing to a first target sequence in a genomic locus of interest in the cell; a second sgRNA molecule comprising a second guide sequence capable of hybridizing to a second target sequence in the genomic locus of interest in the cell; and wherein upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to the first and second target sequences and edits the genomic locus of the cell by removing a DNA segment located between the first and second target sequences from the genome of the cell. . The system of clause 232 or 233, wherein the dynamic range of the system is enhanced upon introduction of one or more of: a.) a ligand that controls the destabilizing domain; b.) an agent that activates the inducible degradation domain; and c.) an agent that activates the ligand inducible alternative splicing switch. . An isolated, non-naturally occurring polynucleotide encoding: a Cas protein optionally fused to a destabilizing domain and operably linked to an inducible promoter element; one or more sgRNA molecules comprising one or more guide sequences capable of hybridizing to a target sequence in a genomic locus of interest in a cell; and optionally an anti-CRISPR. . The polynucleotide of clause 235, wherein the destabilizing domain comprises a ligand controllable destabilizing domain. . The polynucleotide of clause 236, wherein the ligand controllable destabilizing domain comprises a FK506 binding protein-12 (FKBP12), ecDHFR, and any derivatives thereof. . The polynucleotide of clause 237, wherein ligand controllable destabilizing domain comprises a modified or mutant cytosolic signaling protein FKBP12.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The polynucleotide of clause 238, wherein the modified or mutant FKBP12 comprises one or more mutations or modifications that create an enlarged binding pocket for FKBP12 ligands. . The polynucleotide of clause 239, wherein the modified or mutant FKBP12 comprises a mutation of the phenylalanine (F) at amino acid position 36 to valine (V) (F36V) (referred to interchangeably herein as FKBP* or FKBP12*). . The polynucleotide of clause 240, wherein the modified or mutant FKBP12 comprises FKBPF36VL106P (SEQ ID NO: 40). . The polynucleotide of clause 241, wherein the ligand that controls the destabilizing domain comprises Shield1. . The polynucleotide of clause 237, wherein ligand controllable destabilizing domain comprises ecDHFR (SEQ ID NO: 41) or ecDHFR (SEQ ID NO: 42). . The polynucleotide of clause 243, wherein the ligand that controls the destabilizing domain comprises TMP. . The polynucleotide of any one of clauses 235-244, wherein the Cas protein is selected from Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12, Cas13, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, Csf3, and Csf4. . The polynucleotide of clause 245, wherein the Cas protein is a homolog or modified versions of the Cas protein. . The polynucleotide of clause 246, wherein the homolog or modified version of the Cas protein is nCas9 or dCas9. . The polynucleotide of clause 245, wherein the Cas protein is Cas9. . The polynucleotide of any one of clauses 235-248, wherein the inducible promoter element comprises a TET-responsive promoter element (TRE). . The polynucleotide of clause 249, wherein the TRE comprises pTET. . The polynucleotide of clause 250, wherein the inducible promoter element is induced by a tetracycline molecule. . The polynucleotide of clause 251, wherein the inducible promoter element is induced by a derivative of the tetracycline molecule. . The polynucleotide of clause 252, wherein the derivative of the tetracycline molecule is selected from the group consisting of: doxycycline, chlortetracycline, demeclocycline, oxytetracycline, and minocycline. . The polynucleotide of clause 253, wherein the inducible promoter element is induced by doxycycline.Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The polynucleotide of any one of clauses 235-254, wherein one or more of the guide RNA molecules comprises an sgRNA molecule. . The polynucleotide of clause 255, wherein the guide RNA molecule is operably linked to a constitutively expressed Pol III promoter. . The polynucleotide of clause 256, wherein the constitutively expressed promoter comprises a U6 promoter. . The polynucleotide of any one of clauses 255-257, comprising two or more sgRNA molecules. . The polynucleotide of clause 258, wherein each sgRNA molecule is driven by different Pol III promoter. . The polynucleotide of clause 259, wherein the different Pol III promoters are U6 and H1. . The polynucleotide of any one of clauses 255- 260, further comprising an sgRNA molecule library. . The polynucleotide of clause 261, wherein the sgRNA molecule library comprises a Pol III promotor. . The polynucleotide of clause 262, wherein the sgRNA molecule library comprises a plurality of Pol III promoters. . The polynucleotide of any one of clauses 235-263, wherein at least one of the one or more sgRNA molecules targets a gene of interest. . The polynucleotide of any one of clauses 235-264, wherein the polynucleotide further encodes a selectable marker. . The polynucleotide of any one of clauses 235-265, further comprising a ligand inducible alternative splicing switch upstream from the encoded Cas protein. . The polynucleotide of clause 266, wherein the ligand inducible alternative splicing switch comprises an Xon splicing switch modulator. . The polynucleotide of clause 267, wherein activation of the Xon splicing switch modulator by branaplam (LMI070) activates expression of the encoded Cas protein. . The polynucleotide of clause 266, wherein the ligand inducible alternative splicing switch comprises SF3B3. . The polynucleotide of clause 266, wherein the ligand inducible alternative splicing switch comprises a SMN2 splicing switch modulator. . The polynucleotide of clause 266, wherein the SMN2 splicing switch modulator comprises an SMN2 ON-switch selected from pMM596 (SEQ ID NO: 11), pMM569 (SEQ ID NssO: 12), and pLS41 (SEQ ID NO: 13).Attorney Ref.59868.00075WO01 (GNE-0006-WO) . The polynucleotide of clause 266, wherein the SMN2 ON-switch is activated by risdiplam or RG7800, and wherein activation induces expression of the encoded Cas protein. . The polynucleotide of any one of clauses 235-269, wherein: the anti-CRISPR is a fusion protein comprising ACRIIA4 and a ligand inducible domain (LID), wherein the LID is induced by Shield1; the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40); and the ligand that controls the destabilizing domain is Shield1; the Cas protein is a Cas9 protein; the inducible promoter element comprises pTET; and the one or more guide RNA molecules comprises an sgRNA molecule library. . A vector comprising the polynucleotide of any one of clauses 235-273, for delivery of an inducible CRISPR-Cas system to a cell. . A cell comprising the inducible CRISPR-Cas system of any one of clauses 1- 234. . The cell of clause 275, wherein the cell is an eukaryotic host cell. . A cell line comprising a plurality of clonal cells stably transfected with the vector of clause 274. . A transgenic organism stably transfected with the vector of clause 274 or expressing the inducible CRISPR-Cas system of any one of clauses 1-234. . A model organism that constitutively expresses the inducible CRISPR-Cas system of any one of clauses 1-234. . A method of modifying a target nucleic acid in a cell, the method comprising: introducing the inducible CRISPR-Cas system according to any one of clauses 1-234 into the cell; contacting the cell with a ligand that activates the inducible expression element; and contacting the cell with a ligand that deactivates the anti-CRISPR and / or the destabilizing domain, thereby causing the Cas protein to modify the target nucleic acid. . The method of clause 280, wherein the inducible CRISPR-Cas system comprises both an anti-CRISPR and a destabilizing domain, and wherein the method comprises contacting the cell with one or more ligands that deactivate both the anti- CRISPR and the destabilizing domain.Attorney Ref.59868.00075WO01 (GNE-0006-WO) 282. The method of clause 280 or 281, wherein modifying the target nucleic acid in the cell alters expression of a target gene in the cell. 283. The method of clause 282, wherein altering expression of the target gene in the cell comprises decreasing expression of the target gene. 284. The method of clause 282, wherein altering expression of the target gene in the cell comprises increasing expression of the target gene. 285. The method of any one of clauses 280-284, wherein modifying the target nucleic acid in the cell comprises cutting the target nucleic acid at two different locations. 286. The method of clause 285, wherein cutting the target nucleic acid at the two different locations removes a target gene or gene segment from the cell. 287. The method of any one of clauses 280-286, wherein modifying the target nucleic acid in the cell further comprises editing the target nucleic acid. 288. A method of increasing a dynamic range of an inducible expression system in a cell, the method comprising: introducing the inducible CRISPR-Cas system according to any one of clauses 1-234 into the cell; culturing the cell under a first set of conditions in which the inducible CRISPR-Cas system exhibits a first expression level; contacting the cell with a ligand that activates the inducible expression element and a ligand that deactivates the anti-CRISPR and / or the destabilizing domain to cause the inducible CRISPR-Cas system to exhibit a second expression level that is higher than the first expression level, thereby increasing the dynamic range of the inducible expression system. 289. The method of clause 288, wherein the inducible CRISPR-Cas system comprises both an anti-CRISPR and a destabilizing domain, and wherein the method comprises contacting the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain.

