Modified CASCADE components and CASCADE complexes

Abstract

The present disclosure provides an engineered first class I CRISPR-Cas (Cascade) system comprising a multi-protein effector complex, a nucleoprotein complex comprising a I CRISPR-Cas subunit protein and a nucleic acid guide, a polynucleotide encoding the I CRISPR-Cas subunit protein, and a guide polynucleotide. Also disclosed are methods of making and using the engineered first class I type CRISPR-Cas systems of the present invention.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980038867.7. The application date of Chinese patent application No. 201980038867.7 is June 12, 2019, and all the contents are incorporated herein.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] No. 16 / 262,773, filed on January 30, 2019, which is a continuation-in-part of U.S. patent application serial number 16 / 104,875, filed on August 17, 2018, now pending, U.S. Patent No. 10,227,576, which issued on March 12, 2019, and which claims the benefit of U.S. Provisional Patent Application serial number 62 / 684,735, filed on June 13, 2018, now pending, and U.S. Provisional Patent Application serial number 62 / 807,717, filed on February 19, 2019, now pending: the contents of which are incorporated herein by reference in their entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] not applicable.

[0006] Sequence Listing

[0007] This application contains a sequence listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy generated on June 12, 2019 is named CBI032-30_ST25.txt and is 3.1 MB in size. Technical Field

[0008] The present disclosure generally relates to a modified first type I CRISPR-Cas (Cascade) system, comprising a multiprotein effector complex, a nucleoprotein complex comprising a type I CRISPR-Cas subunit protein and a nucleic acid guide, a polynucleotide encoding a type I CRISPR-Cas subunit protein, and a guide polynucleotide. The present disclosure also relates to compositions and methods for preparing and using the modified type I CRISPR-Cas system of the present invention. Background Art

[0009] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute the CRISPR-Cas system. The CRISPR-Cas system provides adaptive immunity against foreign polynucleotides in bacteria and archaea (see, e.g., Barrangou, R., et al., Science 315: 1709-1712 (2007); Makarova, KS, et al., Nature Reviews Microbiology 9: 467-477 (2011); Garneau, JE, et al., Nature 468: 67-71 (2010); Sapranauskas, R., et al., Nucleic Acids Res. 39: 9275-9282 (2011); Koonin, EV, et al., Curr. Opin. Microbiol. 37: 67-78 (2017)). Various CRISPR-Cas systems are capable of targeting DNA (class I, type I; class II, types II and V), RNA (class II, type VI), and combined targeting of DNA and RNA (class I, type III) in their natural hosts (see, e.g., Makarova, KS, et al., Nat. Rev. Microbiol. 13:722-736 (2015); Shmakov, S., et al., Nat. Rev. Microbiol. 15:169-182 (2017); Abudayyeh, OO, et al., Science 353:1-17 (2016)).

[0010] The classification of CRISPR-Cas systems has many subdivisions. Koonin, EV, et al., (Curr. Opin. Microbiol. 37: 67-78 (2017)) proposed a classification system that takes into account the tag cas genes that are specific to each type and subtype of the CRISPR-Cas system. The classification also takes into account the sequence similarity between multiple common Cas proteins, the phylogeny of the best conserved Cas proteins, the gene organization, and the structure of the CRISPR array. This method provides a classification scheme that divides CRISPR-Cas systems into two different types: the first type includes multiprotein effector complexes (type I (CRISPR-associated complex (“Cascade”) effector complex for antiviral defense), type III (Cmr / Csm effector complex) and type IV); and the second type includes single effector proteins (type II (Cas9), type V (Cas12a, formerly known as Cpf1) and type VI (Cas13a, formerly known as C2c2)). In the first type of system, type I is the most common and diverse, type III is more common in archaea than in bacteria, and type IV is the least common.

[0011] Type I systems include tag Cas3 proteins. Cas3 proteins have helicase and DNA enzyme domains responsible for DNA target sequence cutting. So far, 7 subtypes of type I systems have been identified (i.e., IA, IB, IC, ID, IE, IF types (and IF (e.g., I-Fv1, I-Fv2)) and variants of IU), which have a variable number of cas genes. Type I cas genes include, but are not limited to, the following genes: cas7, cas5, cas8, cse2, csa5, cas3, cas2, cas4, cas1, and cas6. Examples of organisms with type I systems are as follows: IA, Archaeoglobus fulgidus; IB, Clostridium kluyveri; IC, Bacillus halodurans; IU, Geobacter sulfurreducens; ID, Cyanothece sp. 8802; IE, E. coli K12; IF, Yersinia pseudo-tuberculosis; IF variant, Shewanella putrefaciens CN-32 (Koonin, EV, et al., Curr. Opin. Microbiol. 37:67-78 (2017)).Features of Cas3 protein-mediated DNA cleavage and progressive degradation have been described (see, e.g., Plagens, A., et al., Nucleic Acids Res. 42:5125-5138 (2014); Maier, L., et al., RNA Biol. 10:865–874 (2013); Hochstrasser, M., et al., Proc. Natl. Acad. Sci. USA 111:6618–6623 (2014); Sinkunas, T., et al., EMBO J. 30:1335–1342 (2011); Westra, E., et al., Mol. Cell 46:595–605 (2012); Mulepati, S., et al., Proc. Natl. Acad. Sci. USA 111:6618–6623 (2014); Sinkunas, T., et al., EMBO J. 30:1335–1342 (2011); Westra, E., et al., Mol. Cell 46:595–605 (2012); Mulepati, S., et al., Proc. Natl. Acad. Sci. USA 111:6618–6623 (2014). al., J. Biol. Chem. 288:22184–22192 (2013); Sinkunas, T., et al., EMBO J. 32: 385–394 (2013); Mulepati, S., et al., J. Biol. Chem. 288: 22184–22192 (2013); Redding, S., et al. al., Cell 163:854–865 (2015); Sinkunas, T., et al., EMBO J. 32: 385–394 (2013); Westra, E., et al., Mol. Cell 46: 595–605 (2012)).

[0012] Type I systems typically encode proteins that combine with CRISPR RNA (crRNA or "guide RNA") to form the Cascade complex. These complexes contain a variety of proteins and crRNAs, all of which are transcribed from the CRISPR site. In type I systems, the initial processing of the crRNA precursor is catalyzed by Cas6. This typically results in the production of crRNA with an 8-nucleotide 5' handle, a spacer, and a 3' handle; both the 5' handle and the 3' handle are derived from repeated sequences. In some systems, the 3' handle forms a stem-loop structure; in other systems, the secondary processing of the 3' end of the crRNA is catalyzed by ribonucleases (see, e.g., van der Oost, J., et al., Nature Reviews Microbiology 12: 479-492 (2014)).

[0013] The Cascade effector complex of the type I CRISPR-Cas system comprises a backbone with paralogous repeat-associated proteins (RAMPs; e.g., Cas7 and Cas5 proteins) containing an RNA recognition motif (RRM) fold and additional "large" and "small" subunit proteins (see, e.g., Koonin, EV, et al., Curr. Opin. Microbiol. 37: 67-78, (2017), Figure 2). These Cascade effector complexes typically have a Cas5 subunit protein and several Cas7 subunit proteins. Such Cascade effector complexes also include a guide RNA. The Cascade effector complex comprises different subunit proteins arranged in an asymmetric form along the length of the guide RNA. The Cas5 subunit protein and the large subunit protein (Cas8 protein) are located at one end of the complex, wrapping around the 5' end of the guide RNA. Several copies of the small subunit protein contact the guide RNA backbone bound to multiple copies of the Cas7 subunit protein. The Cas6 subunit protein (another RAMP protein) primarily binds to the Cascade effector complex by binding to the 3′ handle (repeat region) of crRNA. The Cas6 subunit protein typically acts as a repeat-specific RNase that participates in crRNA precursor processing; however, in the IC-type system, Cas5 acts as a repeat-specific RNase and Cas6 is absent.

[0014] The primary sequences of CRISPR-Cas type I Cascade subunit proteins share little sequence identity; however, the presence of homologous RAMP modules and the overall structural similarity of the multiprotein effector complexes support a common origin of these effector complexes (see, e.g., Koonin, EV, et al., Curr. Opin. Microbiol. 37:67-78 (2017)).

[0015] The adaptive immunity mechanism in type I CRISPR-Cas system mainly involves three stages: adaptation, expression and intervention. In the adaptation stage, foreign DNA or RNA infects the host, and the protein encoded by various cas genes binds to the region of infected DNA or RNA. Such a region is called the protospacer. The protospacer sequence adjacent motif (PAM) is a short nucleotide sequence adjacent to the protospacer (such as a DNA sequence of 2-6 base pairs). The PAM sequence is usually recognized by the Cas1 subunit protein / Cas2 subunit protein complex, wherein the active PAM sensing site is associated with the Cas1 subunit protein (see, for example, Jackson, SA, et al., Science 356:356 (6333) (2017)).

[0016] During the expression phase, the CRISPR array containing multiple spacer repeat elements is transcribed into a single transcript. The single spacer repeat element is processed into a single crRNA by a nuclease (e.g., type I, Cas6 protein; and type IC, Cas5 protein). The Cas subunit protein is expressed and binds to the crRNA to form the Cascade effector complex.

[0017] The Cascad effector complex scans foreign polynucleotides in the infected host to identify DNA complementary to the spacer region. In type I systems, when the effector complex recognizes a sequence complementary to the spacer region adjacent to the PAM, interference occurs, and the Cas3 protein is recruited to the DNA-bound Cascade effector complex to cleave and progressively digest the foreign polynucleotide.

[0018] Makarova, KS, et al., (Cell 168:946(2017)) provide an overview of the genes, homologs, Cascade complexes, and mechanisms of action of type I CRISPR-Cas systems.

[0019] Therefore, type I CRISPR-Cas systems currently have limited use in eukaryotic genome engineering applications, in part due to the difficulty of heterologous expression of the Cascade complex and the way in which type I CRISPR-Cas systems cleave DNA targets. SUMMARY OF THE INVENTION

[0021] The present invention generally relates to compositions comprising engineered type I CRISPR-Cas effector complexes and components thereof, including protein components, modified or differentially altered guide polynucleotides, and combinations thereof.

[0022] One embodiment of the present invention is a composition comprising:

[0023] A first engineered type I CRISPR-Cas effector complex comprising:

[0024] a first Cse2 subunit protein, a first Cas5 subunit protein, a first Cas6 subunit protein, and a first Cas7 subunit protein,

[0025] a first fusion protein comprising a first Cas8 subunit protein and a first FokI, wherein the N-terminus of the first Cas8 subunit protein or the C-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus or N-terminus of the first FokI respectively through a first linker polypeptide, and wherein the first linker polypeptide has a length of 10 to 40 amino acids, and

[0026] a first guide polynucleotide comprising a first spacer capable of binding to a first nucleic acid target sequence; and

[0027] A second engineered type I CRISPR-Cas effector complex comprising:

[0028] a second Cse2 subunit protein, a second Cas5 subunit protein, a second Cas6 subunit protein, and a second Cas7 subunit protein,

[0029] a second fusion protein comprising a second Cas8 subunit protein and a second FokI, wherein the N-terminus of the second Cas8 subunit protein or the C-terminus of the second Cas8 protein is covalently linked to the C-terminus or N-terminus of the second FokI, respectively, through a second linker polypeptide, and wherein the second linker polypeptide has a length of 10 to 40 amino acids, and

[0030] A second guide polynucleotide comprising a second spacer capable of binding to a second nucleic acid target sequence, wherein the protospacer adjacent motif (PAM) of the second nucleic acid target sequence and the PAM of the first nucleic acid target sequence have a spacer spacing of 20 base pairs to 42 base pairs.

[0031] In some embodiments, the length of the first linker polypeptide and / or the second linker polypeptide is 15 amino acids to 30 amino acids or 17 amino acids to 20 amino acids in length. In one embodiment, the length of the first linker polypeptide and the second linker polypeptide is the same.

[0032] The spacing of the spacer between the second nucleic acid target sequence and the first nucleic acid target sequence includes, but is not limited to, 22 base pairs to 40 base pairs, 26 base pairs to 36 base pairs, 29 base pairs to 35 base pairs, or 30 base pairs to 34 base pairs.

[0033] The first FokI and the second FokI can be monomeric subunits capable of combining to form a homodimer, or different subunits capable of combining to form a heterodimer.

[0034] In some embodiments, the N-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus of the first FokI via a first linker polypeptide, the C-terminus of the first Cas8 subunit protein is covalently linked to the N-terminus of the first FokI via a first linker polypeptide, the N-terminus of the second Cas8 subunit protein is covalently linked to the C-terminus of the second FokI via a second linker polypeptide, the C-terminus of the second Cas8 subunit protein is covalently linked to the N-terminus of the second FokI via a second linker polypeptide, and combinations thereof. Each of the first Cas8 subunit protein and the second Cas8 subunit protein can comprise a Cas8 subunit protein having a different sequence, or the first and second Cas8 subunit proteins can comprise the same amino acid sequence.

[0035] Similarly, each of the first Cse2 subunit protein and the second Cse2 subunit protein can contain different or identical Cse2 subunit protein amino acid sequences, each of the first Cas5 subunit protein and the second Cas5 subunit protein can contain different or identical Cas5 subunit protein amino acid sequences, each of the first Cas6 subunit protein and the second Cas6 subunit protein can contain different or identical Cas6 subunit protein amino acid sequences, each of the first Cas7 subunit protein and the second Cas7 subunit protein can contain different or identical Cas7 subunit protein amino acid sequences, and combinations of the above.

[0036] In preferred embodiments, the guide polynucleotide comprises RNA.

[0037] In additional embodiments, the invention includes engineered type I CRISPR Cas3 mutant proteins ("mCas3 proteins") that are capable of reduced movement along DNA relative to wild-type type I CRISPR Cas3 proteins ("wtCas3 proteins").

[0038] The present invention also includes using the above composition to perform genome editing in cells, and a method for preparing the above composition.

[0039] Other embodiments of the present invention will become readily apparent to those of ordinary skill in the art in view of the disclosure herein.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The figures are not drawn to scale, nor are they drawn to scale. The positions of the indicators are approximate.

[0042] Figure 1A A generalized view of the type I CRISPR-Cas effector complex is presented. Figure 1B A generalized view of type I CRISPR-Cas crRNAs is presented.

[0043] Figure 2A 、 Figure 2B and Figure 2C Illustrative examples of two engineered type I CRISPR-Cas effector complexes with fusion domains that bind to adjacent spacer sequences are presented.

[0044] Figure 3A and Figure 3B Examples of circularly arranged proteins are presented.

[0045] Figure 4A 、 Figure 4B 、 Figure 5A 、 Figure 5B 、 Figure 6A 、 Figure 6B 、 Figure 6C 、 Figure 7A 、 Figure 7B 、 Figure 8 、 Figure 9 、 Figure 10A and Figure 10B Various examples of engineered type I CRISPR-Cas effector complexes of the present invention are shown.

[0046] Figure 11A and Figure 11B An example of a substrate channel is shown.

[0047] Figure 12A 、 Figure 12B and Figure 12C A generalized view of the site-directed recruitment of functional protein domains fused to Cascade subunit proteins by the dCas9:NATNA complex is presented.

[0048] Figure 13A 、 Figure 13B 、 Figure 14A 、 Figure 14B and Figure 14C Examples of engineered type I CRISPR-Cas effector complexes of the present invention are shown.

[0049] Figure 15A 、 Figure 15B 、 Figure 15C 、 Figure 16A 、 Figure 16B 、 Figure 16C 、 Figure 17A 、 Figure 17B 、 Figure 17C 、 Figure 18A 、 Figure 18B 、 Figure 18C 、 Figure 18D 、 Figure 19A 、 Figure 19B 、 Figure 20A and Figure 20B Examples of engineered type I CRISPR-Cas effector complexes of the invention and methods of their use are presented.

[0050] Figure 21A 、 Figure 21B 、 Figure 21C 、 Figure 21D 、 Figure 22A 、 Figure 22B 、 Figure 22C and Figure 22D An embodiment of the invention is shown that uses a Cas3 protein comprising active endonuclease activity.

[0051] Figure 23A 、 Figure 23B 、 Figure 23C 、 Figure 23D 、 Figure 23E 、 Figure 24 、 Figure 25 、 Figure 26 and Figure 27 Schematic diagrams of the expression systems for various Cascade components are presented.

[0052] Figure 28 、 Figure 29 、 Figure 30 、 Figure 31A 、 Figure 31B 、 Figure 32 、 Figure 33A 、 Figure 33B and Figure 34 Data related to genome editing using the engineered Cascade system of the present invention are presented.

[0053] Figure 35 Shown are examples of minimal CRISPR arrays containing pairs of guide RNAs (gRNAs).

[0054] Figure 36A 、 Figure 36B 、 Figure 36C and Figure 36D Data related to genome editing in human cells via RNP- and plasmid-based delivery of engineered type I CRISPR-Cas complexes are presented.

[0055] Figure 37A 、 Figure 37B 、 Figure 37C 、 Figure 37D 、 Figure 37E 、 Figure 37F and Figure 37G Data related to the restoration results are presented.

[0056] Figure 38A 、 Figure 38B and Figure 38C Data are presented on how mismatches between gRNAs and target DNA inhibit genome editing by engineered type I CRISPR-Cas complexes.