[0205] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. Examples Example 1: The TET-ON CRISPR / Cas9 system exhibits significant basal editing

[0206] The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activityAttorney Ref.59868.00075WO01 (GNE-0006-WO) when Cas9 endonuclease and its sgRNA are co-expressed. Inducible systems allow temporal and tighter control of CRISPR-Cas9 activity by relying on transcriptional regulation of Cas9 expression. Inducible CRISPR / Cas9 and the guide RNA can be either co-delivered in an “all-in- one” plasmid format, or delivered sequentially on separate vectors, depending on the needs of the experiment. The TET-inducible CRISPR / Cas9 system minimizes toxicity associated with constitutive Cas9 expression and has the potential to precisely time target perturbation in a drug- dependent manner. However, like other conditional systems, background expression is a significant problem with 5%-60% background transcriptional activity in the absence of doxycycline.

[0207] To understand the limitations of the TET-inducible Cas9 system, its functionality was explored across multiple commonly used mammalian cell types. “All-in-one” systems are easier to deliver to cells, however any amount of leaky Cas9 expression can result in target disruption due to the transient influx of Cas9 and guide RNA plasmid in the cells immediately following the transfection. Since this design sets a higher standard for tight control, an “all-in-one” piggyBac plasmid (shown in FIG.1A) was designed with PiggyBac transposase (pBO). Cas9 expression is driven by a Tetracycline-inducible promoter (pTET). A human U6 promoter (pU6) drives constitutive expression of the CD81 sgRNA. Puromycin resistance gene (PuroR) and the reverse tetracycline transactivator (rtTa) are constitutively expressed from an EF1alpha promoter and separated by a self-cleaving T2A linker. Cells that have successfully integrated the cassette are enriched by puromycin selection. CD81 guide RNA was chosen to target a ubiquitously expressed cell surface protein. Such a target facilitates measurement of gene editing efficiency via flow cytometry.

[0208] The pTET plasmid was stably integrated into HEK293T, SW480, HeLa, HAP-1, A549, HCT116 and K562 parental cells. Stable cells were then treated with 2μg / ml Doxycycline (Dox) to induce CD81 knockout, or left untreated as a control, for 10 days. (FIG.1B). Cells were stained with an antibody specific for CD81 and protein levels were monitored by flow cytometry 10 days post-treatment. Flow cytometric analysis and data from all cell lines were analyzed with a similar gating strategy. The percentage CD81 knockout as determined by measuring CD81 negative cells by flow cytometry in both the untreated (gray bars) and treated (striped bars) cell populations (FIG.1C). Data are shown as mean values ± SD from at least three independent experiments.

[0209] Significant Dox-independent editing of CD81 was observed in all the mammalian cells tested as illustrated in the flow cytometry plots of HEK293T pTET and pTET-NTC stable cells with and without Dox treatment (see, e.g., FIG.1C and FIG.1D). SSC-A (side scatter) and FSC-A (forward scatter) profiles were used to isolate the live cell population. Doublets were excluded using FSC-A and FSC-H profiles.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0210] FIG. 1D illustrates representative flow cytometry plots of pTET and pTET-NTC stable HEK293T cells with and without Dox treatment. SSC-A (side scatter) and FSC-A (forward scatter) profiles were used to isolate the live cell population. Doublets were excluded using FSC-A and FSC-H profiles. The pTET-NTC control line was stained in parallel to define the CD81 positive population.

[0211] The pTET-NTC control line was also stained in parallel to define the CD81 positive population. Background editing efficiencies were variable across lines ranging from ~8% in HeLa to as high as 60% in SW480. Upon treatment with Dox, significant CD81 knockout (more than 95% for most of the lines) was observed as expected. These data highlight a pervasive problem of leaky Cas9 expression with the current TET-CRISPR / Cas9 system and underscore a critical need to build a tightly regulated inducible CRISPR system. Example 2: An ‘ultra-tight” TET-CRISPR / Cas9 system demonstrates minimal basal editing and high knockout efficiency

[0212] To minimize background editing, a series of modifications to the TET-CRISPR / Cas9 construct were by adopted by engineering combinations of different strategies that regulate Cas9 in the uninduced state including i.) conditional degradation to control stability, ii.) structural inhibition to control activity, and iii.) alternative splicing to control translation (FIG.2A). The various engineered and tested transgenic circuits, control modules for each, and the specific drug(s) required for functional Cas9 activity are identified and tabulated below (Table 6). Table 6: Transgenic Circuits Control  ds Attorney Ref.59868.00075WO01 (GNE-0006-WO) pTET; AcrIIA4‐LID  CD81  ‐  AcrIIa4‐LID  Dox + Shield1 RNA expressed, the control modules incorporated to reduce uninduced expression of Cas9, and the ligands (drugs) required to turn on Cas9 expression and, in certain cases, activity. Unless otherwise indicated, the drugs were used at the following concentrations: 2μg / ml Dox; 1μM Shield1; 2μM TMP; 1μM dTagv1; 20-150μM Branaplam, depending on the cell line.

[0214] FIG.2A is a schematic representation of the innovative control modules added to the basic pTET system that significantly reduced uninduced Cas9 activity. One aspect of a pTET inducible system provided herein is the provision of control module 1 that comprises an encoded destabilizing domain or a splicing switch upstream from Cas9. In some embodiments, control module 1 is a destabilizing domain, for example, DD: a FKBPF36VL106P destabilizing domain, or DHFR, a destabilizing domain based on a mutant E. coli dihydrofolate reductase. Uninduced expression of the Cas9 fusion protein is degraded by the DD or DHFR destabilizing domains. A second aspect of a pTET system provided herein is the provision of control module II that comprises an anti-CRISPR gene with an NLS fusion expressed constitutively, for example, from a human PGK promoter. In some embodiments, the anti-CRISPR gene encodes AcrIIA4, a protein that inhibits leaky Cas9 activity. In some embodiments, control module 2 encodes a fusion protein fusing the anti-CRISPR to a degron. For example, control module 2 encodes AcrIIA4 fused to a ligand inducible degron (AcrIIA4-LID) or the FKBP12(F36V) degron (AcrIIA4-dTag), optionally, with a GSAGSAAGSG (SEQ ID NO: 46) flexible linker, that can be induced to degrade the anti-CRISPR protein when Cas9 activity is desired.

[0215] FIG.2B is a cartoon depicting conditional degradation of DD or DHFR fused Cas9 in untreated cells. When Cas9 expression is desired, addition of a stabilizing ligand (+Drug) degrades the destabilizing domain and restores protein stability. To reduce uninduced Cas9 protein expression levels, previously described conditional destabilizing domains, DD or DHFR (Banaszynski et al. Cell.2006; 126:995–1004; Iwamoto et al.2010), were genetically fused, rendering the expressed Cas9 protein unstable in cells that are untreated, that is, without addition of the corresponding stabilizing ligand (drug) that causes degradation of the destabilizing domain (FIG.2B, and Table 6). DD is an engineered variant of the FKBP12 protein that is continuously degraded until stabilized by treatment with Shield1. DHFR is an E. coli dihydrofolate reductase mutant that destabilizes tagged proteins and is stabilized by treatment with Trimethoprim (TMP).Attorney Ref.59868.00075WO01 (GNE-0006-WO) In these embodiments, addition of Dox plus Shield1 or TMP stabilizes the induced Cas9 protein achieving target gene knockout.