[0057] Figure 39A 、 Figure 39B 、 Figure 39C and Figure 39D Data related to an expanded screen of PAM selectivity of three Cascade homolog variants are presented.

[0058] Figure 40A 、 Figure 40B 、 Figure 40C 、 Figure 40D 、 Figure 40E and Figure 40FData are presented relating to exemplary changes in editing efficiency of engineered type I CRISPR-Cas complexes.

[0059] Figure 41A 、 Figure 41B and Figure 41C Data related to an expanded screen of FokI-Cas8 linker length and spacer spacing for three Cascade homolog variants are presented.

[0060] Figure 42A and Figure 42B An example of PCR amplification using an oligomer as a template is shown.

[0061] Figure 43 Data are presented for the percentage of genome editing as a function of FokI-Cascade homolog variant and spacer spacing.

[0062] Figure 44 A linear representation of the functional domains of the EcoCas3 protein and the relative positions of the mutants made within the sequence are shown.

[0063] Figure 45A 、 Figure 45B 、 Figure 45C and Figure 45D Data related to genome editing using EcoCascade RNP complexes containing wild-type or mutant EcoCas3 proteins are shown.

[0064] Figure 46A 、 Figure 46B 、 Figure 46C 、 Figure 47A and Figure 47B Data related to dCas9-VP64 / sgRNA RNP complex roadblocks and their impact on target cleavage by the EcoCascade RNP complex are presented.

[0065] Figure 48 Exemplary editing data for Cas3[D452A] / -EcoCascade or mCas3[D452A]-EcoCascade are shown.

[0066] Figure 49 Data are presented for genome editing at eight TRAC target sites using the PseCascade RNP complex.

[0067] Incorporated herein by reference

[0068] All patents, publications, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Detailed Description of the Invention

[0070] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes one or more polynucleotides, and reference to "a vector" includes one or more vectors.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although other methods and materials similar or equivalent to those described herein can be used in the present invention, preferred materials and methods are described herein.

[0072] In view of the teachings of the present specification and examples, one of ordinary skill in the art can apply conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant polynucleotides, as taught, for example, in the following standard texts: Cellular and Molecular Immunology, 9th Edition, A.K. Abbas., et al., Elsevier (2017), ISBN 978-0323479783; Cancer Immunotherapy Principles and Practice, 1st Edition, L.H. Butterfield, et al., Demos Medical (2017), ISBN 978-1620700976; Janeway's Immunobiology, 9th Edition, Kenneth Murphy, Garland Science (2016), ISBN 978-0815345053; Clinical Immunology and Serology: A Laboratory Perspective, 4th Edition, C. Dorresteyn Stevens, et al., F.A. Davis Company(2016),ISBN978-0803644663;Antibodies:A Laboratory Manual,Second Edition,EAGreenfield,ColdSpring Harbor Laboratory Press(2014),ISBN 978-1-936113-81-1;Culture of AnimalCells:A Manual of Basic Technique and Specialized Applications, seventh edition, RIFreshney, Wiley-Blackwell (2016), ISBN 978-1118873656; Transgenic Animal Technology, third edition: ALaboratory Handbook, CAPinkert, Elsevier (2014), ISBN 978-0124104907; The Laboratory Mouse, second edition, H.Hedrich, Academic Press(2012), ISBN978-0123820082; Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition, R.Behringer, et al., Cold Spring Harbor Laboratory Press (2013), ISBN 978-1936113019; PCR 2: A Practical Approach, M.J. McPherson, et al., IRL Press (1995), ISBN 978-0199634248; Methods in Molecular Biology (Series), J.M. Walker, ISSN1064-3745, Humana Press; RNA: A Laboratory Manual, D.C. Rio, et al., Cold Spring Harbor Laboratory Press (2010), ISBN 978-0879698911; Methods in Enzymology (Series), Academic Press; Molecular Cloning: A Laboratory Manual (Fourth Edition), M.R. Green, et al., Cold Spring Harbor Laboratory Press (2012), ISBN 978-1605500560; Bioconjugate Techniques, Third Edition, G.T. Hermanson, Academic Press (2013), ISBN 978-0123822390; Methods in Plant Biochemistry and Molecular Biology, W.V. Dashek, CRC Press (1997), ISBN 978-0849394805; Plant Cell Culture Protocols (Methods in Molecular Biology), V.M. Loyola-Vargas, et al., Humana Press (2012), ISBN 978-1617798177; Plant Transformation Technologies, C.N. Stewart, et al., Wiley-Blackwell (2011), ISBN 978-0813821955; Recombinant Proteins from Plants (Methods in Biotechnology), C. Cunningham, et al.,Humana Press(2010),ISBN 978-1617370212;Plant Genomics:Methods and Protocols(Methods in MolecularBiology),W.Busch,Humana Press(2017),ISBN 978-1493970018;Plant Biotechnology:Methods in Tissue Culture and Gene Transfer,R.Keshavachandran,et al.,OrientBlackswan(2008),ISBN 978-8173716164. .

[0073] Clustered regularly interspaced short palindromic repeats (CRISPR) and associated CRISPR-associated proteins (Cas proteins) constitute the CRISPR-Cas system (see, eg, Barrangou, R., et al., Science 315: 1709-1712 (2007)).

[0074] As used herein, "Cas protein", "CRISPR-Cas protein", "CRISPR-Cas subunit protein", and "Cas subunit protein" refer to the first type I CRISPR-Cas protein, unless otherwise indicated. Typically, for use in aspects of the present invention, the Cas subunit protein is capable of interacting with one or more homologous polynucleotides (most commonly crRNA) to form a type I effector complex (most commonly, an RNP complex).

[0075] Over time, the genes encoding Cascades in type IE CRISPR–Cas systems have been named using various conventions, which can be a point of confusion when comparing recent and older literature. Generally, this specification uses the nomenclature as shown in Koonin, E., et al. (Curr. Opin. Microbiol. 37:67-78 (2017)), where the order of genes in the reference E. coli K12 operon is: cas3, cas8, cas11, cas7, cas5, cas6, cas1, and cas2. For simplicity, the "e" qualifier in cas8e is sometimes used to distinguish cas8 genes between different subtypes in type I systems. The stoichiometry of wild-type E. coli type IE CRISPR-Cas is Cas51-Cas61-Cas76-Cas81-Cas112-gRNA1.

[0076] However, for cross-reference purposes: cas8 was previously known as cse1 and casA, and is also referred to as the "large subunit"; cas11 was previously known as cse2 and casB, and is also referred to as the "small subunit"; cas7 was previously known as cse4 and casC; cas5 was previously known as casD, and is sometimes also given the qualifier cas5e; and cas6 was previously known as cse3 and casE, and is usually given the qualifier cas6e. Table 1 lists the genes encoding the Cas subunit proteins.

[0077]

[0078]

[0079] *As defined in Makarova, KS, et al., Nat. Rev. Microbiol. 13:722-736 (2015); Koonin, EV, et al., Curr Opin Microbiol. 37:67-78 (2017).

[0080] The PAM sequence is typically recognized by the Cas1 subunit protein / Cas2 subunit protein complex, where the active PAM sensing site is associated with the Cas1 subunit protein (see, e.g., Jackson, SA, et al., Science 356:356(6333)(2017)). Cas1 and Cas2 proteins are present in the vast majority of known CRISPR-Cas systems and are sufficient to insert the spacer into the CRISPR box (see, e.g., Yosef, I, et al., Nucleic Acids Res. 40:5569–5576(2012)). The two proteins form a complex for the adaptation process. The endonuclease activity of the Cas1 protein is required for spacer integration, while the Cas2 protein appears to perform non-enzymatic functions (see, e.g., Nunez, J., et al., Nat Struct Mol Biol. 21: 528–534 (2014); Richter, C., et al., PLoS One. 2012; 7: e49549). The Cas1-Cas2 protein complex represents a highly conserved information processing module of the CRISPR-Cas system that appears to be quasi-autonomous relative to the rest of the system (see, e.g., Makarova, K., et al., Methods Mol. Biol. 1311: 47-75 (2015)). The endonuclease Cas1 protein is a Cas protein required to ensure the unique ability of the CRISPR system to maintain memory of previous encounters with infectious agents.

[0081] The terms "Type I CRISPR-Cas effector complex," "Type I CRISPR-Cas nucleoprotein (NP) complex," "Cascade nucleoprotein (NP) complex," and "Type I nucleoprotein (NP) complex" are used interchangeably herein and generally refer to the Cascade protein that forms a complex with a guide polynucleotide. "Cascade complex" and "Type I complex" are generally used when referring to the protein components of the Cascade NP complex. The terms "Cascade RNP complex," "Type I CRISPR-Cas RNP complex," and "Type I RNP complex" refer to the Cascade complex that contains crRNA relative to the more general guide polynucleotide (i.e., as in the Cascade NP complex). An example of a wild-type Type I CRISPR-Cas effector complex is shown in Figure 1A middle. Figure 1A Adapted from Makarova, KS, et al., (Cell 168:946 (2017); Makarova, K., et al., Nature Reviews Microbiology 13:722-736 (2015)). Figure 1A Shown are six Cas7 proteins, Cas5 protein, Cas8 protein, two Cse2 proteins, Cas6 protein and crRNA combined into the Cascade complex ( Figure 1A : Cas7, Cas5, Cas8, Cse2 and Cas6; the dashed box around Cas6 refers to its interaction with the crRNA hairpin; the crRNA is shown as a black line including the hairpin). The complex is able to bind to the nucleic acid target sequence. In the wtCas3 protein ( Figure 1A After binding to the complex, the Cas3 (encircled by the dotted box) can cleave the nucleic acid target sequence. As shown in Table 1, the total number of some Cas subunit proteins may vary in the Cascade complex.

[0082] "Cas3" and "Cas3 protein" are used interchangeably herein to refer to type I CRISPR-Cas3 proteins, their modifications, and variants. Type I CRISPR-Cas effector complexes bind foreign DNA complementary to the crRNA guide and recruit Cas3, a trans-acting nuclease-helicase required for target degradation. Cas3 proteins possess motifs characteristic of helicases from superfamily 2 and contain a DEAD / DEAH box region and a conserved C-terminal domain. Cas3 proteins and variants thereof are known in the art (see, e.g., Westra, ER, et al., Mol. Cell. 46:595–605 (2012); Sinkunas, T., et al., EMBO J. 30:1335-1342 (2011); Beloglazova, N., et al., EMBO J. 30:4616-4627 (2011); Mulepati, S., et al., J. Biol. Chem. 286:31896-31903 (2011)). As used herein, the term "mCas3 protein" refers to a Cas3 protein that comprises one or more mutations relative to its corresponding wtCas3 protein. mCas3 proteins include, but are not limited to, mCas3 proteins (e.g., Example 23A, Example 23B, and Example 23C), dblmCas3 proteins (e.g., Example 26A, Example 26B, and Example 26C), and dCas3* (a mutant Cas3 protein that does not have any nuclease activity and / or helicase activity).

[0083] As used herein, the term "nuclease" refers to an enzyme that is capable of cleaving a phosphodiester bond, such as that connecting two nucleotides, as found in double-stranded (ds) nucleic acids (e.g., dsDNA, genomic DNA (gDNA), dsRNA), single-stranded (ss) nucleic acids (e.g., ssDNA, RNA), or hybrid dsRNA / DNA. An "endonuclease" can generally affect either a ss-(nick) or a ds-nick in its target molecule. An example of a DNA endonuclease is the FokI enzyme. "FokI endonuclease" and "FokI" are used interchangeably herein and refer to the FokI enzyme, FokI homologs, the enzymatically active domain of the FokI enzyme, and variants of the FokI enzyme. FokI dimerization is generally required for DNA cleavage. FokI dimers can comprise two monomeric subunits that bind to form a homodimer or two different monomeric subunits that bind to form a heterodimer (see, e.g., Bitinaite, J., et al., Proc. Natl. Acad. Sci. USA 95: 10570-10575 (1998); Ramalingam, S., et al., J. Mol. Biol. 405: 630-641 (2011)). An example of a FokI variant is the Sharkey variant described by Guo, et al. (Guo, J., et al., J. Mol. Biol. 400: 96-107 (2010)). Additional DNA and RNA nucleases are known in the art.

[0084] As used herein, "CRISPR RNA," "crRNA," and "guide RNA" refer to one or more RNAs with which Cas subunit proteins can interact to form a guide complex that preferentially binds to a nucleic acid target sequence in a polynucleotide relative to a polynucleotide that does not contain a nucleic acid target sequence. As used herein, "guide" and "guide polynucleotide" refer to the polynucleotide component of a Type I effector complex comprising a ribonucleotide base (e.g., RNA) and a ribose sugar, as well as different components and combinations thereof, including but not limited to deoxyribonucleotide bases, nucleotide analogs, modified nucleotides, different nitrogenous bases, fundamentally different nucleotide bases, chemically different molecules, mixtures of bases (e.g., RNA bases, DNA bases, and / or modified bases), and the like, and combinations thereof, as well as synthetic backbones, naturally occurring backbones, non-naturally occurring backbones, fundamentally different backbone residues, chemically different residues or bonds, modified backbones, mixtures (e.g., ribose and deoxyribose components of the backbone), and the like, and combinations thereof. Some examples of guide polynucleotides are described herein. An example of a type I CRISPR-Cas crRNA that binds to a nucleic acid target sequence via a crRNA spacer is shown in Figure 1B middle. Figure 1B Adapted from Hochstrasser, ML, et al., Mol. Cell 63:840-851 (2016). Figure 1B In PAM( Figure 1B , 104) binds to the nucleic acid target sequence and shows the 5' and 3' strands of the double-stranded nucleic acid ( Figure 1B , vertical lines represent hydrogen bonds). Guide polynucleotide ( Figure 1B , 106) typically includes a 5' handle region ( Figure 1B , 101), a spacer region containing a seed region ( Figure 1B , 103), and a 3' hairpin containing two hydrogen-bonding repeat regions ( Figure 1B , 102); horizontal lines represent hydrogen bonds. This article discusses the PAM sequences associated with numerous type I Cascade homologs. The PAM sequence is the adjacent protospacer sequence ( Figure 1B , 105). Figure 1B The Cascade complex spacer region ( Figure 1B , vertical lines represent hydrogen bonds). Figure 1B Also shown is the proseptum ( Figure 1B , pre-spacer). The spacer may include a crRNA region of about 6 to about 56 nucleotides, wherein the spacer is complementary to the nucleic acid target sequence in the polynucleotide. In the IE type CRISPR-Cas system, the spacer length can be changed to finely tune the Cascade activity. The Cascade complex can incorporate additional Cas7 subunits, wherein every 6 nucleotides are added to the crRNA spacer, and incorporate additional Cse2 subunits, wherein every 12 nucleotides are added to the spacer (see, e.g., Luo, ML, et al., Nucleic Acids Res. 44(15): 7385-7394(2016)). The spacer typically includes a region of about 32 to about 36 nucleotides.

[0085] The terms "spacer," "spacer sequence," and "nucleic acid target binding sequence" are used interchangeably herein.

[0086] "Target," "target sequence," "nucleic acid target sequence," and "on-target sequence" are used interchangeably herein to refer to a nucleic acid sequence that is fully or partially complementary to a nucleic acid target binding sequence (e.g., a spacer region of a crRNA) of a guide of a Cascade nucleoprotein complex (e.g., a Cascade RNP complex). Typically, the nucleic acid target binding sequence is selected to be 100% complementary to the nucleic acid target sequence to which the binding of the Cascade nucleoprotein complex is directed; however, a lower percentage of complementarity can be used to attenuate binding to the nucleic acid target sequence. When the target binding sequence is 100% complementary to the target sequence, "off-target" sequence binding refers to binding of the Cascade nucleoprotein complex to a nucleic acid sequence that has less than 100% complementarity with the nucleic acid target binding sequence (spacer region). Double-stranded DNA sequences typically contain a nucleic acid target sequence ( Figure 1B , the portion of hydrogen that binds to the guide RNA). The "target region" comprises the nucleic acid target sequence.

[0087] As used herein, a "stem element" or "stem structure" refers to two nucleic acid chains that are known or predicted to form a double-stranded region ("stem element"). A "stem-loop element" or "stem-loop structure" refers to a stem structure in which the 3' end sequence of one chain is covalently bound to the 5' end sequence of the second chain through a nucleotide sequence that is typically a single-stranded nucleotide ("stem-loop element nucleotide sequence"). In some embodiments, the loop element comprises a loop element nucleotide sequence of about 3 to about 20 nucleotides in length, preferably about 4 to about 10 nucleotides in length. In a preferred embodiment, the loop element nucleotide sequence is a single-stranded nucleotide sequence of unpaired nucleic acid bases that does not interact through hydrogen bond formation to produce a stem element within the loop element nucleotide sequence. The term "hairpin element" is also used herein to refer to a stem-loop structure. Such structures are well known in the art. Base pairing may be exact; however, as known in the art, stem elements do not require exact base pairing. Therefore, the stem element may include one or more base mismatches or unpaired bases. Examples of stem-loop structures in guide polynucleotides are Figure 1B shown.