[0216] Secondly, a system expressing an anti-CRISPR gene was tested to inhibit leaky Cas9 activity by interfering with its ability to bind DNA. AcrIIA4 was previously identified as a highly effective anti-CRISPR (Nakamura et al.2019; Yang and Patel 2017). As an additional layer of control, the system was engineered to produce AcrIIA4 fusions with previously characterized degron tags, LID or FKBP12(F36V). FIG.2C illustrates embodiments where an anti-CRISPR degron tag fusion is expressed, where addition of a degron tag specific ligand (+Drug) degrades the anti-CRISPR protein thereby relieving inhibition of Cas9 activity. The dTag degron (FKBP12(F36V)) is a mutant version of the FKBP12 protein that is selectively degraded upon treatment with the ligand dTagV1 (Nabet et al.2018; Nabet et al.2020). The LID degron is degraded in response to treatment with the Shield1 ligand (Bonger et al.2011). In the OFF state, the constitutively expressed AcrIIA4 inhibits leaky Cas9 protein. Co-treatment with Dox plus dTagV1 or Shield1 induces Cas9 and in parallel degrades the anti-CRISPR protein alleviating the inhibitory effect (FIG.2C, Table 6).

[0217] Finally, it was decided to exploit an alternative splicing X-on system (Monteys 2021) based on a splicing cassette from SF3B3 to reduce leaky translation of Cas9. In the absence of drug treatment, the splicing event excludes the exon that contains an ATG start codon eliminating Cas9 protein translation. FIG.2D is a schematic illustrating the splicing switch mechanism, i.e., drug treatment induces an alternative splicing mechanism that retains the start codon leading to translation of Cas9, thus controlling leaky Cas9 levels. In untreated cells, the splicing isoform of Cas9 is not translated due to lack of the ATG start codon. Upon treatment with Dox and the splicing modifier drug Branaplam, a switch in the splicing mechanism retains the ATG-containing exon, allowing in-frame translation of the Dox-induced Cas9 protein.

[0218] In order to compare the various strategies, stable polyclonal lines were generated with each of these modified genetic circuits in cell lines that exhibited high (293T), mid (A549) and negligible (K562) levels of background editing in the uninduced state. As a control the CD81 sgRNA was replaced within the pTET plasmid with a control sgRNA (pTET-NTC) to define baseline CD81 levels. Each stable cell line was treated with the specific drug combination to induce target gene knockout (Table 6), while an equal number of cells were left untreated as a control. To evaluate editing efficiency, CD81 levels were measured by staining and flow cytometry 10 days post- treatment. To further distinguish variations in efficacy of these methods with the aim of identifying the most tightly controlled system, signal-to-noise ratios were calculated between treated and untreated cells to determine the dynamic range of editing between ON / OFF states.

[0219] FIGS.2E-2F illustrate the various pTET modified PiggyBac constructs as described in FIG. 1A and Table 6 that were stably transfected into 293T (FIG.2E) and A549 (FIG.2F) cells followed by drug treatment and measurement of CD81 levels. The percentage of CD81 negativeAttorney Ref.59868.00075WO01 (GNE-0006-WO) cells was determined in untreated cells (dark gray bar on left) and treated cells (light gray bar on right). Box plots display the dynamic range calculated as the ratio of percentage knockout in treated vs. untreated cells for each method. All the re-engineered systems displayed dramatically reduced CD81 knockout efficiencies (p<0.0001) in untreated cells as compared to pTET for both 293T (FIG.2E) and A549 (FIG.2F). In untreated K562 cells, the background editing for pTET was comparable to pTET-NTC and all the modified systems maintained this low level of background editing (FIG.2I).

[0220] When comparing the CD81 knockout efficiencies in the drug treated ON state there were some notable differences with the various methods. First, editing efficiency was drastically reduced with pTET-Xon-Cas9 in all three cell types, suggesting additional considerations may be needed to make Xon and pTET systems compatible (FIG.2E, 2F, S2A). The pTET-AcrIIA4 line also displayed significantly compromised editing levels (~49%) in drug-treated A549 cells (FIG. 2F). Presumably the induced levels of Cas9 are insufficient to overcome the suppressive effects of the constitutively expressed anti-CRISPR protein in this cell line. Consistent with this notion, editing efficiency was restored in the degron controlled AcrIIA4 lines, pTET-AcrIIa4-LID and pTET-AcrIIa4-dTag, by elimination of Cas9 inhibition through ligand-induced AcrIIA4 degradation (FIG.2F).

[0221] The remaining modified methods maintained knockout efficiencies greater than 80% in all three cell types (FIGS.2E, 2F, 2G).

[0222] Although the destabilizing domain tagged Cas9 versions (pTET-DD-Cas9 and pTET-DHFR- Cas9) displayed comparable percentage knockout in the ON state, the latter system displayed a lower dynamic range of editing efficiency due to elevated background editing, indicating that the DD-tagged Cas9 module achieved relatively tighter control (FIGS.2E, 2F, 2G).

[0223] In HEK293T and A549, there was no significant difference in drug-induced percent CD81 KO between the two degron-tagged AcrIIA4 systems (FIGS.2E, 2F). In K562, despite a ~10% higher editing efficiency (p=0.0060) with the pTET-AcrIIA4-dTag system, this was offset by a modest increase in background editing (FIG.2G). Consequently, in all three cell types, both systems displayed no significant differences in signal-to-noise ratios, suggesting the two degron modules are interchangeable (FIGS.2E, 2F, 2G).

[0224] When comparing the pTET versions that harbor the destabilizing domain tagged Cas9 modules vs. the degron tagged AcrIIA4 modules, no significant differences were detected in HEK293T with respect to percentage knockout or signal-to-noise ratios. In A549, despite the higher editing efficiency displayed by the pTET-DHFR-Cas9 system in comparison to the pTET- AcrIIA4-degron versions, the associated increase in background editing resulted in lower signal- to-noise ratios (FIG.2F). In K562, pTET-DHFR-Cas9 had higher editing efficiency than pTET- AcrIIA4-LID but not pTET-AcrIIA4-dTag. However, differences in signal-to-noise ratios in thisAttorney Ref.59868.00075WO01 (GNE-0006-WO) cell type were insignificant for all four versions indicating modest shifts in background editing levels (FIG.2G).

[0225] In general, among the four control modules, the destabilizing domain tagged Cas9 modules displayed the highest ON state knockout efficiency (>90%) in all three cell types. Specifically, the pTET-DD-Cas9 system demonstrated both high editing efficiency and a relatively high dynamic range.

[0226] Finally, the pTET-DD-Cas9-AcrIIA4-LID system, which combines both the DD-tagged Cas9 and the LID degron tagged anti-CRISPR modules, emerged as an “ultra-tight” system demonstrating the highest dynamic range of Cas9 activity (FIGS.2E, 2F, 2G). In all three cell types, the pTET-DD-Cas9-AcrIIA4-LID system uniquely displayed negligible background editing levels that was indistinguishable from pTET-NTC in untreated cells, while simultaneously sustaining an 80% or higher efficiency of target gene knockout upon drug exposure (FIG.2E, 2F, 2G). Henceforth, this system is referred to as “pTET-Ultra-tight”.

[0227] The “ultra-tight” feature of the optimized TET-CRISPR / Cas9 system is preserved in additional cell lines.

[0228] Evaluation of the pTET-Ultra-tight system was expanded to additional cell lines to explore broad applicability. The pTET-Ultra-tight construct was stably integrated as well as pTET, into SW480, HAP-1, HeLa and HCT116. These four cell lines have a range of drug-independent basal editing ranging from ~8% to ~60%. Stable cells were treated with either Dox (pTET) or Dox + Shield1 (pTET-Ultra-tight) for 10 days followed by flow cytometry to measure CD81 levels. Consistent with our previous observations, the pTET-Ultra-tight lines displayed substantially reduced basal editing in untreated cells relative to pTET in all cell types (FIG.2H). Of the tested lines, SW480 cells represented an outlying context, where pTET:Ultra-tight was unable to fully reduce background editing. pTET:Ultra-tight was therefore compared to an expanded set of genetic circuits (FIG.2J), revealing that it remained the superior option for balancing on versus off-state editing (FIG.2K). Upon drug exposure, the pTET-Ultra-tight system maintained a knockout efficiency greater than 90% for all lines, with the exception of HCT116 (~55-58%). HCT116 displayed less than 80% CD81 knockout even with the pTET circuit, suggesting the Dox-induced levels of Cas9 are insufficient to achieve a complete knockout in this particular cell type (FIG.2H). Despite this reduction in percent knockout, when comparing signal-to-noise ratios between the two systems, pTET-Ultra-tight was still favored over pTET in all four cell lines, as it displayed a significantly higher dynamic range of editing efficiency, indicating tighter regulation (FIG.2I). Taken together, these results demonstrate the broad utility of our pTET-Ultra tight system to minimize to background editing levels while maintaining significant target gene disruption in multiple cell lines.Attorney Ref.59868.00075WO01 (GNE-0006-WO) Example 3: The pTET-Ultra-tight system demonstrates negligible background editing at additional target genes

[0229] To investigate if the pTET-Ultra-tight system maintains stringent regulation at other target loci besides CD81, the CD81 sgRNA in pTET and pTET-Ultra-tight constructs was replaced with sgRNA targeting non-essential cell surface expressing markers, β2-microglobulin (B2M), CD298 or CD9. These constructs were integrated into HEK293T cells, stable cells were left untreated or treated for 10 days with 2μg / ml Dox (pTET) or 2μg / ml Dox + 1μM Shield1 (pTET-Ultra-tight) to induce Cas9 expression and evaluated target protein levels 10 days later by staining and flow cytometry (FIGS. 3A, 3C, and 3D). Bar graphs are shown as mean values + / − SD from triplicate experiments. Statistical comparison between the methods was performed with either Two-way ANOVA with Tukey’s multiple comparisons test (A-C) or unpaired two-tailed t-test (D-F). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (FIGS.3B, 3D, and 3F). Untreated cells were included as a control to determine background editing. Consistent with our observations for CD81, variable but substantial background editing was detected in untreated cells at all three target loci with the pTET system (FIGS.3A, 3C, and 3E). In contrast, pTET-Ultra-tight displayed strikingly improved reduction in background editing for all three targets (FIGS. 3A, 3C, and 3E). Importantly, upon drug exposure, a knockout efficiency greater than 90% was observed for all three target genes (FIGS.3B, 3D, and 3F).

[0230] Evaluation of ratios of CRISPR activity in the treated vs. treated states indicated a significant increase in dynamic range with the pTET-Ultra-tight system, underscoring the “ultra-tight” nature of this method. In conclusion, the pTET-Ultra-tight system not only exhibits strict editing control across multiple cell lines but demonstrates negligible basal editing while maintaining high drug induced knockout efficiency across several target loci. Example 4: “Ultra-tight” Dox independent inducible CRISPR / Cas9 system

[0231] While the Dox inducible system is widely used, it has limited compatibility with cell models and biological process. For instance, doxycycline has been shown to interfere with mitochondrial activities which renders the dox inducible system undesirable for studies on mitochondria (Moullan et al.2015; Chatzispyrou et al.2015). Furthermore, the ability to orthogonally control two processes in the same cell model is a common need that necessitates exploration of an alternative inducible system for controlling CRISPR activity.

[0232] Previous studies have reported inducible control of Cas9 using conditional degradation as well as alternative splicing (Maji et al.2016; Senturk et al.2017; Monteys et al.2021).

[0233] To test the feasibility of Dox-independent inducible Cas9, dox independent inducible Cas9 systems were investigated as illustrated in FIG.4A. Cas9-DD and DD-Cas9 systems were engineered where the DD domain is fused to Cas9 at the C- or N- terminus, respectively. In theAttorney Ref.59868.00075WO01 (GNE-0006-WO) DHFR-Cas9 system, the DHFR domain is fused to the Cas9 at the N-terminus. In the Xon-Cas9 system, ATG is deleted from the Cas9 coding sequence and provided in the alternatively spliced exon.

[0234] Plasmid constructs were delivered into HEK293T cells via the piggybac transposon system to generate stable cell lines. After antibiotic selection, cells were cultured for 10 days with or without drug treatment. FIG.4B illustrates the CD81 knockout percentage in HEK293T cells that stably express the inducible Cas9 systems with a single regulatory module showing the results in untreated cells in the bar on the left and in treated cells in the bar on the right for each system. Surprisingly, both DD and DHFR fused Cas9 led to 100% background expression of CD81 in the untreated group, and placing the DD tag at the C-terminus of Cas9 did not yield tighter control. By contrast, the Xon system demonstrated inducible control of Cas9 activity. However, this system displayed nearly 20% leaky cutting in our test, leaving room for further improvement (FIG.4B).

[0235] Given the success in suppressing background (uninduced) Cas9 activity within the pTET system, similar strategies were deployed to improve the Xon-Cas9 system (FIG.4C). The DD and alternatively, the DHFR domain, were added to the N- or C-terminus, respectively, of Cas9 under control of Xon. Our data with the optimized TET systems suggested that N-terminal fusion of DD and DHFR provides tight control. However, in the context of Xonswitch, N-terminal fusion led to 100% expression in the uninduced state (FIG.4D). It was reasoned that this could be due to an internal start codon provided by DD or DHFR for Cas9 which bypassed the requirement of the middle exon inclusion (FIG.4E).

[0236] Xonswitch systems where the destabilizing domain DD or DHFR was fused to the C- terminus of Cas9 were generated and confirmed to reduce the background activity of Cas9 in HEK293T cells (FIGS.4F and 4G). Xonsystems with an anti-CRISPR protein AcrIIA4 were engineered with or without FKBP12(F36V) (the dTag degron) (FIG.4A). The addition of an AcrIIA4 module dramatically reduced the leakiness of Xon-Cas9 in HEK293T cells, but at the cost of significantly compromised inducible cutting (FIG.4F). In contrast, addition of the AcrIIA4-dTag module not only reduced the background activity of Cas9 but also maintained the inducibility of the system, offering the best dynamic range (FIG.4F, 4G).

[0237] The Xon-Cas9 + AcrIIA4-dTag combination was then tested in a different cell line, K562. Similar to our observations in HEK293T, the unmodified Xon-Cas9 system displayed a 19% basal editing in the absence of induction (FIG.4H). To our surprise, although the Xon-Cas9 + AcrIIA4- dTag combination reduced basal editing in the absence of induction, it also resulted in poor induction of Cas9 cutting (FIG.4H) as characterized by a low dynamic range. In this design, the AcrIIA4 protein is under control of a PGK promoter (FIG.4A). It was hypothesized that the expression of AcrIIA4 is too high in K562 to be sufficiently degraded by the dTag upon drug treatment.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0238] Strategies to reduce the expression level of AcrIIA4 were considered with the idea that this would maintain suppression of Cas9 in the uninduced state but would be completely degraded in the induced state. Therefore, it was decided to test another weak promoter UBC (Qin et al.2010) in addition to an IRES element to express AcrIIA4 (FIG.4J and FIG.4H). IRES elements are commonly used to express multiple genes simultaneously and have been reported to drive lower expression of the downstream gene. Surprisingly, the UBC promoter version led to an even poorer induction of cutting and lower basal editing in the absence of induction, suggesting UBC promoter is stronger than PGK in K562 cells (FIG.4H). In contrast, IRES-AcrIIA4-dTag led to significantly reduced basal editing in the absence of induction with no compromise in induced cutting (FIG.4H). Box plots display the dynamic range for each method. Bar graphs are presented as mean values + / − SD from triplicate experiments (FIG.4I). Statistical significance was determined with either Two-way ANOVA with Tukey’s multiple comparisons test (B, D) or unpaired two-tailed t-test (C, F). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These results demonstrate that variability in Cas9 expression levels and inducible system activity across cell types will necessitate empirical testing for optimal performance. Example 5: pTET Ultra-tight system exhibits tight control over essential gene knockout

[0239] Inducible Cas9 systems enable the study of essential genes. However, a system with a high level of uninduced Cas9 activity can result in a suboptimal phenotype due to premature disruption of the target, leading to cell death and elimination of these cells from the population. Although clonal selection can overcome this issue, it is time consuming, laborious, and can misrepresent heterogeneity within the population. An ideal inducible system should exercise tight control over target perturbation resulting in a clear-cut phenotype at the population level.

[0240] Given the minimal uninduced Cas9 activity of our pTET-Ultra-tight system, it was sought to test inducible knockout of an essential gene. pTET and pTET-Ultra-tight all-in-one plasmids were constructed containing sgRNA targeting the Polo-like kinase 1 (PLK1) gene. PLK1 is a serine / threonine kinase and a master regulator of cell cycle and mitosis. Disruption of PLK1 results in proliferation defects. PLK1 is overexpressed in several cancers and is considered a potential target for cancer therapy.