[0088] “Linker element nucleotide sequence,” “linker nucleotide sequence,” and “linker polynucleotide” are used interchangeably herein and refer to a single-stranded or double-stranded nucleic acid sequence of one or more nucleotides covalently attached to a first nucleic acid sequence (e.g., 5′-linker nucleotide sequence-first nucleic acid sequence-3′). In some embodiments, a linker nucleotide sequence connects two different nucleic acid sequences to form a single polynucleotide (e.g., 5′-first nucleic acid sequence-linker nucleotide sequence-second nucleic acid sequence-3′). Other examples of linker nucleotide sequences include, but are not limited to, 5′-first nucleic acid sequence-linker nucleotide sequence-3′ and 5′-linker nucleotide sequence-first nucleic acid sequence-linker nucleotide sequence-3′. In some embodiments, a linker element nucleotide sequence can be a single-stranded nucleotide sequence of unpaired nucleic acid bases that do not interact with each other through hydrogen bond formation and produce a secondary structure (e.g., a stem-loop structure) within the linker element nucleotide sequence. In some embodiments, two linker element nucleotide sequences can interact with each other through hydrogen bonds between the two linker element nucleotide sequences. In some embodiments, a linker polynucleotide encodes a “linker polypeptide.” Such a linker polynucleotide typically connects the 3' end of the first polynucleotide encoding the first polypeptide to the 5' end of the second polynucleotide encoding the second polypeptide to form a single polynucleotide encoding a fusion protein comprising N-first polypeptide-linker polypeptide-second polypeptide-C. In some embodiments of the present invention, more than two polypeptide sequences can be connected in series via a linker polypeptide (e.g., N-first polypeptide-first linker polypeptide-second polypeptide-second linker polypeptide-third polypeptide-C). "Linker polypeptide," "linker polypeptide sequence," "amino acid linker sequence," and "linker sequence" are also used interchangeably herein.

[0089] As used herein, "linking nucleotide sequence" refers to a single-stranded nucleic acid sequence linker sequence that covalently links a first nucleic acid sequence to a second nucleic acid sequence.

[0090] As used herein, the terms "interspacer," "interspacer region," and "interspacer spacing" are used interchangeably and refer to the distance, typically in the PAM-in direction, between a PAM of a first nucleic acid target sequence (e.g., a first DNA target sequence) and a PAM of a second nucleic acid target sequence (e.g., a second DNA target sequence), wherein a first type I CRISPR-Cas effector complex comprises a first spacer capable of binding to the first nucleic acid target sequence, and a second type I CRISPR-Cas effector complex comprises a second spacer capable of binding to the second nucleic acid target sequence. Figure 2A 、 Figure 2B and Figure 2CIllustrative examples of two type I CRISPR-Cas effector complexes comprising fusion proteins are presented ( Figure 2A : "Cascade1", solid outline box, including "crRNA1"; and "Cascade2", dashed outline box, including "crRNA2") Figure 2A , "FP1" and "FP2" are represented as circled parts; for example, FP1 and FP can be FokI), which is connected to each Cascade complex via a linker polynucleotide ( Figure 2A , "Linker 1" and "Linker 2"), where the CRISPR-Cas effector complex binds to adjacent nucleic acid target sequences on double-stranded DNA ( Figure 2A The PAM sequences associated with each nucleic acid target sequence are indicated ( Figure 2A , “PAM1”, open box, and “PAM2”, open box)). Figure 2A The spacer spacing between two target sites in a PAM-containing (PAM-containing / PAM-containing) configuration is shown (shown as Figure 2A horizontal double-arrow line at the top). Figure 2B The spacer spacing between two target sites in a PAM-containing / non-PAM configuration is shown (shown as Figure 2B horizontal double-arrow line at the top). Figure 2C The spacer spacing between two target sites in the PAM-free (no PAM / no PAM) configuration is shown (shown as Figure 2C horizontal double-arrow line at the top). Figure 2A 、 Figure 2B and Figure 2C Separation of the two strands of dsDNA is also shown. The Cascade complex recognizes the dsDNA target sequence adjacent to the PAM. The PAM sequence is recognized by Cse1. Base pairing between the crRNA and the complementary target DNA strand results in the generation of an R-loop with the displaced non-complementary target DNA strand (see, e.g., Beloglazova, N., et al., Nucleic Acids Res. 43:530–543 (2015)).

[0091] As used herein, the term "homologous" refers to interacting biological molecules, such as cell surface receptors (e.g., chemokine receptors) and their ligands (e.g., chemokines expressed on tumor cells or in the tumor microenvironment); site-directed polypeptides and their guides; site-directed polypeptide / guide complexes (i.e., nucleoprotein complexes) capable of site-directed binding to a nucleic acid target sequence complementary to the guide binding sequence, etc. In addition, the term "homologous" refers to a group of Cas subunit proteins (e.g., Cse2, Cas5, Cas6, Cas7, and Cas8), and one or more guide polynucleotides (e.g., type I CRISPR-Cas RNA) capable of forming a nucleoprotein complex capable of site-directed binding to a nucleic acid target sequence complementary to a spacer present in one of the one or more guide polynucleotides.

[0092] The terms "wild-type," "naturally occurring," and "unmodified" are used herein to refer to the typical (or most common) form, appearance, phenotype, or strain found in nature; for example, the typical form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome in which it occurs and which can be isolated from a natural source. The wild-type form, appearance, phenotype, or strain serves as the original parent prior to intended modification, alteration, mutation, and / or significantly different structural changes. Thus, mutants, variants, engineered, recombinant, and modified forms are not wild-type forms.

[0093] The terms "engineered," "genetically engineered," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-natural" refer to the intentional human or machine manipulation of the genome of an organism or cell. The term encompasses methods of genome modification, which include genome editing as defined herein, as well as techniques for altering gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art.

[0094] "Covalent bond," "covalently attached," "covalently bound," "covalently linked," "covalently connected," and "molecular bond" are used interchangeably herein and refer to a chemical bond involving the sharing of electron pairs between atoms. Examples of covalent bonds include, but are not limited to, phosphodiester bonds, phosphorothioate bonds, disulfide bonds, and peptide bonds (-CO-NH-).

[0095] "Non-covalent bond," "non-covalent attachment," "non-covalent binding," "non-covalent linking," "non-covalent interaction," and "non-covalent connection" are used interchangeably herein and refer to any relatively weak chemical bond that does not involve the sharing of electron pairs. Multiple non-covalent bonds generally stabilize the conformation of macromolecules and mediate specific interactions between molecules. Examples of non-covalent bonds include, but are not limited to, hydrogen bonds, ionic interactions (e.g., Na + Cl - ), van der Waals interactions and hydrophobic bonds.

[0096] As used herein, "hydrogen bonding," "hydrogen-base pairing," and "hydrogen bonding" are used interchangeably and refer to both typical and non-typical hydrogen bonding, including, but not limited to: "Watson-Crick hydrogen bonding base pairs" (WC-hydrogen bonding base pairs or WC hydrogen bonding); "Hoogsteen hydrogen bonding base pairs" (Hoogsteen hydrogen bonding); and "wobble hydrogen bonding base pairs" (wobble hydrogen bonding). WC hydrogen bonding, including reversed WC hydrogen bonding, refers to purine-pyrimidine base pairing, e.g., adenine:thymine, guanine:cytosine, and uracil:adenine. Hoogsteen hydrogen bonding, including reversed Hoogsteen hydrogen bonding, refers to a variation of base pairing in nucleic acids in which two nucleic acid bases of one kind on each chain are held together by hydrogen bonds in the major groove. This non-WC hydrogen bonding can allow a third chain to wrap around the double strand and form a triple-stranded helix. Wobble hydrogen bonding, including reverse wobble hydrogen bonding, refers to the pairing of two nucleotides in RNA molecules that does not follow the Watson-Crick pairing rules. There are four main types of wobble base pairs: guanine:cytosine, inosine (hypoxanthine):cytosine, inosine-adenine, and inosine-cytosine.The rules of canonical and atypical hydrogen bonding are known to those skilled in the art (see, e.g., The RNA World, 3rd Edition (Cold Spring Harbor Monograph Series), R.F. Gesteland, Cold Spring Harbor Laboratory Press (2005), ISBN 978-0879697396; The RNA World, 2nd Edition (Cold Spring Harbor Monograph Series), R.F. Gesteland, et al., Cold Spring Harbor Laboratory Press (1999), ISBN 978-0879695613; The RNA World (Cold Spring Harbor Monograph Series), R.F. Gesteland, et al., Cold Spring Harbor Laboratory Press (1993), ISBN 978-0879694562 (see, e.g., Appendix 1: Structures of Base Pairs Involving at Least Two Hydrogen Bonds, I. Tinoco); Principles of Nucleic Acid Structure, W. Saenger, Springer International Publishing AG (1988), ISBN 978-0-387-90761-1; Principles of Nucleic Acid Structure, first edition, S. Neidle, Academic Press (2007), ISBN 978-01236950791).

[0097] "Connect," "connected," and "connecting" are used interchangeably herein and refer to a covalent or non-covalent bond between two macromolecules (eg, polynucleotides, proteins, etc.).

[0098] As used herein, the terms "nucleic acid sequence," "nucleotide sequence," and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides having a 5' end and a 3' end, and may comprise one or more nucleic acid sequences. A "circular polynucleotide" refers to a polynucleotide having a covalent bond between its 5' end and its 3' end, thereby forming a circular polynucleotide. Nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof (e.g., as described above in the context of guide polynucleotides), and may have any length. Polynucleotides may perform any function and may have various secondary and tertiary structures. The term includes known analogs of natural nucleotides and modified nucleotides in base, sugar, and / or phosphate moieties. Analogs of specific nucleotides have the same base pairing specificity (e.g., analogs of A base pairs with T). A polynucleotide may comprise one modified nucleotide or a plurality of modified nucleotides. Examples of modified nucleotides include, but are not limited to, fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. Nucleotide structure can be modified before or after polymer assembly.After polymerization, polynucleotide can be modified via, for example, being puted together with labeling component or target binding component in addition.The nucleotide sequence can comprise non-nucleotide components.Also comprise the backbone residue or connection comprising modification, i.e. synthetic, naturally occurring and / or non-natural existence, and with the nucleic acid of reference polynucleotide (for example, DNA or RNA) having similar binding characteristics.The example of such analogues includes but is not limited to phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNAs), locked nucleic acid (LNA TM ) (Exiqon, Inc., Woburn, MA) nucleosides, glycol nucleic acids, bridged nucleic acids, and morpholino structures.

[0099] Peptide-nucleic acids (PNAs) are synthetic analogs of nucleic acids in which the polynucleotide sugar-phosphate backbone is replaced by a flexible pseudo-peptide polymer and the nucleobases are linked to the polymer. PNAs have the ability to hybridize with complementary sequences of RNA and DNA with high affinity and specificity.

[0100] In phosphorothioate nucleic acids, phosphorothioate (PS) bonds replace sulfur atoms with non-bridging oxygens in the polynucleotide phosphate backbone. This modification renders internucleotide linkages resistant to nuclease degradation. In some embodiments, phosphorothioate bonds are introduced between the last 3-5 nucleotides at the 5' or 3' end of a polynucleotide sequence to inhibit exonuclease degradation. Placing phosphorothioate bonds throughout the oligonucleotide also helps reduce endonuclease degradation.

[0101] Threonose nucleic acid (TNA) is an artificial genetic polymer. Its backbone consists of repeating threose sugars linked by phosphodiester bonds. TNA polymers are resistant to nuclease degradation and can self-assemble into double-stranded structures through base-pair hydrogen bonding.

[0102] The linkage can be reversed by using a "reverse phosphoramidite" (see, for example, www.ucalgary.ca / dnalab / synthesis / -modifications / linkages). The 3'-3' linkage at the end of a polynucleotide stabilizes the polynucleotide against exonuclease degradation by producing an oligonucleotide with two 5'-OH ends but lacking a 3'-OH end. Typically, such polynucleotides have a phosphoramidite group at the 5'-OH position and a dimethoxytrityl (DMT) protecting group at the 3'-OH position. Typically, the DMT protecting group is on the 5'-OH and the phosphoramidite is on the 3'-OH.

[0103] Polynucleotide sequences are shown herein in the conventional 5'-3' orientation unless otherwise indicated.

[0104] As used herein, "sequence identity" generally refers to the percent identity of nucleotide bases or amino acids derived by comparing a first polynucleotide or polypeptide with a second polynucleotide or polypeptide using algorithms with different weight parameters. The sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment using various methods and computer parameters (e.g., BLAST, CS-BLAST, PSI-BLAST, FASTA, HMMER, L-ALIGN, etc.) available on the World Wide Web, including but not limited to GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk) websites. The standard default parameters of various methods or computer programs are generally used to calculate the sequence identity between two polynucleotides or two polypeptide sequences. As used herein, the height of sequence identity between two polynucleotides or two polypeptides is generally from about 90% identity to 100% identity, for example, about 90% identity or higher, preferably about 95% identity or higher, more preferably about 98% identity or higher. As used herein, the moderate sequence identity between two polynucleotides or two polypeptides is generally about 80% identity to about 85% identity, for example, about 80% identity or higher, preferably about 85% identity. As used herein, the low sequence identity between two polynucleotides or two polypeptides is generally about 50% identity to 75% identity, for example, about 50% identity, preferably about 60% identity, more preferably about 75% identity. For example, the Cas protein (for example, IE type Cse2, Cas5, Cas6, Cas7 and / or Cas8) comprising amino acid substitutions can have a low sequence identity, a moderate sequence identity or a high sequence identity in its length with a reference to Cas protein (for example, wild type IE type Cse2, Cas5, Cas6, Cas7 and / or Cas8, respectively). As another example, a guide polynucleotide can have a low degree of sequence identity, a moderate degree of sequence identity, or a high degree of sequence identity over its length compared to a reference wild-type guide polynucleotide complexed with a reference Cas protein (e.g., a guide polynucleotide complexed with type IE Cse2, Cas5, Cas6, Cas7, and / or Cas8).

[0105] As used herein, "hybridization," "hybridize," or "hybridizing" is the process of combining two complementary single-stranded DNA or RNA molecules by hydrogen base pairing to form a single double-stranded molecule (DNA / DNA, DNA / RNA, RNA / RNA). Hybridization stringency is generally determined by the hybridization temperature and the salt concentration of the hybridization buffer; for example, high temperature and low salt provide high stringency hybridization conditions. Examples of salt concentration ranges and temperature ranges for different hybridization conditions are as follows: high stringency, about 0.01 M to about 0.05 M salt, hybridization temperature 5°C-10°C below Tm; moderate stringency, about 0.16 M to about 0.33 M salt, hybridization temperature 20°C-29°C below Tm; and low stringency, about 0.33 M to about 0.82 M salt, hybridization temperature 40°C-48°C below Tm. The Tm of double-stranded nucleic acid sequences is calculated by standard methods well known in the art (see, for example, Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: New York (1982); Casey, J., et al., Nucleic Acids Res. 4: 1539-1552 (1977); Bodkin, DK, et al., J. Virological Methods 10: 45-52 (1985); Wallace, RB, et al., Nucleic Acids Res. 9: 879-894 (1981)). Algorithmic prediction tools for estimating Tm are also widely available. High stringency conditions for hybridization generally refer to conditions under which a polynucleotide complementary to a target sequence primarily hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Typically, hybridization conditions are of moderate stringency, preferably high stringency.

[0106] As used herein, "complementarity" refers to the ability of a nucleic acid sequence to form hydrogen bonds with another nucleic acid sequence (e.g., by canonical Watson-Crick base pairing). Percent complementarity represents the percentage of residues in a nucleic acid sequence that can form hydrogen bonds with a second nucleic acid sequence. If two nucleic acid sequences have 100% complementarity, then the two sequences are fully complementary, i.e., all contiguous residues of the first polynucleotide hydrogen bond with the same number of contiguous residues in the second polynucleotide.

[0107] As used herein, “binding” refers to non-covalent interactions between macromolecules (e.g., between proteins and polynucleotides, between polynucleotides and polynucleotides, between proteins and proteins, etc.). Such non-covalent interactions are also referred to as “binding” or “interaction” (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule binds to the second macromolecule in a non-covalent manner). Some parts of the binding interaction may be sequence-specific (the terms “sequence-specific binding,” “sequence-specific binding,” “site-specific binding,” and “site-specific binding” are used interchangeably herein). Sequence-specific binding as used herein generally refers to the ability to form a complex with a type I CRISPR-Cas subunit protein (e.g., Cse2, Cas5, Cas6, Cas7, and Cas8), and preferably with respect to a second nucleic acid sequence (e.g., a second DNA sequence) without a nucleic acid target binding sequence (e.g., a DNA target binding sequence), causing the protein to bind to one or more guide polynucleotides of a nucleic acid sequence (e.g., a DNA sequence) comprising a nucleic acid target sequence (e.g., a DNA target sequence). All components of the binding interaction do not need to be sequence-specific, such as contacts of proteins with phosphate residues in the DNA backbone. A binding interaction can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of a binding interaction. Increased binding affinity is associated with a lower Kd.

[0108] As used herein, an effector complex is said to "target" a polynucleotide if such a complex binds to or cleaves a polynucleotide at a nucleic acid target sequence within the polynucleotide.

[0109] As used herein, a "double-strand break" (DSB) refers to a double-stranded DNA fragment in which both strands are severed. In some cases, if such a break occurs, one strand may be said to have a "sticky end," in which nucleotides are exposed and do not hydrogen bond with nucleotides on the other strand. In other cases, a "blunt end" may occur, in which both strands remain fully base-paired with each other.

[0110] "Donor polynucleotide," "donor oligonucleotide," and "donor template" are used interchangeably herein and can be a double-stranded polynucleotide (e.g., DNA), a single-stranded polynucleotide (e.g., DNA or RNA), or a combination thereof. The donor polynucleotide can be included in homology arms on both sides of the insertion sequence (e.g., DSBs in DNA). The length of the homology arms on each side can be different (e.g., 1-50 bases, 50-100 bases, 100-200 bases, 200-300 bases, 300-500 bases, 500-1000 bases). The homology arms can be symmetrical or asymmetrical in length. Parameters for designing or constructing donor polynucleotides are well known in the art (see, e.g., Ran, F., et al., Nature Protocols 8:2281-2308 (2013); Smithies, O., et al., Nature 317:230-234 (1985); Thomas, K., et al., Cell 44:419-428 (1986); Wu, S., et al., Nature Protocols 3:1056-1076 (2008); Singer, B., et al., Cell 31:25-33 (1982); Shen, P., et al., Genetics 112:441-457 (1986); Watt, V., et al., Proc. Natl. Acad. Sci. USA In some embodiments, the donor polynucleotide comprises a chimeric antigen receptor (e.g., CAR).

[0111] The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein and refer to polypeptide molecules created in the laboratory that typically contain at least two components: an extracellular antigen recognition domain (also called a target binding domain or an extracellular ligand binding domain) and an intracellular activation domain (e.g., containing one or more intracellular signaling domains, and typically containing one or more co-stimulatory signaling domains). CAR may also contain a hinge domain and a transmembrane domain. The structure of a typical CAR polypeptide is as follows: N-terminus - extracellular - [antigen recognition domain - hinge domain] - transmembrane - [transmembrane domain] - intracellular - [intracellular activation domain] - C-terminus; or N-terminus - intracellular - [intracellular activation domain] - transmembrane - [transmembrane domain] - extracellular - [antigen recognition domain - hinge domain] - C-terminus.

[0112] Examples of extracellular antigen recognition domains include portions that bind to antigens and include, but are not limited to, single-chain immunoglobulin variable fragments (scFv), antigen-binding fragments (Fab; typically the region of an antibody that binds to an antigen and consists of one constant domain and one variable domain of each heavy and light chain), nanobodies, single-chain antibodies of camelid or shark origin, engineered protein binding scaffolds (e.g., DARPins and Centyrins), or natural ligands that bind to their cognate receptors.

[0113] Examples of hinge domains include, but are not limited to, polypeptide hinges of variable length (e.g., one or more amino acids), the hinge region of CD8α, the hinge region of CD28, the hinge region of IgG4, and combinations thereof.

[0114] Examples of transmembrane domains include, but are not limited to, transmembrane regions derived from transmembrane proteins such as CD8α, CD28, DAP10, DAP12, NKG2D, and combinations thereof.

[0115] Examples of intracellular activation domains include, but are not limited to, intracellular signaling domains of CD28, 4-1BB, CD3ζ, OX40, 2B4, DAP10, DAP12, truncated and mutated signaling domains (e.g., mutations and truncations in the three ITAM domains of CD3ζ), or other intracellular signaling domains, and combinations thereof.

[0116] When the extracellular ligand binding domain binds to the cognate ligand, the extracellular signaling domain of the CAR activates lymphocytes (for descriptions of CAR-T cells, see, e.g., Brudno, J., et al., Nature Rev. Clin. Oncol. 15:31-46 (2018); Maude, S., et al., N. Engl. J. Med. 371:1507-1517 (2014); Sadelain, M., et al., Cancer Disc. 3:388-398 (2013); U.S. Pat. No. 7,446,190; U.S. Pat. No. 8,399,645) (for descriptions of CAR-NK cells, see, e.g., Rezvani, K., et al., Mol. Ther., 25:1769-1781 (2017); Siegler, E., et al., Cell Stem Cell. 25:1769-1781 (2017); Cell.23:160-161(2018); Li,Y.,et al.,Cell StemCell.23:181-192(2018); Lin,C.,et al.,Biochim.Biophys.Acta.Rev.Cancer.1869:200-215(2018); Hu,Y.,et al., Acta. Pharmacol. Sin. 39: 167-176 (2018); Fang, F., et al., Semin. Immunol. 31: 37-54 (2017); Glienke, W., et al., Front Pharmacol. 6: 21 (2015)).

[0117] Table 2 presents exemplary cellular targets and scFvs / binding proteins that bind to the cellular targets. Such scFvs / binding proteins or portions thereof can be incorporated into CAR constructs.

[0118]

[0119]

[0120]

[0121] As used herein, " homology-directed repair " (HDR) refers to the DNA repair that occurs in the cell, for example, during the DSB repair in gDNA. HDR requires nucleotide sequence homology and uses donor or template polynucleotide to repair sequence, wherein DSB (for example, in DNA target sequence) occurs. Donor polynucleotides generally have the sequence homology required with the sequence on both sides of DSB, so that donor polynucleotides can be used as a suitable template for repair. HDR causes genetic information to be transferred to the DNA target sequence from, for example, donor polynucleotides. If the donor polynucleotide sequence is different from the DNA target sequence, and part or all of the donor polynucleotides are incorporated into the DNA target sequence, HDR can cause changes (for example, insertions, deletions or mutations) in the DNA target sequence. In some embodiments, all donor polynucleotides, a portion of the donor polynucleotides or a copy of the donor polynucleotides are integrated into the site of the DNA target sequence. For example, donor polynucleotides can be used for the repair of fracture in the DNA target sequence, wherein repair causes the transmission of genetic information at the break site or close to the fracture in the DNA of the donor polynucleotides. Thus, new genetic information can be inserted or copied at the DNA target sequence.

[0122] A "genomic region" is a chromosomal segment in the genome of a host cell that is present on either side of a nucleic acid target sequence site, or optionally includes a portion of a nucleic acid target sequence site. The homology arms of the donor polynucleotide have sufficient homology to undergo homologous recombination with the corresponding genomic region. In some embodiments, the homology arms of the donor polynucleotide have a large amount of sequence homology with the genomic regions immediately adjacent to the nucleic acid target sequence site on both sides; it is recognized that the homology arms can be designed to have sufficient homology with genomic regions away from the nucleic acid target sequence site.

[0123] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of DSBs in DNA by directly joining one end of the break to the other end of the break without the need for a donor polynucleotide. NHEJ is a DNA repair pathway that can be used by cells to repair DNA without the use of a repair template. In the absence of a donor polynucleotide, NHEJ typically results in random insertions or deletions of nucleotides at the DSB site.

[0124] "Microhomology-mediated end joining" (MMEJ) is a way to repair DSBs in gDNA. MMEJ involves deletions on both sides of the DSB and alignment of microhomology sequences within the break site before joining. MMEJ is defined by genes and requires, for example, the activity of CtIP, poly (ADP-ribose) polymerase 1 (PARP1), DNA polymerase θ (Pol θ), DNA ligase 1 (Lig 1), or DNA ligase 3 (Lig 3). Additional genetic components are known in the art (see, e.g., Sfeir, A., et al., Trends in Biochemical Sciences 40: 701-714 (2015)).

[0125] As used herein, "DNA repair" includes any process by which cellular machinery repairs damage to DNA molecules contained in cells. The damage repaired can include single-strand breaks or DSBs. There are at least three mechanisms for repairing DSBs: HDR, NHEJ, and MMEJ. "DNA repair" is also used herein to refer to DNA repair produced by human or robotic manipulation, wherein the target site is modified, for example, by insertion, deletion, or substitution of nucleotides, all of which represent forms of genome editing.

[0126] As used herein, "recombination" refers to the process by which genetic information is exchanged between two polynucleotides.

[0127] As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are interchangeable and refer to polynucleotide sequences to be expressed upstream (5' non-coding sequences), internal, or downstream (3' non-translated sequences) of a polynucleotide target. Regulatory sequences influence, for example, the timing of transcription; the amount or level of transcription; RNA processing or stability; and / or translation of associated structural nucleotide sequences. Regulatory sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, transcription start sites, repressor binding sequences, stem-loop structures, translation start sequences, internal ribosome entry sites (IRES), translation leader sequences, transcription termination sequences (e.g., polyadenylation signals and poly-U sequences), translation termination sequences, primer binding sites, and the like.

[0128] Regulatory elements include those that direct the constitutive, inducible and repressible expression of the nucleotide sequence in many types of host cells, and those that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). In some embodiments, the vector comprises one or more pol III promoters, one or more pol II promoters, one or more pol I promoters, or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, retroviral Rous sarcoma virus (RSV) LTR promoters (optionally with RSV enhancers), cytomegalovirus (CMV) promoters (optionally with CMV enhancers; see, e.g., Boshart, M., et al., Cell 41:521-530 (1985)), SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, phosphoglycerate kinase (PGK) promoters and EF1α promoters, and artificial promoters (e.g., MND promoters and CAG promoters) of transformation. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. The vector can be introduced into a host cell to produce RNA transcripts, proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acid sequences as described herein.

[0129] As used herein, "gene" refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may also comprise introns and / or untranslated regions (UTRs).

[0130] As used herein, the term "operably connected" refers to a polynucleotide sequence or amino acid sequence that is functionally associated with each other. For example, if a regulatory sequence regulates or contributes to the transcriptional regulation of a polynucleotide, a regulatory sequence (e.g., a promoter or enhancer) is "operably connected" to the polynucleotide encoding the gene product. The regulatory element operably connected is usually adjacent to the coding sequence. However, if no more than several thousand bases or more are separated from the promoter, the enhancer can function. In addition, a polycistronic construct can include multiple coding sequences that only use one promoter by including 2A self-cleaving peptides, IRES elements, etc. Therefore, some regulatory elements can be operably connected to a polynucleotide sequence, but are not adjacent to the polynucleotide sequence. Similarly, translational regulatory elements contribute to regulating protein expression from a polynucleotide.

[0131] As used herein, "expression" refers to the transcription of a polynucleotide from a DNA template to produce, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding, such as structural or scaffold RNAs). The term also refers to the process by which transcribed mRNA is translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from gDNA, expression in eukaryotic cells may include splicing of the mRNA.

[0132] A "coding sequence," or a sequence "encoding" a selected polypeptide, is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminus and a translation stop codon at the 3' terminus.

[0133] As used herein, by "artificial transcriptional activator (ATA)" or "artificial transcription factor (ATF)" is meant a complex capable of recruiting RNA polymerase II holoenzyme to its associated gene, thereby causing ectopic expression of the target gene. Such activators include at least two components: (1) a catalytically inactive polynucleotide binding domain that directly recognizes homologous nucleotide sequences and is capable of binding to these sequences, or a polynucleotide binding domain that directs such sequences for binding (e.g., a nucleoprotein complex comprising a nucleic acid binding domain and a guide as described herein); and (2) an activation domain (also referred to as an "effector domain") that interacts with various proteins that constitute the transcriptional machinery to upregulate transcription.

[0134] By "catalytically inactive polynucleotide binding domain," it is meant a molecule that binds but does not cleave the nucleic acid target site to which the binding domain is bound. Representative examples of such domains are described in detail herein.

[0135] As used herein, the term "modulate" refers to a change in the quantity, degree, or amount of a function. For example, the type I CRISPR nucleoprotein complex disclosed herein can modulate the activity of a promoter sequence by binding to a nucleic acid target sequence at or near a promoter or transcription start site or regulatory site. Depending on the effects that occur upon binding, the type I CRISPR nucleoprotein complex can induce, enhance, repress, or inhibit transcription of a gene operably linked to a promoter sequence. Thus, "modulation" of gene expression includes both gene activation and gene repression.

[0136] Regulation can be analyzed by determining any characteristic that is directly or indirectly affected by the expression of the target gene. These characteristics include, for example, changes in RNA or protein levels, protein activity, product levels, expression of a gene, or activity levels of a reporter gene. Thus, the terms "regulate the expression of a gene," "inhibit the expression of a gene," and "activate the expression of a gene" can refer to the ability of a type I CRISPR nucleoprotein complex to alter, activate, or inhibit gene transcription.

[0137] Function (e.g., enzymatic function) can be upregulated (e.g., increased, enhanced, amplified, or strengthened function) or downregulated (e.g., decreased, weakened, reduced, or reduced function). In one embodiment, mCas3 protein binding to single-stranded DNA (ssDNA) or ATP binding / hydrolysis by mCas3 protein can be upregulated or downregulated relative to the corresponding wtCas3 protein.

[0138] As used herein, "vector" and "plasmid" refer to polynucleotide vectors that introduce genetic material into cells. Vectors can be linear or circular. A vector can include a replication sequence that enables replication of the vector in a suitable host cell (e.g., an origin of replication). After transforming a suitable host, the vector can replicate and function independently of the host genome, or it can be integrated into the host genome. Among other things, vector design depends on the intended use of the vector and the host cell, and the design of the vector of the present invention for specific uses and host cells is within the technical level of this art. The four main types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Typically, a vector includes an origin of replication, a multiple cloning site, and / or a selectable marker. An expression vector typically includes an expression cassette. By "recombinant virus," it is meant a virus to which a heterologous nucleic acid construct has been, for example, added or inserted into a viral genome or a portion thereof and genetically altered.

[0139] As used herein, "expression cassette" refers to a polynucleotide construct produced using recombinant methods or by synthetic means and comprising regulatory sequences operably linked to a selected polynucleotide to promote expression of the selected polynucleotide in a host cell. For example, the regulatory sequences can promote transcription of the selected polynucleotide in the host cell, or transcription and translation of the selected polynucleotide in the host cell. For example, the expression cassette can be integrated into the genome of the host cell, or can be present in a vector to form an expression vector.

[0140] As used herein, "targeting vector" is a recombinant DNA construct that typically includes a specially made DNA arm homologous to gDNA, located on both sides of a target gene or nucleic acid target sequence (e.g., DSB). The targeting vector comprises a donor polynucleotide. The elements of the target gene can be modified in a variety of ways, including deletion and / or insertion. Defective target genes can be replaced with functional target genes, or alternatively, functional genes can be knocked out. The donor polynucleotide of the targeting vector comprises a selection cassette that includes a selectable marker introduced into the target gene. The targeting region (comprising a nucleic acid target sequence) adjacent to or within the target gene can be used to achieve regulation of gene expression.

[0141] As used herein, the term "between" includes the endpoints within a given range (e.g., a length between 1-50 nucleotides includes 1 nucleotide and 50 nucleotides; a length between 5 amino acids and 50 amino acids includes 5 amino acids and 50 amino acids).

[0142] As used herein, the term "amino acid" (aa) refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and the D or L optical isomers.

[0143] As used herein, the terms "peptide," "polypeptide," "protein," and "subunit protein" are interchangeable and refer to polymers of amino acids. A polypeptide can be of any length. It can be branched or linear, it can be interrupted by non-amino acids, and it can contain modified amino acids. The term also refers to amino acid polymers that have been modified, for example, by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling component or ligand). Unless otherwise indicated, polypeptide sequences herein are shown in a conventional N-terminal to C-terminal direction.

[0144] Polypeptides and polynucleotides can be prepared using conventional techniques in the field of molecular biology (see, for example, the standard texts listed above). In addition, essentially any polypeptide or polynucleotide can be obtained from commercial sources.

[0145] As used herein, the terms "fusion protein" and "chimeric protein" refer to a single protein produced by connecting two or more proteins, protein domains, protein fragments, or circularly arranged polypeptides that are not naturally present together in a single protein. In some embodiments, a linker polynucleotide can be used to connect a first protein, protein domain or protein fragment or a circularly arranged polypeptide to a second protein, protein domain, protein fragment, or a circularly arranged polypeptide. For example, a fusion protein can comprise a type I CRISPR-Cas protein (e.g., Cas8, Cas3) and a functional domain from another protein (e.g., FokI; see, e.g., U.S. Patent No. 9,885,026). Modification to include such a domain in the fusion protein can confer additional activity to the modified type I CRISPR-Cas protein. Such activities can include nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming 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, adenylylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, and / or myristoylation activity or demyristoylation activity that modifies a polypeptide associated with a nucleic acid target sequence (e.g., a histone).

[0146] In some embodiments, the fusion protein may comprise an epitope tag (e.g., a histidine tag, an HA tag, (Sigma Aldrich, St. Louis, MO) tag, Myc tag, nuclear localization signal (NLS) tag, SunTag), reporter protein sequence (e.g., glutathione-S-transferase, β-galactosidase, luciferase, green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein), and / or nucleic acid sequence binding domain (e.g., DNA binding domain or RNA binding domain).