[0241] The pTET and pTET-Ultra-tight system was stably integrated into 293T cells and tested inducible knockout of PLK1 with two different sgRNAs (sgRNA1 and sgRNA2). An equal number of cells were left untreated or exposed to inducing drugs. Stable lines were generated in HEK293T with pTET and pTET-Ultra-tight lines expressing a PLK1 guide RNA, while pTET- NTC expresses a control guide RNA. Equal number of cells were plated with or without drug treatment and imaged in an Incucyte over 5 days in 4 h intervals. Cell growth was monitored over five days by periodic imaging in an Incucyte as a readout of PLK1 knockout (FIG.5A). FIG.5BAttorney Ref.59868.00075WO01 (GNE-0006-WO) (sgRNA1) and FIG.5D (sgRNA2) illustrate the growth curves shown for each line in the presence and absence of drug treatment. Cells were treated with 2ug / ml Dox (pTET and pTET-NTC), 2μg / ml Dox + 1μM Shield1 (pTET-Ultra-tight), or left untreated. Data are shown as mean values ±SEM of triplicate experiments. FIGS.5C (sgRNA1) and 5E (sgRNA2) are box plots that display fold change in % confluence for each line. Statistical significance was determined with ordinary one-way ANOVA. ****p < 0.0001. (D) Top: Schematic of the human H3-3A locus showing the sgRNA cut sites and expected amplicon products from wild-type and deleted alleles. Blue squares indicate exons with exons 2 and 3 depicted as dark blue squares. Half arrows represent PCR primers. Bottom: PCR gel image of amplicon products from the indicated cell lines are shown. Cells were either left untreated or treated with 2ug / ml Dox (parental, pTET, pTET- NTC) or 2μg / ml Dox + 1μM Shield1 (pTET-Ultra-tight) for 72 h.

[0242] The pTET-NTC line was similarly treated and monitored as a control. Although stable lines were successfully generated, there were notable differences in growth rates in the presence and absence of drug treatment. In the untreated state, the pTET line targeting PLK1 demonstrated growth retardation with both sgRNAs, indicating knockout of the target gene in the absence of induction (FIG.5B (sgRNA1) and FIG.5D (sgRNA2)). Gratifyingly, the growth of the pTET- Ultra-tight line was similar to the pTET-NTC control line, suggesting the Ultra-tight circuit is able to minimize leaky cutting of PLK1. Upon drug treatment, while growth was suppressed in both pTET and pTET-Ultra-tight lines, the latter demonstrated a greater reduction in growth rate. The pTET-Ultra-tight system had significantly higher dynamic range between treated and untreated states, as compared to pTET, due to elimination of leaky editing, while maintaining high knockout efficiency in the ON state (FIGS.5C (sgRNA1) and 5E (sgRNA2)). Consistent with our earlier observations, the pTET-Ultra-tight system retains superiority over pTET and is more suited for the study of essential genes due to the ability to implement precise control over target gene perturbation.

[0243] Efficient target gene knockout with a single guide RNA relies on repair of the double- stranded break by the cell’s error prone non-homologous end joining (NHEJ) pathway, which can introduce indels disrupting the coding region of the gene. However, this process can be incomplete if it doesn’t result in a frameshift mutation or if there are alternative start codons. Moreover, a single guide RNA (sgRNA) strategy cannot be used to create genomic deletions. On the other hand, paired sgRNAs can facilitate the deletion of larger genomic regions. By designing sgRNAs to target sites flanking the region of interest, the intervening sequence can be efficiently removed. This is particularly useful for studying the function of non-coding regions, regulatory elements, or large gene segments. Furthermore, using paired sgRNA results in complete loss of function by removal of several exons within a gene or the entire gene. Multiplexing guide RNAs can also target more than one gene simultaneously.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0244] As shown in FIG.5F, it was confirmed that the pTET-Ultra-tight system can be combined with dual gRNAs to inducibly delete a ~2.5kb genomic locus by introducing dual sgRNA into the pTET and pTET-Ultra-tight constructs to eliminate exons 2 and 3 within the H3-3A gene. Stable lines were generated in 293T and cells were either left untreated or exposed to inducing drug(s) for 72 hours followed by PCR genotyping with primers spanning the target sites. An amplicon size of 2949bp indicates presence of wild-type alleles, while successful deletion of the target locus should result in a shorter amplicon of 416bp with the same primers. (A) pTET and pTET- Ultra-tight stable HEK293T lines expressing a different PLK1 sgRNA were generated. Growth curves are shown for each stable line in the presence and absence of drug treatment. Cells were treated with 2μg / ml Dox (pTET and pTET-NTC), 2μg / ml Dox + 1μM Shield1 (pTET-Ultra-tight), or left untreated. Essential gene knockout was confirmed with the pTET-ultra-tight system. (A) pTET and pTET-Ultra-tight stable HEK293T lines expressing a different PLK1 sgRNA were generated. Growth curves are shown for each stable line in the presence and absence of drug treatment. Cells were treated with 2μg / ml Dox (pTET and pTET-NTC), 2μg / ml Dox + 1μM Shield1 (pTET-Ultra-tight), or left untreated. Data are shown as mean values ±SEM of triplicate experiments. (B) Box plot displays fold change in percent confluence for each line. Statistical significance was determined by ordinary one-way ANOVA. *p < 0.05.

[0245] In the absence of drug treatment, a 2949bp band corresponding to the wild-type allele was detected in parental, pTET-NTC control, pTET and pTET-Ultra-tight cells. However, a 416bp band was also detected in untreated pTET cells indicating leaky editing in this cell line. Conversely, no dropout band was observed in the pTET-Ultra-tight cells. Upon drug treatment, a 416bp deletion product was observed for both pTET and pTET-Ultra-tight systems with a concomitant reduction in signal for the 2949 wild-type amplicon. As expected, a deletion product was not detected for either parental or pTET-NTC control cells. Taken together, the pTET-Ultra- tight system is preferable over pTET for a dual guide strategy due to lack of leaky editing and efficient deletion of the ~2.5kb target locus in the treated state. Example 6: Ultra-tight inducible CRISPRi system

[0246] While CRISPR knockout is widely used to study gene functions, it creates double stranded DNA breaks which could be undesirable for certain applications. CRISPR interference (CRISPRi) emerged as another powerful tool, in which the inactive Cas9 (dCas9) is often fused to the Krüppel-associated box (KRAB) repressor and blocks the transcription of the target guided by the sgRNA. The commonly used KRAB domain is derived from KOX1 gene but recent studies have reported ZIM3’s KRAB domain as a superior alternative. CRISPRi can be particularly advantageous for large scale pooled library screens that are combined with single cell RNA-seq, as the knockdown efficacy of each guide RNA can be directly measured from the screen.Attorney Ref.59868.00075WO01 (GNE-0006-WO) Inducible CRISPRi is generally preferred to study genes that are essential or whose phenotype is time sensitive. Given that the silencing effect of CRISPRi is generally reversible, it is possible that the leaky expression of the Cas9 protein is better tolerated than it is for inducible CRISPR knockout systems.

[0247] Here, an objective was to test the TET-inducible CRISPRi systems first. KRAB domain from either KOX1 or ZIM3 was fused to the dCas9 that is driven by the TRE3G promoter (FIG.6A). The inducible dCas9KRAB constructs were integrated into the cells via lentivirus transduction. Cell line was established first and then the sgRNA was introduced by a second round of transduction. The stable cell line is then transduced with lentivirus expressing guides targeting CD81 promoter. FIG.6B shows the CD81 silencing in the inducible CRISPRi cells as measured by flow cytometry. pTET-KOX1, TET-on dCas9 fused with KRAB domain from KOX1; pTET- ZIM3, TET-on dCas9 fused with KRAB domain from ZIM3; pTET-DD-ZIM3, TET-on system expressing a fusion protein containing DD, KRAB from ZIM3 and dCas9. CD81 expression was measured after 7 days of treatment or non-treatment. Stable inducible ZIM3 and DD-ZIM3 lines from (A) were transduced with lentivirus expressing sgRNA targeting PLK1 or a non-targeting control. The cells were selected and then treated or untreated for 5 days before measurement by Cell Titer Glo assay. FIG.6C shows that CD81 silencing with KOX1 vs. ZIM3 based inducible CRISPRi systems was confirmed by analysis of representative flow cytometry result of the CD81 stained cells with inducible CRISPRi systems. DLD1 cells expressing sgCD81 and pTET-KOX1 or pTET-ZIM3 were treated with 250ng / ml Dox or untreated for 7 days. The cells were stained with CD81 antibody conjugated with PE. CD81 positive and negative cells were separated by the dashed line. Control: parental DLD1.As expected, the KOX1 KRAB exhibited a minimal leaky effect on CD81 in the untreated condition, suggesting the leaky expression of dCas9- KOX1KRAB fusion protein from the TET system is well tolerated (FIG.6B, 6C). However, upon dox treatment, KOX1KRAB only showed a moderate silencing activity for CD81 (FIG.6B, 6C). On the contrary, ZIM3KRAB drastically outperformed the KOX1KRAB in silencing CD81, agreeing with the previous report, but as a tradeoff, it suffered from a massive leaky effect, leading to silencing of CD81 in 40% of the cells without induction (FIG.6B, 6C). This prompted us to optimize the ZIM3 based CRISPRi system to reduce its leakiness and maintain its activity.