[0147] The fusion protein can also include an activator domain (e.g., a heat shock transcription factor NFKB activator) or a repressor domain (e.g., a KRAB domain). As described in Lupo, A., et al., Current Genomics 14:268-278 (2013), the KRAB domain is an effective transcriptional repression module and is located in the amino-terminal sequence of most C2H2 zinc finger proteins (see, e.g., Margolin, J., et al., Proc. Natl. Acad. Sci. USA 91:4509-4513 (1994); Witzgall, R., et al., Proc. Natl. Acad. Sci. USA 91:4514-4518 (1994)). The KRAB domain typically binds to co-repressor proteins and / or transcription factors via protein-protein interactions, resulting in transcriptional repression of genes bound to KRAB zinc finger proteins (KRAB-ZFPs) (see, e.g., Friedman, JR, et al., Genes & Development 10: 2067-2678 (1996)). In some embodiments, a linker nucleic acid sequence is used to connect two or more proteins, protein domains, or protein fragments.

[0148] As used herein, "CASCADEa" (Cascade activation) is a CRISPR method or system wherein the method or system activates the expression of a gene associated with a site of a target nucleic acid sequence of the Cascade RNP complex. In some embodiments, one or more proteins of the Cascade complex are fused to an effector domain (e.g., VP16 or VP64), and the Cascade RNP complex comprising the fusion and a guide polynucleotide is used to recruit endogenous transcription factors. In some embodiments, the guide polynucleotide can be fused 5' or 3' to a nucleotide effector domain, such as an MS2-binding RNA that also recruits transcription factors.

[0149] As used herein, "CASCADEi" (Cascade inhibition) is a CRISPR method or system that downregulates the expression of a gene associated with the site of the target nucleic acid sequence of the Cascade RNP complex (i.e., the Cascade RNP complex is used to downregulate the expression of a gene). For recruitment of endogenous repressors, one or more proteins in the Cascade complex are typically fused to an effector domain (e.g., KRAB). In some embodiments, the guide polynucleotide can be fused 5' or 3' to a nucleotide effector domain that also recruits endogenous transcriptional repressor effector proteins.

[0150] As used herein, "moiety" refers to a portion of a molecule. A moiety can be a functional group, or it can describe a portion of a molecule that has multiple functional groups (e.g., sharing common structural aspects). The terms "moiety" and "functional group" are generally used interchangeably herein; however, "functional group" can more specifically refer to a portion of a molecule that includes some common chemical behavior. "Moiety" is generally used as a structural description. In some embodiments, the 5' end, the 3' end, or both the 5' end and the 3' end (e.g., a non-natural 5' end and / or a non-natural 3' end in the first stem element) can comprise one or more moieties.

[0151] As used herein, "adoptive cells" refer to cells that can be genetically modified for use in cell therapy, such as for the treatment of cancer and / or prevention of graft-versus-host disease (GvHD) and other adverse side effects of cell therapy, such as, but not limited to, cytokine storm, oncogenic transformation of the administered genetically modified material, neurological disorders, etc. Adoptive cells include, but are not limited to, stem cells, induced pluripotent stem cells (iPSCs), umbilical cord blood stem cells, lymphocytes, macrophages, erythrocytes, fibroblasts, endothelial cells, epithelial cells, and pancreatic precursor cells.

[0152] As used herein, "cell therapy" refers to the use of genetically modified cells to treat a disease or condition. Genetic modifications can be introduced using methods described herein, such as methods including viral vectors, nucleofection, gene gun delivery, sonication, cell squeezing, lipofection, or the use of other chemicals, cell penetrating peptides, and the like.

[0153] As used herein, "adoptive cell therapy (ACT)" refers to a therapy that uses genetically modified cells derived from a specific patient (autologous cell therapy) or a third-party donor (allogeneic cell therapy) returned to the patient to treat the patient. ACTs include, but are not limited to, bone marrow transplantation, stem cell transplantation, T cell therapy, CAR-T cell therapy, and natural killer (NK) cell therapy.

[0154] As used herein, "lymphocyte" refers to a leukocyte (white blood cell) that is part of the vertebrate immune system. The term "lymphocyte" also includes hematopoietic stem cells or induced pluripotent stem cells (iPSCs) that produce lymphoid cells. Lymphocytes include T cells for cell-mediated cytotoxic adaptive immunity, such as CD4+ and / or CD8+ cytotoxic T cells; α / βT cells and γ / δT cells; regulatory T cells, such as Treg cells; NK cells that play a role in cell-mediated cytotoxic innate immunity; B cells for humoral, antibody-driven adaptive immunity; NK / T cells; cytokine-induced killer cells (CIK cells); and antigen presenting cells (APCs), such as dendritic cells. Lymphocytes can be mammalian cells, such as human (Homo sapiens; H. sapiens) cells. The term "lymphocyte" also includes genetically modified T cells and NK cells that are modified to produce chimeric antigen receptors (CARs) on the surface of T or NK cells (CAR-T cells and CAR-NK cells). These CAR-T cells recognize specific soluble antigens or antigens on the surface of target cells, such as tumor cells, or cells in the tumor microenvironment.

[0155] As used herein, the term "lymphocyte" also includes T cell receptor-engineered T cells (TCRs), which are genetically modified to express one or more specific naturally occurring or engineered T cell receptors that can recognize protein or (glyco) lipid antigens of target cells presented by the major histocompatibility complex (MHC). Small fragments of these antigens, such as peptides or fatty acids, are shuttled to the surface of target cells and presented to the T cell receptor as part of the MHC. The binding of the T cell receptor to the antigen-loaded MHCs activates the lymphocyte.

[0156] Lymphocyte activation occurs when lymphocytes are triggered through antigen-specific receptors on their cell surfaces. This leads to cell proliferation and differentiation into specialized effector lymphocytes. These "activated" lymphocytes are often characterized by a set of receptors on their surface. Surface markers of activated T cells include CD3, CD4, CD8, PD1, IL2R, and others. Activated cytotoxic lymphocytes can kill target cells after binding to their cognate receptors on the target cell surface.

[0157] As used herein, the term "lymphocyte" also includes tumor-infiltrating lymphocytes (TILs). TILs are immune cells that have penetrated the tumor and its surrounding environment (the "tumor microenvironment"). TILs are typically isolated from tumor cells and the tumor microenvironment and selected in vitro for their high reactivity against tumor antigens. TILs are grown in vitro under conditions that overcome the tolerance effects present in vivo and then introduced into the subject for treatment.

[0158] T cells typically present many subtypes, such as "naive T cells" (Tn), "stem cell memory T cells" (Tscm), "central memory T cells" (Tcm), "effector memory T cells" (Tem), "effector T cells" (Teff), and "regulatory T cells" (Treg). Each T cell subset is characterized by a set of cell surface markers.

[0159] As used herein, the term "affinity tag" generally refers to one or more parts that increase the binding affinity of a macromolecule to another macromolecule, for example, to promote the formation of a modified type I CRISPR-Cas nucleoprotein complex. In some embodiments, an affinity tag can be used to increase the binding affinity of one Cas subunit protein to another Cas subunit protein (for example, a first Cas7 protein to a second Cas7 protein). In some embodiments, an affinity tag can be used to increase the binding affinity of one or more Cas subunit proteins to a homologous guide polynucleotide. Some embodiments of the present invention introduce one or more affinity tags into the N-terminus of the Cas subunit protein sequence, the C-terminus of the Cas subunit protein sequence, a position between the N-terminus and the C-terminus of the Cas subunit protein sequence, or a combination thereof. In some embodiments of the present invention, one or more guide polynucleotides comprise an affinity tag that increases the binding affinity of the guide polynucleotide to one or more Cas subunit proteins. A variety of affinity tags are disclosed in U.S. Published Patent Application No. 2014-0315985, published on October 23, 2014. The ligand and the ligand-binding moiety are paired affinity tags.

[0160] As used herein, "crosslinking" is the bonding of one polymer chain (e.g., a polynucleotide or polypeptide) to another. Such bonding can be a covalent bond or an ionic bond. In some embodiments, one polynucleotide can be bound to another polynucleotide by crosslinking the polynucleotides. In other embodiments, a polynucleotide can be crosslinked to a polypeptide. In yet other embodiments, a polypeptide can be crosslinked to a polypeptide.

[0161] As used herein, the term "cross-linking moiety" generally refers to a moiety suitable for providing a cross-link between two macromolecules. A cross-linking moiety is another example of an affinity tag.

[0162] As used herein, "host cell" generally refers to a biological cell. A cell is the basic structural, functional, and / or biological unit of an organism. A cell can be derived from any organism that has one or more cells. Examples of host cells include, but are not limited to, prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, cells of eukaryotic organisms, protozoan cells, cells from plants, algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nanonochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. agardh, etc.), seaweed (e.g., macroalgae), fungal cells (e.g., yeast cells or cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates including mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). In addition, the host cell can be a stem or progenitor cell, as well as an immune cell, such as any immune cell described herein. The host cell can be a human cell. In some embodiments, the human cell is outside the human body. In some embodiments, the somatic cells of a living organism (e.g., a human body) are manipulated ex vivo (i.e., outside the body). Ex vivo generally refers to a medical procedure in which an organ, cell, or tissue is removed from a living organism (e.g., a human body) for treatment or surgery and then returned to the living organism.

[0163] As used herein, "stem cell" refers to a cell that has the ability to self-renew, i.e., undergo many cycles of cell division while remaining in an undifferentiated state. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells can be embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.

[0164] As used herein, "induced pluripotent stem cells" refers to a class of pluripotent stem cells artificially derived from non-pluripotent cells, typically somatic cells. In some embodiments, the somatic cells are human somatic cells. Examples of somatic cells include, but are not limited to, dermal fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Other examples of somatic cells include cells of the immune system, including but not limited to B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells.

[0165] As used herein, "hematopoietic stem cells" refer to undifferentiated cells that have the ability to differentiate into hematopoietic cells such as lymphocytes.

[0166] As used herein, "plant" refers to whole plants, plant organs, plant tissues, germplasm, seeds, plant cells and their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, branches, gametophytes, sporophytes, pollen and microspores. Plant parts include differentiated and undifferentiated tissues, including but not limited to roots, stems, branches, leaves, pollen, seeds, tumor tissues and various forms of cells and cultures (e.g., unicellular, protoplasts, embryos and callus). Plant tissue can be in plants or in plant organs, tissues or cell cultures. "Plant organ" refers to a plant tissue or a group of tissues that constitute the morphologically and functionally different parts of a plant.

[0167] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including but not limited to humans and other primates, including non-human primates such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; birds including domestic, wild, and game birds such as chickens, turkeys, and other galliform birds, ducks, and geese; and the like. The term does not denote a particular age or sex. Thus, the term includes adult, young, and newborn individuals, as well as males and females. In some embodiments, the host cell is derived from the subject (e.g., lymphocytes, stem cells, progenitor cells, or tissue-specific cells). In some embodiments, the subject is a non-human subject. In some embodiments, the subject is a human (Homo sapiens) subject.

[0168] The term "effective amount" or "therapeutically effective amount" of a composition or reagent, such as the genetically modified adoptive cells provided herein, refers to an amount of the composition or reagent sufficient to provide a desired response, such as preventing or eliminating one or more harmful side effects associated with allogeneic adoptive cell therapy. Such a response will depend on the specific target disease. For example, in patients using adoptive cell therapy to treat cancer, the desired response includes, but is not limited to, treating or preventing GvHD, host-versus-graft rejection, cytokine release syndrome (CRS), cytokine storm, and reducing the effects of oncogenic transformation of the administered genetically modified cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific modified lymphocytes used, the mode of administration, etc. In any individual case, one of ordinary skill in the art can determine the appropriate "effective" amount using routine experiments.

[0169] "Treatment" or "treating" a particular disease, such as a cancerous condition or GvHD, includes: (1) preventing the disease, e.g., preventing the development of the disease or causing the disease to occur at a lesser intensity in a subject who may be susceptible to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, e.g., reducing the rate of progression, arresting progression, or reversing the disease state; and / or (3) alleviating the symptoms of the disease, e.g., reducing the number of symptoms experienced by a subject.

[0170] As used herein, by "gene editing" or "genome editing" is meant a type of genetic engineering that results in genetic modification, such as the insertion, deletion, or substitution of a nucleotide sequence at a specific site in the cell genome, or even a single base. The term includes, but is not limited to, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, and destructive genetic modification as defined herein.

[0171] By "epitope" is meant the site on a molecule to which specific B cells and T cells respond. In the spatial configuration of the epitope uniqueness, an epitope can comprise 3 or more amino acids. Typically, an epitope is composed of at least five such amino acids, and more typically, of at least 8-10 such amino acids. Methods for determining the spatial configuration of amino acids are known in the art and include, for example, x-ray crystallography, electron microscopy, and two-dimensional nuclear magnetic resonance. In addition, the identification of epitopes in a given protein can be easily accomplished using techniques well known in the art, such as by using hydrophobicity studies and by site-directed serology.

[0172] A "mimotope" is a macromolecule, such as a peptide, that mimics the structure of an epitope. Due to this property, it elicits an antibody response similar to that elicited by the epitope. Antibodies specific for a given epitope will recognize a mimotope that mimics that epitope. Mimotopes are typically obtained from phage display libraries through biopanning.

[0173] "Antibody" means a molecule that "recognizes," i.e., specifically binds to, a target epitope presented in a polypeptide, such as a ligand binding domain. By "specifically binds," it is meant that the antibody interacts with the epitope in a "lock and key" type interaction to form a complex between the antigen and the antibody. As used herein, the term "antibody" includes antibodies obtained from monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules; F(ab')2 and F(ab) fragments; Fv molecules (non-covalent heterodimers; single-chain Fv molecules (scFv); dimeric and trimeric antibody fragment constructs; minibodies; humanized antibody molecules; single-chain antibodies; nanobodies (Ablynx NV, Zwijnaarde, Belgium) antibodies; and any functional fragments obtained from these molecules, wherein such fragments retain the immunological binding properties of the parent antibody molecule. Antibodies can be derived from different species, such as humans, mice, rats, rabbits, camels, chickens, etc. Antibodies and antibody portions can then be further obtained by in vitro techniques, such as phage display and yeast display. Fully humanized antibodies can be obtained from human plasma, human B cell clones, mice, rats, rabbits, chickens, etc., which have engineered humanized B cell repertoires. Antibodies can then be further modified by affinity maturation and other methods, such as fucosylation or IgG Fc engineering.

[0174] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited with respect to the species or source of the antibody, nor is it intended to be limited by the manner in which it is prepared. The term includes intact immunoglobulins as well as fragments such as Fab, F(ab')2, Fv and other fragments, as well as chimeric and humanized homogeneous antibody populations, all of which exhibit the immunological binding properties of the parent monoclonal antibody molecules of the invention.

[0175] "Antibody-dependent cell-mediated cytotoxicity (ADCC)", also known as "antibody-dependent cellular cytotoxicity", refers to a mechanism by which effector cells of the immune system, such as adoptive cells, actively lyse target cells when the membrane surface ligand binding domain has been bound by a specific antibody. Effector cells are usually natural killer (NK) cells. However, macrophages, neutrophils, and eosinophils can also mediate ADCC. ADCC is independent of complement-dependent cytotoxicity (CDC), which can also lyse targets by disrupting membranes without the involvement of antibodies or immune system cells.

[0176] As used herein, "transformation" refers to the insertion of exogenous polynucleotides into a host cell, regardless of the method used to insert. For example, transformation can be carried out by direct uptake, transfection, infection, etc. Exogenous polynucleotides can be maintained as non-integrated vectors, such as episomes, or alternatively, can be integrated into the host genome. As used herein, "transgenic organism" refers to an organism containing genetic material, into which DNA from an unrelated organism has been artificially introduced. The term includes the offspring (any generation) of a transgenic organism, as long as the offspring has genetic modification. In some embodiments, the transgenic organism is a non-human transgenic organism.

[0177] As used herein, "isolated" can refer to a molecule (e.g., a polynucleotide or polypeptide) that exists outside of its natural environment through human intervention and is therefore not a product of nature. When referring to a polypeptide, isolated means that the molecule is separated and discontinuous from the intact organism with which it is found in nature, or occurs in the substantial absence of other biological macromolecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule that lacks, in whole or in part, the sequence normally associated with it in nature; or a sequence, when it occurs in nature, but with heterologous sequences associated with it; or a molecule separated from a chromosome.

[0178] As used herein, the term "purified" preferably means that at least 75% by weight, more preferably at least 85% by weight, still more preferably at least 95% by weight, and most preferably at least 98% by weight of the same molecule is present.

[0179] As used herein, "substrate channeling" refers to the direct transfer of reactants from one enzymatic reaction to another without first diffusing into the bulk environment (see, e.g., Wheeldon, I., et al., Nat. Chem. 8: 299-309 (2016)). The intermediates of these enzymatic steps are not in equilibrium with the bulk solution, which improves the efficiency and yield of the enzymatic process. Typically, in naturally occurring metabolic processes, enzymes have developed ways to co-localize and assemble into controlled aggregates.

[0180] As used herein, "substrate access element" refers to a component of a metabolic pathway. In some embodiments, a substrate access element is an enzyme that catalyzes a chemical reaction.

[0181] As used herein, "substrate access complex" refers to multiple substrate access elements that are co-localized together by some means.

[0182] As used herein, "RNA scaffold" refers to an RNA molecule to which a peptide can serve as a binding substrate.