[0248] It was reasoned that a similar strategy from the above study by adding a destabilizing domain to the ZIM3KRAB-dCas9 would achieve such goals. The DD domain was selected based on its superior performance over DHFR. The undesired leaky silencing of CD81 was suppressed to a minimal level, while the desired silencing upon treatment remained uncompromised (FIG.6B). As a result, the dynamic range of the inducible CRISPRi system is increased over 13 folds than the original ZIM3 (FIG.6B). As a further test, the system was used to knockdown an essential gene PLK1, silencing of which leads to cell death (FIG.6C). As expected, a high cell death was observed overall by Cell Titer Glo assay in the untreated inducible ZIM3-dCas9 (FIG.6D). PLK1Attorney Ref.59868.00075WO01 (GNE-0006-WO) silencing shown as normalized cell death. Normalized cell death is calculated as (cells with sgNTC - cells with sgPLK1) / (cells with sgNTC) in each condition. Bar graphs are shown as mean values + / − SD from triplicate experiments. Statistical comparison between the methods was performed with unpaired two-tailed t-test (B, D). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001. The leaky effect is nearly completely suppressed by the fusion of DD domain to the ZIM3-dCas9 (FIG.6D). The silencing activity upon treatment is not compromised by the DD domain incorporation compared to ZIM3-dCas9 alone (FIG.6D). In summary, combination of DD destabilizing domain with the ZIM3 based CRISPRi system offers an ultra-tight and yet highly inducible tool for gene silencing. Example 7: Promoter hijacking strategy for stable integration of the splice switch based inducible Cas9 into a genome

[0249] FIG.7 is a schematic diagram of an exemplary promoter hijacking strategy for CRISPR knock- in for stable integration of a splice switch based inducible Cas9 into a genome.

[0250] To prevent transgene silencing, the splice switch based inducible Cas9 cassette is inserted downstream of a promoter of a housekeeping gene (such as ACTB, as shown as an example in FIG. 7). The inducible Cas9 is then driven by the endogenous promoter that is less susceptible to silencing. Only one allele is hijacked to maintain the housekeeping gene’s function. The insertion can be achieved by CRISPR mediated HDR (Homology Directed Repair) or HITI (Homology Independent Target Integration). Example 8: The Tet-On CRISPR / Cas9 system exhibits significant basal editing

[0251] To establish a baseline for background versus drug-induced editing with a foundational Tet-On Cas9 variant, piggyBac was used to stably integrate an “all-in-one” transgene across a panel of seven human cell lines. Here, S. pyogenes (Sp) Cas9 was placed under the control of the TRE3G Tet-inducible promoter upstream of a U6 promoter for constitutive expression of an sgRNA targeting CD81. A separate constitutive promoter (EF1α) was incorporated to drive expression of the reverse tetracycline transactivator (rtTA) and puromycin resistance (PuroR) gene, each separated by a T2A sequence to enable co-expression as distinct polypeptides. Hereafter, this construct, inclusive of the CD81 sgRNA, is referred to as “pTET”.

[0252] Disruption of CD81, encoding a ubiquitously-expressed cell surface protein, can be easily detected by a highly specific antibody and quantified through flow cytometry (see FIGS.1A-1B). To confirm antibody specificity, a CD81 knockout HEK293T line (pCAG-Cas9) was generated with constitutive Cas9 and coincident CD81 sgRNA expression from the CAG and U6 promoters, respectively (FIG.8). Wild-type and knockout cells were stained with anti-CD81 or an IgG isotype control, alongside unstained samples. Minimal signal in the isotype and unstained samplesAttorney Ref.59868.00075WO01 (GNE-0006-WO) confirmed the specificity of anti-CD81 staining, which was detected in wild-type but not knockout cells. After generation of stable polyclonal lines with pTET, cell populations were either treated with Dox to induce CD81 knockout or left untreated as a control. To ensure complete Cas9 cutting upon drug treatment and to capture cumulative leaky cutting in the untreated cells, loss of CD81 was measured via flow cytometry ten days after induction, with flow cytometry plots shown in FIG. 1D. A representative gating strategy is shown for HEK293T (FIG. 1D). Significant and reproducible Dox-independent loss of CD81 was observed in all tested cell lines (FIG. 1C). Background knockout levels ranged from a low of ~4% in K562 (bone marrow) cells to as high as 60% in SW480 (colorectal) cells. Upon treatment with Dox, robust (≥95%) depletion of CD81 was observed in all but one cell line (HCT116, ~80% depletion). Despite potent target disruption, these data highlight a pervasive problem of leaky Cas9 activity in the context of a conventional Tet-On system.

[0253] FIG.8 shows representative flow cytometry results of HEK293T wild-type and CD81 knockout cells stained with APC-conjugated CD81 antibody or an IgG isotype control antibody, with unstained samples included as negative controls. The knockout line was generated by stably integrating a plasmid in which Cas9 and CD81 sgRNA are constitutively expressed from the CAG and U6 promoters, respectively (schematic shown). FIG.1D shows representative flow cytometry plots of pTET and pTET-NTC stable HEK293T cells with and without Dox treatment. SSC-A (side scatter) and FSC-A (forward scatter) profiles were used to isolate the live cell population. Doublets were excluded using FSC-A and FSC-H profiles. The pTET-NTC control line was stained in parallel to define the CD81 positive population. Example 9: Cas9 expression levels upon module induction and constitutive Cas9 disruption of CD81

[0254] To further validate the function of the control modules, western immunoblotting was performed to assess Cas9 protein expression in pTET and pTET:Ultra-tight systems following drug treatments (FIG. 9A). HEK293T cells transfected with each construct were treated with the indicated small molecules for 72 hours, followed by protein extraction and immunoblotting for Cas9. As expected, Cas9 protein was undetectable in untreated pTET:Ultra-tight cells, reflecting tight regulation. Interestingly, Cas9 protein levels remained below the detection limit in pTET cells despite the significant background editing observed by flow cytometry, suggesting that functional activity can occur at levels undetectable by western blot analysis. As such, target gene depletion is a more sensitive readout of leaky Cas9 activity relative to Cas9 protein detection. Cas9 protein was readily observed upon Dox treatment of both pTET and pTET:Ultra-tight cells. Expression was lower in the pTET:Ultra-tight condition, likely due to degradation via the DD tag in the absence of Shield1. Cas9 protein was not seen when pTET:Ultra-tight cells were treated with Shield1 alone. Co-Attorney Ref.59868.00075WO01 (GNE-0006-WO) treatment of pTET:Ultra-tight cells with Dox and Shield1 increased Cas9 protein levels, though to a lesser extent than in pTET cells. However, this expression level was sufficient to drive efficient target gene knockout as indicated by flow cytometry results (FIGS.2E-2G).

[0255] Attempts to detect Anti-CRISPR protein by immunoblotting were unsuccessful due to limitations of available antibodies. Despite this, changes in CD81 protein levels were consistent with the expected regulation of the degron-tagged anti-CRISPR in the presence or absence of the specific drugs (FIGS.2E-2F).