[0183] The data presented herein demonstrate that fusion between Cascade components and a nuclease domain (e.g., a dimerization-dependent, nonspecific FokI nuclease domain; see, e.g., Urnov, FD, et al., Nature Reviews Genetics 11:636–646 (2010); Joung, JK, et al., Nat. Rev. Mol. Cell Biol. 14:49–55 (2013); Guilinger, JP, et al., Nat. Biotechnol. 32:577–582 (2014); Tsai, SQ, et al., Nat. Biotechnol. 32:569–576 (2014)) mediates efficient, programmable RNA-guided gene editing in human cells using a type I system. The data demonstrate that modified type I CRISPR-Cas systems (e.g., including FokI-Cascade component fusions) can be directly transfected as complete ribonucleoprotein (RNP) complexes or assembled in cells via delivery of components encoded by a single plasmid. As described herein, all CRISPR-associated (Cas) genes are assembled on a single polycistronic vector, resulting in a simplified two-component Cas protein-guided RNA expression system. In addition, the length / composition design of the nuclease (e.g., FokI) / Cascade component linker sequence and appropriate DNA geometry settings, as well as the selection of selective Cascade homologs, provide modified type I CRISPR-Cas complexes with editing efficiencies of up to approximately 50%. Key features of modified type I CRISPR-Cas systems (e.g., including FokI–Cascade component fusion proteins) involving PAM requirements and mismatch sensitivity during DNA targeting were determined.

[0184] In a first aspect, the present invention relates to engineered polynucleotides encoding Cascade components including, but not limited to, Cascade subunit proteins and Cascade guide polynucleotides.

[0185] In one embodiment, the present invention relates to polynucleotides encoding the transformation of Cascade components derived from Cascade IE type systems. In Example 1, exemplary polynucleotide constructs comprising Cascade proteins and Cascade crRNAs are presented. Example 1, Table 15, and SEQ ID NO: 1 to SEQ ID NO: 20 present polynucleotide DNA sequences encoding genes encoding five subunit proteins of IE type Cascade specifically from Escherichia coli strain K-12MG1655, as well as amino acid sequences of the resulting protein components. The polynucleotide sequence is derived from Escherichia coli gDNA, and is specifically codon-optimized for expression in Escherichia coli, and / or specifically codon-optimized for expression in eukaryotic cells (e.g., human cells). When the polynucleotide is transcribed into precursor crRNA and treated with Cascade RNA endonuclease, a mature crRNA used as a guide RNA is produced to target the complementary DNA sequence in the genome. The minimal CRISPR array is comprised of two repeat sequences (underlined in the CRISPR array sequence presented in Example 1) on both sides of the exemplary spacer sequence, which represent the guide portion of the crRNA. Processing of RNA by Cascade endonucleases produces crRNA with repeat sequences on both sides of the guide sequence at the 5' and 3' ends. One of ordinary skill in the art, with reference to the present specification and the examples, can select an appropriate spacer sequence to target the Cascade complex to the selected target sequence (e.g., in gDNA).

[0186] Polynucleotide sequences encoding Cascade components from additional bacterial and archaeal species can be identified and designed according to the guidance of this specification and using bioinformatics tools such as BLAST and PSI-BLAST to locate, for example, homologs of the Cascade subunit genes from E. coli strain K-12MG1655, and then examine the genomic proximity of the two sides of the Cascade gene to locate and identify the genes for the remaining Cascade subunit proteins (see, for example, Examples 14A, 14B, 15A, and 15B). Because Cascade genes occur together as conserved operons, they are typically arranged in the same type I subtype in a consistent order, thereby facilitating their identification and selection for subsequent analysis and experiments. For example, additional type IE systems can be identified by locating Cas8 homologs, identifying promising bacterial species for homologous Cascade testing, and then obtaining or designing polynucleotide sequences encoding Cas8 and other protein components of Cascade from those homologous CRISPR-Cas systems.

[0187] The polynucleotide DNA sequences of genes encoding subunit proteins of Cascade from a number of species (some with Cascade complexes homologous to those derived from E. coli strain K-12 MG1655) (listed in Tables 3 and 4), and the amino acid sequences of the resulting protein components, and an exemplary minimal CRISPR array, are presented as SEQ ID NO: 22 to SEQ ID NO: 213 (Table 3).

[0188] Table 3 Polynucleotide sequences of genes encoding Cascade proteins from 12 species

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] The polynucleotide sequence of the protein is derived from the gDNA of the host bacteria and has been codon-optimized specifically for expression in Escherichia coli and / or codon-optimized specifically for expression in eukaryotic cells (e.g., human cells). The polynucleotide DNA sequence encoding the corresponding minimal CRISPR array is based on a repetitive sequence derived from 12 species and can be used to generate mature crRNA for use as guide RNAs. In Table 4, the minimal CRISPR array is included in two repetitive sequences (lowercase, with underscores) on both sides of the exemplary "spacer" sequence, which represent the guide portion of crRNA. CrRNA with repetitive sequences on both sides of the guide sequence is generated by processing RNA with the endonuclease Cascade subunit.

[0200]

[0201]

[0202] In another embodiment, the present invention relates to modified polynucleotide sequences encoding Cascade components from additional bacterial or archaeal species with other type I phenotypes; including but not limited to IB, IC, IF, and variant types of IF, which can be identified and designed according to the guidance of the present specification and by using bioinformatics tools such as BLAST and PSI-BLAST to locate homologs of Cascade genes from marker systems representing each subtype (see, for example, Makarova, KS, et al., Nat. Rev. Microbiol. 13:722-736 (2015); Koonin, EV, et al., Curr. Opin. Microbiol. 37:67-78 (2017)). After the desired homologs are identified, the genomic neighborhood on both sides of the Cascade gene can be examined to locate and identify the genes for the remaining Cascade subunit proteins disclosed herein. For example, additional IF-type systems can be identified by locating Cas8 homologs (and additional IF-type variant 2 systems can be identified by locating Cas5 homologs) and identifying promising bacterial species for homologous Cascade testing, and then obtaining or designing polynucleotide sequences encoding Cas8, Cas5, and other protein components of Cascade from those homologous CRISPR-Cas systems.

[0203] SEQ ID NO: 214 to SEQ ID NO: 351 show the polynucleotide DNA sequences of genes encoding 3, 4 or 5 subunit proteins of the second type of Cascade from 12 other homologous Cascade complexes, IC, IF and IF variants, and the amino acid sequences of the resulting protein components, as well as exemplary minimal CRISPR arrays (Table 3). The polynucleotide sequences of the subunit proteins are derived from the gDNA of the host bacteria and are specifically codon-optimized for expression in Escherichia coli, and / or specifically codon-optimized for expression in eukaryotic cells (e.g., human cells). The polynucleotide DNA sequences encoding the corresponding minimal CRISPR arrays are based on repetitive sequences derived from 12 species and can be used to generate mature crRNAs for use as guide RNAs. In Table 5, the minimal CRISPR array is included in two repetitive sequences (lowercase, underlined) on both sides of the exemplary "spacer" sequence, which represent the guide portion of the crRNA. The Cascade subunits process RNA to produce crRNAs with repetitive sequences on both sides of the guide sequence at 5' and 3' ends.

[0204]

[0205]

[0206] Examples 19A to 19I and 22A to 22C describe the design and testing of multiple Cascade complex homologs, each of which contains a Cas subunit protein-FokI fusion protein to evaluate the efficiency of genome editing of each Cascade complex. The highest editing was observed using a variant from Pseudomonas S-6-2, while other homologs (i.e., Salmonella enterica, Bacillus geothermophilus EPR-M, Methanospora oryzae MRE50, and Streptococcus thermophilus (strain ND07)) showed editing approximately equal to that of E. coli. Editing was also observed using modified Vibrio cholera strain L15 (IF type) FokI-Cascade complex and Vibrio cholerae strain HE48 (I-Fv2 type) FokI-Cascade complex. In one embodiment, the different PAM requirements of these different homologs can increase the target density in the target polynucleotide (e.g., gDNA in a cell). Thus, the collection of Cascade complex homologs provides greater flexibility in the selection of nucleic acid target sequences in a target polynucleotide (e.g., gDNA in a cell).

[0207] In a second aspect, the present invention relates to modified Cascade subunit proteins. Suitable modified Cascade subunit proteins include, but are not limited to, Cascade subunit proteins of the species described herein.

[0208] In one embodiment, the present invention relates to a modified circularly permuted Cascade subunit protein. Such circularly permuted Cascade subunit proteins result in the production of a protein structure having an original linear sequence of different connectivity of the amino acids of the subunit protein, but having an overall similar three-dimensional shape (see, e.g., Bliven, S., et al., PLoS Comput. Biol. 8: e1002445 (2012)). Circularly permuted Cascade subunit proteins can have many advantages. For example, circularly permuted Cas7 subunit proteins can produce new N-termini and new C-termini that are designed to be placed for connection with another polypeptide sequence to form a fusion protein or linker region without disrupting Cas7 protein folding or Cascade complex assembly. Figure 3A and Figure 3B Three examples of circularly arranged Cas7 (circularly arranged Cas7, cpCas7) are shown in FIG. Figure 3A and Figure 3B In the figure, three parts of the protein are shown: the N-terminal part of the native protein ( Figure 3A, vertical stripes, e.g., Cas7 protein), the central portion of the native protein ( Figure 3A , grey shading) and the C-terminal portion of the native protein ( Figure 3A , no shadow). Figure 3A The repositioning of the N-terminal portion of the native protein to the C-terminal position of the native protein to produce a circularly arranged protein ( Figure 3A , cpCas7), where the N-terminal portion of the native protein is now at the N-terminus of cpCas7 and is connected to the central portion of the native protein via a linker polypeptide ( Figure 3A , linker). Figure 3B The C-terminal portion of the native protein ( Figure 3B , Cas7) to the N-terminal position of the native protein ( Figure 3B , cpCas7), where the C-terminal portion of the native protein is now at the N-terminus of cpCas7 and is connected to the central portion of the native protein ( Figure 3B , linker).

[0209] The data presented in Example 10A, Example 10B, and Example 10 show that the purification of Cascade complexes containing circularly permuted Cas7 subunit protein variants demonstrates that circularly permuted type IE CRISPR-Cas subunit proteins can be successfully used to form Cascade complexes having substantially the same composition (based on molecular weight) as Cascade complexes containing wild-type proteins.

[0210] In another embodiment, the present invention relates to fused to other polypeptide sequences to produce Cascade subunit proteins of fusion proteins, and polynucleotides encoding such fusion proteins. Other polypeptide sequences may include but are not limited to proteins, protein domains, protein fragments, and functional domains. Examples of such other polypeptide sequences include but are not limited to sequences derived from transcriptional activators or repressor domains and nucleotide deaminases (e.g., cytidine deaminases or adenine deaminases, such as Komor, et.al., Nature 553: 420-424 (2016); Koblan, et.al., Nat. Biotechnol. doi: 10.1038 / nbt.4172 (May 29, 2018)). The other functional domains of fusion proteins are shown herein.

[0211] Additional polypeptide sequences can be fused to any Cascade subunit protein, wherein the additional polypeptide sequence is encoded by an additional polynucleotide sequence that is typically attached to the 5' or 3' end of the polynucleotide comprising the coding sequence of the Cascade subunit protein. In some embodiments, the additional polynucleotide sequence encoding the amino acid linker connects the Cascade subunit protein to the additional target polypeptide sequence. In some embodiments, the polynucleotide sequences of the fusion protein partner and the linker sequence can be derived from naturally occurring gDNA sequences, or can be codon optimized for bacterial expression in Escherichia coli or eukaryotic expression in mammalian cells (e.g., human cells). In Example 1, a method for fusion protein partners comprising affinity tags (e.g., His6, Strep- II(IBA GMBH LLC, Germany)), a nuclear localization signal (NLS) of the sequence, maltose binding protein and FokI. Example 1 also discloses an exemplary amino acid linker sequence.

[0212] Example 11A describes the fusion of Cascade subunit protein-FokI, and the fusion of Cascade subunit protein with cytidine deaminase, endonuclease, restriction enzyme, nuclease / helicase or the above domain. Example 11B describes the fusion of Cascade subunit protein with other Cascade subunit protein, and the fusion of Cascade subunit protein with other Cascade subunit fusion protein and enzymatic protein domain (Example 11D). In some embodiments, the ability of type I CRISPR subunit protein to produce protein fusion at a position between the N-terminus, C-terminus, or N-terminus and C-terminus can be evaluated in computer. In some embodiments, one or more polypeptide linkers can be used to connect type I CRISPR subunit protein to one or more fusion domains at a position between the N-terminus, C-terminus, or N-terminus and C-terminus. In some embodiments, the Cascade subunit protein can be fused to a single-chain FokI (e.g., a single-chain FokI is fused to an EcoCascade RNP complex; nucleotide sequence, SEQ ID NO: 1926; protein sequence, SEQ ID NO: 1927). Exemplary polypeptide linkers are shown in Examples 1, 11, 18, and 19.

[0213] Figure 4A and Figure 4B Shown are proteins comprising a Cas8 subunit fused to an additional protein sequence (e.g., FokI) Figure 4A , Figure 4B, Cas7, Cas5, Cas8, Cse2, Cas6, with the dashed box around Cas6 indicating its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin; and Cas8, with the Cascade complex indicated ("C" C-terminus, "N" N-terminus). Figure 4A The C-terminus of the Cas8 subunit protein is connected to the linker polypeptide ( Figure 4A , black curve) additional protein sequences ( Figure 4A , FP) instance. Figure 4B The N-terminus of the Cas8 subunit protein is connected to the linker polypeptide ( Figure 4B , black curve) additional protein sequences ( Figure 4B Example 11A describes the computer design, cloning, expression, and purification of an IE-type Cas8 fused to the N-terminus of the FokI nuclease domain.

[0214] Figure 5A and Figure 5B Additional examples of Cascade complexes comprising Cascade subunit proteins fused to additional protein sequences are shown. Figure 5A and Figure 5B In the figure, cRNA is shown as a black line including a hairpin, and the relative positions of the Cas proteins of the Cascade complex are shown ( Figure 5A , Figure 5B : Cas7, Cas5, Cas8, Cse2, Cas6; the dotted box around Cas6 indicates its interaction with the crRNA hairpin). Figure 5A Each linker polypeptide ( Figure 5A , black curves) fused to a detectable moiety (e.g., green fluorescent protein; Figure 5A Such Cascade complexes can be used to detect binding of the complex to a nucleic acid target sequence by providing significant signal amplification due to the presence of multiple detectable moieties associated with the Cascade complex. Figure 5B Shown is the use of linker polypeptides connected to Cas6 subunit proteins ( Figure 5B , black curve) additional protein sequences ( Figure 5B , FP) instance.

[0215] Examples of fusion proteins containing E. coli type IE Cascade subunit proteins include, but are not limited to, the following: identical subunits (e.g., Cse2_linker_Cse2), circularly arranged subunits (e.g., cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7), type IE Cascade proteins fused to nucleases (e.g., FokI_linker_Cas8, Cas3_linker_Cas8, Cas6_linker_FokI, S1 nuclease_linker_Cse2_linker_Cse2), type IE Cascade proteins fused to cytidine deaminase (e.g., Cas8_linker_AID, Cse2_linker_Cse2_ Linker_APOBEC3G), and one or more other IE type Cascade proteins fused to IE type Cascade proteins (e.g., Cas6_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7, cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_Cas5, Cas6_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_cpCas7_linker_Cas5).

[0216] Figure 6A 、 Figure 6B and Figure 6C A schematic representation of an engineered type I CRISPR-Cas effector complex containing cpCas7 is provided. Figure 6A 、 Figure 6B and Figure 6C In the Cas7 protein, "cpCas7" is a circular arrangement of Cas7 proteins ( Figure 6A , Figure 6B , Figure 6C : cpCas7, Cas5, Cas8, Cse2, and Cas6; the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin; for cpCas7, the shading corresponds to Figure 3A ) and shows the relative positions of the Cas proteins of the Cascade complex. Figure 6A The Cascade complex, comprising six individual cpCas7 subunit proteins, is shown ( Figure 6A , cpCas7). Figure 6BThe Cascade complex comprising six fused cpCas7 subunit proteins is shown, wherein the C-terminus of the cpCas7 subunit protein ( Figure 6B , cpCas7) utilizes a linker polypeptide to connect to the N-terminus of the adjacent cpCas7 subunit protein ( Figure 6B , linker polypeptides are shown as dark black lines connecting the cpCas7 subunit proteins). Figure 6C An embodiment is shown in which the Cascade complex comprises six fused cpCas7 subunit proteins ("skeleton"), wherein the C-terminus of the first cpCas7 subunit protein is linked to the N-terminus of the second cpCas7 subunit protein using a linker polypeptide ( Figure 6C , the linker polypeptide is shown as a dark black line connecting the cpCas7 subunit proteins), and the C-terminus of the second cpCas7 subunit protein utilizes a linker polypeptide ( Figure 6C , the black straight line connecting cpCas7 and FP) and different protein sequences ( Figure 6C The N-terminus of the first cpCas7 subunit protein is connected to the N-terminus of the first cpCas7 subunit protein (e.g., FP) and the C-terminus of the protein coding sequence is connected to the N-terminus of the third cpCas7 using a linker polypeptide. One advantage of such a fusion backbone of the cpCas7 subunit protein is that additional protein sequences can be introduced at specific positions along the backbone to provide additional protein sequences close to different positions along the length of the guide-directed binding nucleic acid target sequence to the Cascade complex.

[0217] Figure 7A and Figure 7B Other embodiments of engineered type I CRISPR-Cas effector complexes comprising fusion proteins are shown. Figure 7A and Figure 7B The relative positions of the Cas proteins in the Cascade complex are shown in Figure 2. Figure 7A , Figure 7B : Cas7, Cas5, Cas8, Cse2 and Cas6; the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin). Figure 7A The Cascade complex including the Cse2-Cse2 fusion protein is shown ( Figure 7A , the two Cse2 proteins are connected by a black curved line.) The computer design, cloning, expression, purification, and electrophoretic mobility shift assay of the Cascade complex comprising a Cse2-Cse2 fusion protein are described in Examples 11B and 11C. Figure 7B shows a polypeptide comprising a polypeptide connected via a linker ( Figure 7B , the black curve connecting Cse2 protein and FP) and another protein sequence ( Figure 7BExample 11D describes the computer design, cloning, expression, and purification of a Cse2-Cse2 protein fused to a cytidine deaminase.

[0218] In some embodiments, one or more nuclear localization signals can be added to the engineered N-terminus or C-terminus of a Cascade protein subunit (e.g., a Cas8-FokI fusion protein, a cpCas7 protein, or a Cse2-Cse2 fusion protein).

[0219] In some embodiments of fusion polypeptide, connexon polypeptide connects two or more protein coding sequences.Exemplary connexon polypeptide length is described in the examples.Generally, connexon length includes but is not limited to about 10 amino acids to about 40 amino acids, about 15 amino acids to about 30 amino acids, and about 17 amino acids to about 20 amino acids.The amino acid composition of connexon polypeptide generally comprises polar, little and / or charged amino acid (for example, Gly, Ala, Leu, Val, Gln, Ser, Thr, Pro, Glu, Asp, Lys, Arg, His, Asn, Cys, Tyr).In other embodiments, connexon polypeptide is designed so that they do not contain methionine, and designed to merge to avoid hidden translation initiation site. Following the guidance of this specification, linker polypeptides were designed to provide appropriate spacing and positions of functional domains and Cascade proteins within the fusion protein (see, e.g., Chichili, C., et al., Protein Science 22: 153-167 (2013); Chen, X., et al., 65: 1357-1369 (2013); George, R., et al., Protein Engineering, Design and Selection 15: 871–879 (2002)). Additional examples of linker polypeptides useful in the practice of the present invention are linker polypeptides identified in organisms comprising the Cascade system that connect the coding sequences of Cascade proteins to each other (e.g., linker polypeptides connecting Cas8 to Cas3 in Streptomyces griseus, as described by Westra, ER, et al., Mol, Cell. 46: 595–605 (2012)).

[0220] The fusion protein encoding DNA sequence can be codon optimized for expression in selected organisms such as bacteria, archaea, plants, fungi, or mammalian cells. Codon optimization programs are widely available, such as on the Integrated DNA Technologies website (www.idtdna.com / CodonOpt) or through To facilitate cloning into receptor expression vectors, additional sequences overlapping with vectors compatible with SLIC cloning can be attached at the 5' and 3' ends of the DNA sequence (see, eg, Li, M., et al., Methods Mol. Biol. 852:51-59 (2012)).

[0221] In other embodiments, Cascade subunit protein can be fused to transcriptional activation and / or inhibition domain. In some embodiments, fusion protein can include activator domain (for example, heat shock transcription factor, NFKB activator, VP16 and VP64 (see, for example, Eguchi, A.et.al., Proc.Natl.Acad.Sci.USA 113:E8257-E8266 (2016); Perez-Pinera, P.et.al., Nature Methods 10:973-6 (2013); Gilbert, LA, et.al.Cell 159:647-61 (2014)) or repressor domain (for example, KRAB domain). In some embodiments, linker nucleic acid sequence is used to connect two or more coding sequences of protein, protein domain or protein fragment.

[0222] The Cascade complex, comprising a type I CRISPR-Cas subunit protein fused to a transcriptional activator, can be used to activate gene expression. The target site may comprise a transcription start site (TSS), which typically has one or more binding sites for the cell's transcriptional activation machinery (factors). Figure 8 Shown is a Cascade complex comprising six fusion proteins comprising a polypeptide linked to a linker ( Figure 8 , black curve connecting cpCas7 to VP64) cpCas7 connected to the transcriptional activator VP64 (with Figure 3A Compared to). Figure 8 In the figure, crRNA is shown as a dark black line including a hairpin, and the relative positions of the Cas proteins of the Cascade complex are shown ( Figure 8: cpCas7, Cas5, Cas8, Cse2 and Cas6; the dotted box around Cas6 indicates its interaction with the crRNA hairpin). The engineering design of this Cascade complex converts the complex into a flexible tool (CASCADEa) for transcriptional activation of genes, wherein targeting of the selected gene is achieved by selecting a guide sequence that directs the binding of the Cascade complex to one or more regulatory elements (e.g., TSS) of the selected gene. Example 12 describes the design of Escherichia coli type IE cp-Cas7 proteins fused to the VP64 activation domain to confer transcriptional activation activity on the Cascade complex. Transcriptional activators include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, leucine zipper proteins, helix-loop-helix proteins, heterodimeric transcription factors, activation domains, and transcription factors that bind enhancers (see, e.g., Molecular Cell Biology, Harvey Lodish, et al., WH Freeman & Co; (2002) ISBN 978-0849394805).

[0223] In addition, the Cascade complex comprising a type I CRISPR-Cas subunit protein fused to a transcriptional repressor can be used to inhibit gene expression. The target site can include a transcriptional regulatory element. In one embodiment, the Cascade subunit protein can be connected to the KRAB domain via a linker polypeptide. The Cascade complex comprising a Cascade subunit protein / KRAB domain fusion can convert the complex into a flexible tool (CASCADEi) for gene transcription inhibition, wherein targeting of the selected gene is achieved by selecting a guide sequence that directs the binding of the Cascade complex to one or more regulatory elements of the selected gene. Transcriptional repressors include, but are not limited to, passive transcriptional repressors, the bzip family of transcription factors, sp1-like transcriptional repressors, active transcriptional repressors (e.g., transcriptional repression via recruitment of histone deacetylases, histone deacetylation, and bispecific repressors (see, e.g., Thiel, G., et al., Eur. J. Biochem. 271:2855–2862 (2004); Nicola Reynolds, N., et al., Development 140:505-512 (2013); Gaston, K., et al., Cell Mol. Life Sci., 60:721-741 (2003)).

[0224] In additional embodiments, the Cascade subunit protein can be fused to an affinity tag.

[0225] In other embodiments of the invention, Type I CRISPR-Cas guide polynucleotides can be modified by inserting selected polynucleotide elements or nucleotide changes at selected positions within the guide polynucleotide (e.g., changes from a DNA portion to a fundamentally different RNA portion, as well as other changes to the guide polynucleotides described above). Such embodiments include, but are not limited to, fusions 5', 3', or internally to one or more nucleotide effector domains (e.g., MS2 or MS2-P65-HSF1 aptamers that bind RNA or recruit transcription factors) to the Type I CRISPR-Cas guide polynucleotide. Figure 9 Type I CRISPR guide polynucleotides are shown, and the relative positions of the Cas proteins of the Cascade complex are shown ( Figure 9 : Cas7, Cas5, Cas8, Cse2 and Cas6; the dotted box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin within the dotted box). Figure 9 In the present invention, the crRNA further comprises an RNA aptamer hairpin introduced into the 3' hairpin of the guide polynucleotide ( Figure 9 , positions indicated by arrows).

[0226] The length of type I CRISPR-Cas guides can also be modified, typically by lengthening or shortening the Cas7 and Cse2 binding regions. Figure 10A The Cascade complex with three Cas7 subunits, one Cse2 subunit and a shortened crRNA is shown ( Figure 10A : Cas7, Cas5, Cas8, Cse2 and Cas6; the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin). Figure 10B The Cascade complex with 9 Cas7 subunits, 3 Cse2 subunits and an extended crRNA is shown ( Figure 10B : Cas7, Cas5, Cas8, Cse2 and Cas6; the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin).

[0227] Example 16 describes the generation and testing of modified type I CRISPR-Cas guide crRNAs and the suitability of the modified guides for constructing engineered type I CRISPR-Cas effector complexes.

[0228] In a third aspect, the present invention relates to nucleic acid sequences encoding one or more modified Cascade components, and expression cassettes, vectors, and recombinant cells comprising nucleic acid sequences encoding one or more modified Cascade components. Some embodiments of the third aspect of the invention include one or more polypeptides (e.g., Cse2, Cas5, Cas6, Cas7, and Cas8 proteins, and one or more homologous guides), wherein the components are capable of forming an effector complex. Typically, when more than one homologous guide is expressed, the guides have different spacer sequences to guide binding to different nucleic acid target sequences. Such embodiments include, but are not limited to, expression cassettes, vectors, and recombinant cells.

[0229] In one embodiment, the present invention relates to one or more expression cassettes comprising one or more nucleic acid sequences encoding one or more modified Cascade components. Expression cassettes typically contain regulatory sequences involved in one or more of the following: transcriptional regulation, post-transcriptional regulation, or translational regulation. Expression cassettes can be introduced into a variety of organisms, including but not limited to bacterial cells, yeast cells, plant cells, and mammalian cells (including human cells). Expression cassettes typically contain functional regulatory sequences corresponding to the organism into which they are introduced.

[0230] Other embodiments of the present invention relate to vectors, including expression vectors, comprising one or more nucleic acid sequences encoding one or more modified Cascade components. The vector may also include a sequence encoding a selectable or screenable marker. In addition, a nuclear targeting sequence may be added to, for example, a Cascade subunit protein. The vector may also include a polynucleotide encoding a protein tag (e.g., a poly-His tag, a hemagglutinin tag, a fluorescent protein tag, and a bioluminescent tag). The coding sequence of such a protein tag may be fused to, for example, one or more nucleic acid sequences encoding a Cascade subunit protein.

[0231] The general method for constructing expression vector is known in the art.Expression vector for host cell is commercially available.There are several commercial software products to promote the selection and construction of appropriate carriers, such as insect cell vectors for gene expression in insect cell transformation and insect cells, bacterial plasmids for gene expression in bacterial transformation and bacterial cells, yeast plasmids for cell transformation and gene expression in yeast and other fungi, mammalian vectors for gene expression in mammalian cell transformation and mammalian cells or mammals, and viral vectors for cell transformation and gene expression (including but not limited to slow virus, retrovirus, adenovirus, I type or II type herpes simplex virus, parvovirus, reticuloendotheliosis virus and adeno-associated virus (AAV) vector), for cell transformation and gene expression and the method that is easy to allow cloning of such polynucleotides.

[0232] AAV-based vectors (rAAV) are examples of viral vectors that can be used for the methods of the present invention. AAV is a single-stranded DNA member of the Parvoviridae family and is a naturally replication-defective virus. AAV vectors are the most commonly used viral vectors for gene therapy. Twelve human AAV serotypes are known (AAV serotype 1 [AAV-1] to AAV-12) and more than 100 serotypes from non-humans.

[0233] Lentiviral vectors are another example of viral vectors that can be used for the implementation of the methods of the present invention. Lentiviral is a member of the Retroviridae family and is a single-stranded RNA virus that can infect both dividing and non-dividing cells and can provide stable expression by integrating into the genome. In order to increase the safety of lentiviral vectors, the components necessary for producing viral vectors are divided into multiple plasmids. Transfer vectors are generally not replication-competent and may additionally contain deletions in the 3'LTR, which causes the virus to inactivate itself after integration. Packaging and envelope plasmids are generally used in conjunction with transfer vectors. For example, a packaging plasmid can encode a combination of Gag, Pol, Rev, and Tat genes. The transfer plasmid may contain viral LTRs and psi packaging signals. The envelope plasmid generally contains an envelope protein (generally vesicular stomatitis virus glycoprotein, VSV-GP, because of its wide range of infection).

[0234] Exemplary plant transformation vectors include those derived from the Ti plasmid of Agrobacterium tumefaciens (see, Lee, LY, et al., Plant Physiology 146:325-332 (2008)). Also useful and known in the art are Agrobacterium rhizogenes plasmids. For example, SNAPGENE™ (GSL Biotech LLC, Chicago, IL; snapgene.com / resources / plasmid_files / your_time_is_valuable / ) provides an extensive list of vectors, individual vector sequences and vector maps, and commercial sources of many such vectors.

[0235] In order to express and purify recombinant Cascade in a bacterial expression system, vectors encoding Cascade subunit proteins and a minimal CRISPR array containing a target guide sequence can be designed. Therefore, one aspect of the present invention includes such an expression system. In one embodiment, the Cascade complex is expressed by three different plasmid vectors, which together encode the following components: Cas8 protein; Cse2, Cas7, Cas5 and Cas6 proteins; and CRISPR RNA. In some embodiments, the expression plasmid encoding Cas8 contains a natural gDNA gene sequence, and in other embodiments, the expression plasmid can encode a codon-optimized Cas8 for expression in a selected cell type. Similarly, the expression plasmids encoding Cse2, Cas7, Cas5 and Cas6 can contain a natural gDNA gene sequence, or can contain a gene sequence that has been codon-optimized for expression in a selected cell type. In some embodiments, the entire Cascade subunit protein-encoding operon can be located downstream of a single transcriptional promoter, so that the different proteins are translated from a single polycistronic transcript. In other embodiments, the genes encoding the Cascade subunit proteins can be separated from each other with an intervening transcription terminator and promoter.

[0236] The expression plasmid encoding crRNA can contain as few as two repeats on both sides of a single spacer sequence, downstream of an appropriate transcription promoter, or can contain many repeats of the same guide sequence or multiple different guide sequences on both sides of multiple spacer sequences. The coordinated expression of CRISPR and Cascade subunits, especially the Cas6 subunit, results in the processing of longer precursor crRNAs into mature-length crRNAs, each of which contains a single repeat fragment on the 5' and 3' ends of the crRNA, and a single spacer sequence in the middle.

[0237] An alternative strategy for expressing the complete Cascade complex in E. coli uses two plasmids: one plasmid encodes the entire Cas8–Cse2–Cas7–Cas5–Cas6 operon on a single expression plasmid, and one plasmid encodes the CRISPR RNA. In this case, the 5' end of the Cse2 gene, which normally overlaps with the 3' end of the Cas8 gene, is spatially separated from the 3' end of the Cas8 gene to facilitate the attachment of polynucleotide sequences encoding affinity tags and / or protease recognition sequences.

[0238] Example 2 describes two types of bacterial expression plasmid systems for Cascade proteins: the first type contains two plasmids, the first plasmid encodes the Cas8 protein and the second encodes the four subunit proteins of the CasBCDE complex (cse2–cas7–cas5–cas6 operon); and the second type contains expression plasmids encoding all five subunit proteins of the Cascade complex (cas8–cse2–cas7–cas5–cas6 operon). Homologous CRISPR arrays are also described.

[0239] In order to promote the purification of the Cascade complex, an affinity tag can be attached to the Cse2 subunit, such as an N-terminal Strep-II tag or a six-histidine (His6) tag. In addition, an amino acid sequence recognized by a protease such as a TEV protease or HRV3C protease can be inserted between the natural N-terminus of the affinity tag and the Cse2 subunit, so that after initial purification, the sequence is biochemically cut with a protease and the affinity tag is released from the final recombinant Cascade complex. The affinity tag can also be placed on other subunits, or remain on the Cse2 subunit and be combined with other affinity tags on other subunits. Exemplary Cascade subunit proteins comprising affinity tags are shown in Example 1, Example 2, Example 3A, Example 3B and Example 3C.

[0240] For the IE type Cascade system, a plasmid encoding CRISPR RNA and the cse2-cas7-cas5-cas6 gene can be used to transform Escherichia coli strains, induce protein expression, and produce a Cascade complex lacking the Cas8 subunit. The Cascade complex is commonly referred to as the Cas8-negative Cascade complex, or alternatively, the CasBCDE complex (see, for example, Jore, M., et al., Nat. Struct. Mol. Biol. 18: 529-536 (2011)). The purified complex can be combined with separately purified Cas8 biochemistry to reconstruct a complete Cascade (see, for example, Sashital, DG, et al., Mol. Cell 46: 606-615 (2012)).

[0241] Table 6 shows exemplary sequences of bacterial expression plasmids encoding minimal CRISPR arrays, cas8, cse2–cas7–cas5–cas6 constructs, and cas8–cse2–cas7–cas5–cas6 constructs, containing different tags and designs. Plasmids encoding the Cascade complex and the Cascade complex from the homologous type I system can be designed similarly to the exemplary expression plasmid sequences for type IE found in Escherichia coli K-12 MG1655, following the guidance of this specification. Table 6 also contains sequences of expression plasmids expressing the Cas8–Cse2–Cas7–Cas5–Cas6 proteins, as well as FokI fusions to the cas8 or cas6 genes, for generating nuclease–Cascade fusions for gene editing experiments.