[0256] FIG.9A shows western immunoblotting performed on HEK293T cells transfected with either pTET or pTET:Ultra-tight constructs, followed by 72-hour treatment with the indicated drugs. Untreated cells and wild-type (WT) controls were included. The upper panel shows Cas9 detection; the lower panel shows Tubulin as a loading control. FIGS.9B-9C show bar graphs depicting CD81 knockout by flow cytometry (B) and targeted amplicon sequencing (C) in the constitutive pCAG- Cas9 line at the indicated time points after stable line generation. Data are presented as mean values + / − SD from triplicate experiments. Statistical significance was determined by unpaired two-tailed Welch’s t-test. ns, not significant. Example 10: pTET:Ultra-tight performance in transformed cell lines and assessment of basal iPSC phenotypes engineered with pTET or pTET:Ultra-tight

[0257] Having established pTET:Ultra-tight as a more tightly regulated system than pTET, next the kinetics of editing for each system was compared over a period of 10 days (FIG.10A). Untreated pTET cells exhibited ~50% CD81 depletion, which did not increase further over time. In contrast, untreated pTET:Ultra-tight displayed minimal basal editing throughout the time course. In drug treated pTET cells, maximal CD81 depletion occurred by 72 hours. pTET:Ultra-tight cells took longer than pTET cells to achieve maximal CD81 depletion, but reached equivalent levels by 144- 240 hours.

[0258] To confirm that flow cytometry results reflected underlying genomic changes, targeted amplicon sequencing of CD81 was performed on the same samples to quantify indel frequencies over time (FIG. 10B). The amplicon sequencing data closely paralleled target protein levels. Untreated pTET cells displayed ~50% indel generation at the CD81 locus throughout the time course. In pTET:Ultra-tight cells, indels remained minimal without treatment, confirming suppression of leaky Cas9 activity. For both systems, between 6 and 48 hours after treatment, indel frequencies exceeded the percentage of CD81-depleted cells, suggesting a lag between genomic editing and observable protein loss, likely due to transcriptional / translational rates and / or protein half-life. Similar to flow cytometry results, upon induction, as before, pTET:Ultra-tight cells took longer than pTET cells to achieve maximal editing, but indel frequencies plateaued in both systems by 144 hours of drug exposure.Attorney Ref.59868.00075WO01 (GNE-0006-WO)

[0259] pTET and pTET:Ultra-tight (pTET-DD-Cas9;AcrIIA4-LID) HEK293T cells were either left untreated or drug-treated for the indicated times followed by measurement of CD81 knockout by flow cytometry (FIG.10A) and targeted amplicon sequencing (FIG.10B). Data represent mean ± SEM from triplicate experiments, except for the pTET:Ultra-tight untreated sample at 240 h in FIG. 10B, which has two replicates.

[0260] A CD81 knockout control line (FIG. 8) was established by stable genomic integration of a constitutive Cas9 and CD81 sgRNA-expressing transgene. This line exhibited complete protein depletion and genomic indel formation by 24 hours after removal of puromycin (used for selecting stable integrants) and these levels remained constant through the duration of our assay (~240 hours). (FIGS.9B-9C).

[0261] FIG.11A shows a bar graph depicting percentage indel frequency in untreated vs. treated HAP- 1 cells stably expressing pTET or pTET:Ultra-tight transgenes. Mean ± SD from triplicate experiments are shown, except for pTET-treated and pTET:Ultra-tight untreated conditions, which include two replicates. Statistical significance was determined by either Two-way ANOVA (FIG. 2J) or ordinary one-way ANOVA (FIG.2K), adjusted with Tukey’s multiple comparisons test. ns, not significant. **p < 0.01, ****p < 0.0001.

[0262] FIG.11B shows representative brightfield images depicting colony morphology of KOLF2.1J iPSC stably expressing pTET or pTET:Ultra-tight transgenes, either left untreated or drug-treated for 10 days. Scale bars indicate 500 μm.

[0263] FIG. 11C shows histogram plots displaying flow cytometry results of SSEA-4 marker expression in untreated and treated KOLF2.1J stable lines. Wild-type unstained and FITC- conjugated SSEA-4 stained samples are included as controls. Bar graphs show percent SSEA-4 positive cell populations as mean values + / − SD from triplicate experiments.

[0264] Additionally, editing at the B2M locus showed close alignment between indel frequencies and protein loss (FIG.11D). FIG.11D shows assessment of indel generation at the B2M locus in stable HEK293T cells before and after induction of Cas9 from pTET vs. pTET:Ultra-tight. pTET or pTET:Ultra-tight constructs expressing sgRNA targeting B2M were stably integrated into HEK293T cells. Stable cells were left untreated or treated for 10 days with 2μg / ml Dox (pTET) or 2μg / ml Dox + 1μM Shield1 (pTET:Ultra-tight) to induce Cas9 expression. Percentage indels at the B2M locus were measured by NGS. Mean ± SD from triplicate experiments are shown. Statistical comparison was performed using two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant. ****p < 0.0001.

[0265] Together, these time-course experiments confirmed the exceptionally low background editing and high efficiency with the pTET:Ultra-tight system as quantified by both indel generation and protein depletion.

[0266] While the initial assays focused on a 5–10 day window post-induction, the ability to detect robust editing at earlier timepoints expands the utility of the system, allowing researchers to captureAttorney Ref.59868.00075WO01 (GNE-0006-WO) transient phenotypes, resolve early molecular events, or study genes whose prolonged loss impairs cell viability. Example 11: Confirmation of protein-level changes at genomic locus with indel generation by targeted amplicon sequencing

[0267] To confirm that protein-level changes were reflected at the genomic locus, indel generation in HAP-1 cells was evaluated by targeted amplicon sequencing. Strong agreement between protein- level and genomic changes was observed (FIG.11A), mirroring the concordance seen in our kinetic analysis.

[0268] In addition to immortalized cell lines, the evaluation was extended to include a more sensitive, non-transformed line, KOLF2.1J, an induced pluripotent stem cell (iPSC) line that has been extensively characterized and favored for its genomic stability, efficient CRISPR / Cas9 editing, and robust differentiation capacity (Pantazis, C. B. et al. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell 29, 1685–1702.e22 (2022); Dobner, J. et al. Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J. Stem Cell Reports 19, 343–350 (2024)). Stable, polyclonal populations were engineered with pTET or pTET:Ultra-tight and treated with either Dox (pTET) or Dox + Shield1 (pTET:Ultra-tight) for 10 days followed by flow cytometry to measure CD81 levels. Both treated and untreated KOLF2.1J stable cell lines exhibited morphological features characteristic of healthy iPSCs, including prominent nuclei, tightly packed colony growth with well-defined borders, and minimal evidence of spontaneous differentiation (FIG. 11B). Additionally, SSEA-4, a well-established marker of iPSC pluripotency, was highly expressed across all conditions, confirming that the cells maintained a pluripotent state irrespective of transgene or drug treatment (FIG.11C). While it was observed that a modest decrease in targeting efficiency occurred when comparing pTET (~93%) to pTET:Ultra-tight (~78%), background editing was completely absent, resulting in the highest dynamic range of editing for all tested cell types (>200-fold difference between the on vs. off-state). Notably, the ~78% editing efficiency observed with pTET:Ultra-tight in KOLF2.1J represents a substantial improvement over published inducible systems in stem cells, which report efficiencies closer to ~40% 5,6. Together, these results suggest pTET:Ultra-tight consistently outperforms pTET across multiple cellular contexts. Example 12: Evaluation of editing kinetics in Xon-Cas9 and Xon-Cas9;AcrIIA4-FKBP systems

[0269] To evaluate editing kinetics in the Xon-Cas9 and Xon-Cas9;AcrIIA4-FKBP systems, a time- course analysis was performed with both flow cytometry (FIG. 12A) and targeted amplicon sequencing (FIG.12B). In the absence of drug treatment, Xon-Cas9 cells exhibited progressive loss of CD81 protein and increasing indel formation, whereas Xon-Cas9;AcrIIA4-FKBP cellsAttorney Ref.59868.00075WO01 (GNE-0006-WO) maintained minimal background editing throughout the time course. Upon drug induction, Xon-Cas9 cells showed time-dependent indel formation and depletion of CD81 protein, plateauing by 240 hours. Drug-treated Xon-Cas9;AcrIIA4-FKBP cells exhibited slower editing relative to Xon-Cas9, but comparable knockout levels by 144 hours. These time course results confirm that Xon- Cas9;AcrIIA4-FKBP is a superior system for controlling Cas9 activity.

[0270] FIG.12B shows a time-course analysis of CD81 knockout in Xon-Cas9 vs. Xon-Cas9;AcrIIA4- FKBP HEK293T cells that were left untreated or treated with either 20nM Branaplam (Xon-Cas9) or 20nM Branaplam + 1μM dTagv1 (Xon-Cas9;AcrIIA4-FKBP). Percentage indel frequencies at the CD81 locus were quantified by targeted amplicon sequencing at the indicated time points from triplicate experiments, with the exception of Xon-Cas9-untreated 6 h timepoint, which has two replicates.

[0271] For the time-course analysis of CD81 knockout in Xon-Cas9 vs. Xon-Cas9;AcrIIA4-FKBP HEK293T cells, cells were left untreated or treated with either 20nM Branaplam (Xon-Cas9) or 20nM Branaplam + 1μM dTagV-1 (Xon-Cas9;AcrIIA4-FKBP). Percentage CD81-negative cells were quantified by flow cytometry at the indicated time points. Data represent mean ± SEM from triplicate experiments.

[0272] The Xon-Cas9;AcrIIA4-FKBP combination was then tested in K562 cells. Although background editing was nearly absent in this cell line with the pTET system, the unmodified Xon- Cas9 system displayed ~19% leaky target disruption (Fig 6E). Use of the Xon-Cas9;AcrIIA4-FKBP vector reduced leakiness in this cellular context, but it also resulted in poor induction of Cas9 activity (FIGS.4H-4I). Within this vector design, AcrIIA4 expression is under the control of a PGK promoter (FIG. 4C). Separate studies have observed slightly higher or more stable transgene expression from the PGK promoter relative to other constitutive promoters, albeit with context- dependent results (Qin, J. Y. et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5, e10611 (2010); Norrman, K. et al. Quantitative comparison of constitutive promoters in human ES cells. PLoS One 5, e12413 (2010)). Therefore, it was hypothesized that the PGK-driven levels of AcrIIA4 protein were too high, even with dTag- mediated degradation, to fully reverse inhibition of Cas9 function, and that weaker AcrIIA4 expression was needed. To this end, two distinct strategies were evaluated aimed at lowering the baseline expression of AcrIIA4 such that it would maintain suppression of Cas9 in the uninduced state but allow for more complete reversal of this effect when cultured with a dTag molecule. This included exchange of the PGK promoter for the generally less-expressive UBC promoter (Qin et al., 2010; Norrman et al., 2010) or construction of an extended, polycistronic transgene with AcrIIA4 expressed by way of an IRES downstream of the CMV-Xon-Cas9 cassette (FIG.4J). IRES elements are commonly used to express multiple genes simultaneously and have been reported to drive lower expression of the downstream gene compared to the upstream, cap-dependent translated segment (Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. & Hayakawa, T. IRES-dependentAttorney Ref.59868.00075WO01 (GNE-0006-WO) second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376–382 (2000)). Both background and induced editing were greatly reduced with the UBC promoter driving the anti-CRISPR module suggesting that UBC is stronger than PGK in our tested K562 cell line (FIG.4H). By contrast, IRES-AcrIIA4-FKBP led to significantly-reduced leakiness with no compromise in induced cutting and displayed the best dynamic range of editing (FIGS.4H-4I).

[0273] Together, these results demonstrate that non-Dox-inducible Cas9 expression systems can enable high efficiency and low background editing, aided through co-expression of degradable anti- CRISPR proteins, but cell type-dependent variability necessitates empirical testing for optimal performance.

[0274] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Ref.59868.00075WO01 (GNE-0006-WO) CLAIMS 1. An inducible CRISPR-Cas system comprising a polynucleotide encoding: a Cas protein configured to bind a guide RNA and operably linked to at least one inducible expression element; and an anti-CRISPR protein.

2. The system of claim 1, wherein at least one inducible expression element comprises a ligand inducible alternative splicing switch upstream from the encoded Cas protein.

3. The system of claim 1 or claim 2, wherein the ligand inducible alternative splicing switch is an Xon splicing switch modulator, or a SMN2 splicing switch modulator.

4. The system of any one of claims 1-3, wherein: the Cas protein comprises a Cas9 protein, or a homolog or modified version thereof; which is controlled by at least one inducible expression element comprising: (i) a ligand inducible alternative splicing switch; or (ii) an inducible promoter element.

5. The system of claim 4, wherein the inducible promoter element comprises pTET (SEQ ID NO: 14) or pTET:Ultra-tight.

6. The system of any one of claims 1-5, wherein the Cas protein is fused to a destabilizing domain.

7. The system of claim 6, wherein the destabilizing domain comprises a ligand controllable destabilizing domain selected from the group consisting of: FK506 binding protein-12 (FKBP12), FKBPF36VL106P, ecDHFR, and any derivatives thereof.

8. The system of any one of claims 1-7, wherein the anti-CRISPR is a fusion protein comprising ACRIIA4.

9. The system of any one of claims 1-8, wherein the guide RNA comprises an sgRNA molecule library.Attorney Ref.59868.00075WO01 (GNE-0006-WO) 10. The system of any one of claims 1-9, wherein, upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to a target sequence and, optionally, edits the genomic locus to alter gene expression.

11. The system of claim 10, wherein the system comprises: a first sgRNA molecule comprising a first guide sequence capable of hybridizing to a first target sequence in a genomic locus of interest in the cell; a second sgRNA molecule comprising a second guide sequence capable of hybridizing to a second target sequence in the genomic locus of interest in the cell; and wherein upon introduction of an agent that induces the inducible promoter element, the CRISPR-Cas system binds to the first and second target sequences and edits the genomic locus of the cell by removing a DNA segment located between the first and second target sequences from the genome of the cell.

12. The system of any one of claims 1-11, wherein the Cas protein is a splice switch based inducible Cas9, and wherein the splice switch based inducible Cas9 protein sis stably integrated into a genome.

13. The system of claim 12, wherein the Cas9 comprises a Cas9 cassette, and wherein transgene silencing is prevented by the Cas9 cassette being inserted downstream of a promoter of a housekeeping gene within the genome.

14. The system of any one of claims 1-13, wherein the system is expressed in an inducible pluripotent stem cell line.

15. An isolated, non-naturally occurring polynucleotide encoding: a Cas protein optionally fused to a destabilizing domain and operably linked to an inducible promoter element; one or more sgRNA molecules comprising one or more guide sequences capable of hybridizing to a target sequence in a genomic locus of interest in a cell; and optionally an anti-CRISPR.

16. The polynucleotide of claim 15, wherein:Attorney Ref.59868.00075WO01 (GNE-0006-WO) the anti-CRISPR is a fusion protein comprising ACRIIA4 and a ligand inducible domain (LID), wherein the LID is induced by Shield1; the ligand controllable destabilizing domain comprises FKBPF36VL106P (SEQ ID NO: 40), FK506 binding protein-12 (FKBP12), ecDHFR, or any derivatives thereof; and the ligand that controls the destabilizing domain is Shield1; the Cas protein is a Cas9 protein; the inducible promoter element comprises pTET or pTET:Ultra-tight; and the one or more guide RNA molecules comprises an sgRNA molecule library.

17. A method of modifying a target nucleic acid in a cell, the method comprising: introducing the inducible CRISPR-Cas system according to any one of claims 1-14 into the cell; contacting the cell with a ligand that activates the inducible expression element; and contacting the cell with a ligand that deactivates the anti-CRISPR and / or the destabilizing domain, thereby causing the Cas protein to modify the target nucleic acid.

18. The method of claim 17, wherein the inducible CRISPR-Cas system comprises both an anti- CRISPR and a destabilizing domain, and wherein the method comprises contacting the cell with one or more ligands that deactivate both the anti-CRISPR and the destabilizing domain.

19. The method of claim 17, wherein the Cas protein is a splice switch based inducible Cas9, wherein the Cas9 comprises a Cas9 cassette, and the method further comprising: inserting the Cas9 cassette downstream of a promoter of a housekeeping gene within a genome; and integrating the Cas9 protein into the genome.

20. The method of any one of claims 17-19, wherein modifying the target nucleic acid in the cell alters expression of a target gene in the cell.

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