[0242]

[0243] Table 7 contains a single polymeric promoter bacterial expression plasmid encoding all five subunit proteins and the sequence of the crRNA from a single bacterial expression plasmid. In this design, each gene is separated from other genes with transcriptional promoters and terminators on both sides upstream and downstream. Additional sequences encoding affinity tags and / or protease recognition tags can be introduced, as well as fusions with nuclease proteins to generate Cascade-nuclease fusions for gene editing.

[0244]

[0245] Additional bacterial expression plasmids encoding homologous Cascade complexes from other type I subtypes and other bacterial or archaeal organisms can be designed based on the design criteria herein. Such expression plasmids can be designed using the gDNA sequence of the Cascade gene, or they can be designed using gene sequences that have been codon-optimized for expression in E. coli or other bacterial strains.

[0246] In order to express Cascade in mammalian cells such as human cells or to fuse to Cascade effectors, eukaryotic expression plasmid vectors are designed to enable expression of related proteins and RNA components by eukaryotic transcription and translation mechanisms. In one embodiment, each protein component can be encoded on a separate expression vector driven by a eukaryotic promoter (e.g., cytomegalovirus (CMV) promoter) and crRNA can be encoded on a separate expression vector driven by an RNA polymerase III promoter (e.g., human U6 promoter), thereby producing Cascade in mammalian cells. CRISPR RNA can be encoded with a minimal CRISPR array containing at least two repeat sequences on both sides of one or more spacer sequences, which serve as guide portions of mature crRNA. A construct producing CRISPR RNA can be designed that has additional sequences on both sides of the outermost repeat sequence in the minimal array. The processing of CRISPR RNA precursors is achieved by the RNA processing subunits of the Cascade complex (Cas6 subunit protein), which can be expressed from a separate plasmid. Precursor CRISPR RNA can be processed by the RNA processing subunits (Cas6 subunit protein) of the Cascade complex that can be expressed from a separate plasmid.

[0247] Table 8 contains the sequence of a single eukaryotic expression plasmid for each protein of the Escherichia coli IE type Cascade complex. The Cas8 subunit can be fused to another effector nuclease domain, such as FokI nuclease (Example 1, Example 3A, Example 3B and Example 3C). Table 8 also contains the sequence of the expression plasmid of the crRNA component of Cascade, which encodes two separate crRNAs so that 3 repeats are located on both sides of the two spacer spacers. Each protein-coding gene can be attached to a polynucleotide sequence of a linker sequence to which a nuclear localization signal (NLS), an affinity tag, and those tags are attached. Other fusions with any Cascade subunit protein can be encoded by generally attaching to a 5' or 3' coding sequence, including encoding an additional polynucleotide sequence connecting an amino acid linker connecting a Cascade subunit protein to another target polypeptide sequence. Examples of candidate fusion proteins are described herein.

[0248]

[0249] In order to express the components of the Cascade complex on fewer expression vectors, a polycistronic expression vector can be constructed so that a single promoter (e.g., CMV promoter) simultaneously drives the expression of multiple coding sequences separated by the Thata asigna virus 2A sequence. Sequence. The 2A viral peptide sequence induces ribosome jumping, so that multiple protein-coding genes can be connected in series in a single polycistronic construct for expression in eukaryotic cells. Therefore, a polycistronic vector can be designed that encodes 4 or 5 protein subunits of the Cascade complex on a single transcript driven by a single promoter. Table 9 contains the sequence of a eukaryotic polycistronic expression plasmid that can be combined with a CRISPR RNA expression plasmid to produce a functional Cascade in mammalian cells.

[0250]

[0251] In some embodiments, CRISPR RNA is encoded in the 3' untranslated region (UTR) of protein coding gene, and its expression is driven by RNA polymerase II promoter (for example, CMV promoter) to produce transcript.In such embodiment, minimal CRISPR array is designed to be present in the downstream of protein coding gene such as Cas6, Cas7 or reporter gene (for example, enhanced green fluorescent protein, eGFP), and is separated from protein coding sequence by previously having proved that upstream transcript can be given the MALAT1 triplex sequence with stability. Minimal CRISPR array is processed by Cascade's RNA processing subunit (usually using different plasmids to express)--a kind of nuclease that cuts minimal CRISPR array, introduces breakage in transcript, and triplex sequence protects 3' end of upstream protein coding gene from being degraded by exonuclease prematurely.Table 10 contains the sequence of 3 polynucleotide sequences, so that CRISPR sequence is cloned into the downstream of Cas6, Cas7 or eGFP, and the expression of whole fusion sequence is driven by CMV promoter.

[0252]

[0253]

[0254] In some embodiments, the CRISPR RNA array is encoded on the same vector as a polycistronic construct for expression of the five Cascade subunit proteins; the combination of these two elements creates an all-in-one vector that can produce all functional subunits (proteins and RNAs) of the Cascade complex, as well as any nuclease or effector domain fused to a Cascade subunit. Table 11 contains two representative sequences of these all-in-one polynucleotide sequences, which encode all respective components to produce functional FokI-Cascade RNPs in mammalian cells.

[0255]

[0256] Example 3A, Example 3B, and Example 3C describe expression systems using separate plasmids expressing each Cascade subunit protein and a minimal CRISPR array, an expression system in which multiple Cascade subunit protein coding sequences are expressed from a single promoter, and an expression system in which a single plasmid Cascade expression system is constructed to express the entire cas8–cse2–cas7–cas5–cas6 operon and a minimal CRISPR array for use in mammalian cells.

[0257] Following the guidance of this specification, one of ordinary skill in the art can design additional mammalian expression vectors encoding other Cascade complexes similar to the examples provided for the E. coli type IE Cascade complex.

[0258] In a fourth aspect, the present invention relates to producing a modified type I CRISPR-Cas effector complex by introducing a plasmid encoding one or more components of the modified type I CRISPR-Cas effector complex into a host cell. The transformed host cell (or recombinant cell) or a cell progeny transfected or transfected using recombinant DNA technology may contain one or more nucleic acid sequences encoding one or more components of the modified type I CRISPR-Cas effector complex. Methods for introducing polynucleotides (e.g., expression vectors) into host cells are known in the art and are generally selected according to the type of host cell. Such methods include, for example, viral or phage infection, transfection, binding, electroporation, calcium phosphate precipitation, polyethyleneimine-mediated transfection, DEAE-dextran-mediated transfection, protoplast fusion, lipofection, liposome-mediated transfection, particle gun technology, microparticle bombardment, direct microinjection, and nanoparticle-mediated delivery. In one embodiment of the invention, a polynucleotide encoding a component of a modified type I CRISPR-Cas effector complex is introduced into a bacterial cell (e.g., Escherichia coli).

[0259] Examples 4A and 4B describe methods for introducing and expressing Cas8 protein coding sequences, as well as coding sequences for engineered type I CRISPR-Cas effector complex components, for bacterial production of such complexes using an E. coli expression system.

[0260] Various exemplary host cells disclosed herein can be used to generate recombinant cells using the engineered Cascade effector complex. Such host cells include, but are not limited to, plant cells, yeast cells, bacterial cells, insect cells, algae cells, and mammalian cells.

[0261] For ease of discussion, "transfection" is used below to refer to any method of introducing a polynucleotide into a host cell.

[0262] In some embodiments, host cells are transiently or non-transiently transfected with nucleic acid sequences encoding one or more components of a type I CRISPR-Cas effector complex. In some embodiments, cells are transfected as they would naturally occur in a subject. In some embodiments, the transfected cells are first removed from a subject, e.g., primary cells or progenitor cells. In some embodiments, primary cells or progenitor cells are cultured and / or returned to the same subject or a different subject following ex vivo transfection.

[0263] The expression and purification of engineered type I CRISPR-Cas effector complexes is labor-intensive, so a higher-throughput plasmid-based delivery system was designed to facilitate screening of large numbers of guide polynucleotides or effector complex variants. Each of the five Cas genes was human codon-optimized and cloned into a CMV-driven expression plasmid as an N-terminal NLS fusion, and a minimal CRISPR array containing paired gRNAs targeting the TRAJ27 exon of the T cell receptor α locus (UCSC genome browser, hg38) was cloned into a sixth plasmid downstream of the human U6 promoter (Example 3A; Figure 35 ).exist Figure 35 In the figure, the order of elements from left to right is as follows: hu6 promoter, gray rectangle with diamond ends; repeat region 1, open diamond, (white); spacer region 1, gray waffle rectangle; repeat region 2, gray diamond; spacer region 2, gray stippled rectangle; and repeat region 3, black diamond. Figure 35 In the figure, the brackets show the regions encoding the two gRNAs. In some embodiments, the two guide RNAs can be the same (e.g., targeting the same nucleic acid target sequence), and in other embodiments, the two guide RNAs can be different (e.g., targeting two different nucleic acid target sequences).

[0264] Most gRNAs in type I systems are naturally catalyzed by the Cas6 ribonuclease present in Cascade (see, e.g., Brouns, SJ, et al., Science 321:960-964 (2008); Hochstrasser, M., et al., Trends Biochem. Sci. 40:58–66 (2015), obviating the need for multiple promoter approaches with paired gRNAs as presented herein. Thus, one embodiment of the invention includes vectors comprising paired guide polynucleotides operably linked to regulatory elements to provide expression of guide polynucleotides (e.g., gRNAs). At the TRAJ27 site, six plasmid co-transfections gave yields as high as ~3% editing, and removal of any one component abolished genome editing, with the sole exception of Cas11, which is not absolutely required for DNA binding by the E. coli Cascade effector complex (see, e.g., Westra, E., et al., RNA Biol. 9: 1134-1138 (2012)).

[0265] In another embodiment of the present invention, a minimal CRISPR array, typically comprising two guide sequences, is introduced into a cell or biochemical reaction as a DNA template. The DNA template is generated by PCR amplification (e.g., Figure 42A ; Example 20A). Such a minimal CRISPR array can be introduced into a cell with one or more plasmids encoding Cascade complex protein components. In some embodiments, both the minimal CRISPR array and the vector comprising the paired guide polynucleotides can be introduced into a cell or biochemical reaction. In methods using two Cascade RNP complexes (e.g., methods for binding to a nucleic acid target sequence or methods for cleaving a nucleic acid target sequence; see, e.g., Figure 15A 、 Figure 15B 、 Figure 15C ), the minimal CRISPR array can encode two different guides. Thus, in some embodiments, the two guide RNAs can be different (e.g., targeting two different nucleic acid target sequences). In methods using a single Cascade RNP complex (e.g., when using a type I CRISPR-Cas effector complex associated with an mCas3 protein or a type I CRISPR-Cas effector complex in which a Cas3 fusion protein is associated with the complex; for example, see, e.g., Figure 16A 、 Figure 17B 、 Figure 17C 、 Figure 21A 、 Figure 21B 、 Figure 21C 、 Figure 21D), a minimal CRISPR array can encode two copies of the same guide sequence. Thus, in some embodiments, the two guide RNAs can be identical (e.g., targeting the same nucleic acid target sequence).

[0266] In yet another embodiment, a polynucleotide encoding a guide sequence that is also recognized by the Cas6 protein for processing the crRNA precursor endonucleoside into a sequence and structure of mature guide RNAs can be introduced into a cell or biochemical reaction. In other embodiments, mature guide polynucleotides that do not require processing can be used for assembly of the Cascade complex. Such mature guides can contain sequence modifications (e.g., phosphorothioate bonds at the 5' and / or 3' ends to help protect the guide from nuclease digestion, such as by RNase digestion). Additional guide modifications include those described herein for nucleotide sequences (e.g., nucleotide analogs, etc.).

[0267] Example 9A, Example 9B, Example 9C, and Example 9D illustrate the design and delivery of E. coli type IE Cascade complexes comprising FokI fusion proteins to facilitate genome editing in human cells. Example 9B describes delivery of plasmid vectors expressing Cascade complex components into eukaryotic cells. In a fifth aspect, the present invention relates to the purification of type I CRISPR-Cas effector complexes from cell transformation, and the application of such complexes. The transformed type I CRISPR-Cas effector complexes are produced in host cells. The transformed type I CRISPR-Cas effector complexes (in this case, Cascade RNP complexes) are purified from cell lysates.

[0268] Example 5A and Example 5B describe the purification of E. coli type IE Cascade RNP complexes produced by overexpression in bacteria, as described in Example 4B. The method uses immobilized metal affinity chromatography followed by size exclusion chromatography (SEC). Example 5A and Example 5B describe methods that can be used to assess the quality of purified Cascade RNP products. The examples provided illustrate the purification of Cas8, Cas7, Cas6, Cas5, and Cse2 Cascade RNP complexes, Cascade complexes comprising Cas7, Cas6, Cas5, and Cse2 proteins, and FokI-Cas8 fusion proteins.

[0269] The purified modified type I CRISPR-Cas effector complex can also be used directly in biochemical assays (e.g., binding and / or cleavage assays). Example 6A, Example 6B, and Example 6C describe the generation of dsDNA target sequences for in vitro DNA binding or cleavage assays. Example 6 describes three methods for generating target sequences, including annealing of synthetic ssDNA oligonucleotides, PCR amplification of selected nucleic acid target sequences from gDNA, and cloning of nucleic acid target sequences into bacterial plasmids. dsDNA target sequences are used in Cascade binding or cleavage assays.

[0270] If necessary, site-specific binding and / or cleavage by one or more engineered type I CRISPR-Cas effector complexes can be verified using an electrophoretic mobility shift assay (see, e.g., Garner, M., et al., Nucleic Acids Res. 9:3047-3060 (1981); Fried, M., et al., Nucleic Acids Res. 9:6505-6525 (1981); Fried, M., Electrophoresis 10:366-376 (1989); Fillebeen, C., et al., J. Vis. Exp. (94), e52230, doi: 10.3791 / 52230 (2014)), or the biochemical cleavage assay described in Example 7.

[0271] The data presented in Example 7 demonstrate that engineered type I CRISPR-Cas effector complexes can exhibit near-quantitative DNA cleavage, as demonstrated by conversion of supercoiled, circular plasmid substrates into cleaved linear forms. Following demonstration of robust biochemical activity using engineered type I CRISPR-Cas effector complexes (e.g., comprising FokI-Cascade component fusion proteins), genome editing was performed in cells.

[0272] Examples 8A, 8B, 8C, and 8D illustrate the design and delivery of an E. coli type IE Cascade complex comprising a Cas subunit protein-FokI fusion protein to human cells. The data in Example 8D demonstrates the delivery of preassembled Cascade RNPs to target cells and efficient genome editing in human cells.

[0273] Purified, engineered type I CRISPR-Cas effector complexes can be introduced directly into cells. Methods for introducing components into cells include electroporation, lipofection, particle gun technology, and microparticle bombardment.

[0274] Figure 36A 、 Figure 36B 、 Figure 36C and Figure 36D Comparative data on genome editing in human cells using plasmid-based delivery of engineered Cascade-RNP complexes and engineered type I CRISPR-Cas complexes are presented. Figures 36A-36D , Figure 36A In this study, HEK293 cells were transfected with purified RNPs, and the editing sites were analyzed by next-generation sequencing (NGS). Figure 36A (RNP transfection) shows that the FokI-Cascade RNP complex is targeted to two adjacent sites ( Figure 36A , shown above the left straight line in the figure) were nucleofected into HEK293 cells ( Figure 36A , star-shaped, gray on the left side of the figure) to induce DNA cutting and genome editing. The editing efficiency at 16 unique genomic target sites was calculated (see Example 6C, Table 31, human double Hsa1-16) (n=1). TRAC is the constant region of the T cell receptor. When T cell receptors are produced, they include splicing junctions (ie, "variable" regions and "junction" regions). Some TRAC guides described herein target junction regions (e.g., TRAJ27). The spacing between the spacers for each target is shown below the figure ( Figure 36A , from left to right, 25, 30, 35, 40, 45 base pairs (bp). Figure 36A In the figure, the vertical axis is the percentage editing efficiency ( Figure 36A , editing efficiency (%), the horizontal axis represents targets 1-16, and below the horizontal axis are brackets indicating the length of the interval in base pairs (bp).

[0275] Figure 36B Provided Figure 36A Representative DNA repair results for target 7 in Figure 36B In the figure, the relative positions of the half-sites targeted by the paired gRNAs and their associated PAM sites are shown at the top of the figure. The spacing between the spacers is shown by the top row. In the figure, the expected cleavage site ( Figure 36B , position "0" is shown as a vertical black center line) and bp distance (-50 to 50). Each horizontal gray line represents a different category of sequencing read length observed at the target site. The indicators of these lines are as follows: gray area = sequence match; horizontal black line = deletion; and hollow box = insertion. There is a circle to the right of each line in the graph: black circles are wild-type read lengths; and white hollow circles are mutant read lengths. The expected wild-type read length is shown in the first gray box ("Ref"; i.e., reference sequence). The wild-type read length is shown in the second gray bar (second gray bar; Figure 36B , black circles). The next 11 lines show the mutant read lengths ( Figure 36B, open circles). Insert lengths given in base pairs are shown in the column to the right of the circles. The total percentage of read length is shown in the next column to the right, and the total read length is shown in the last column on the right.

[0276] like Figure 36C As shown in (6-plasmid transfection system), HEK293 cells were transfected with 6 plasmids ( Figure 36C , star-shaped, gray, left side of the figure), 5 plasmids encoding Cas proteins ( Figure 36C , plasmids for FokI-Cas8, Cas11, Cas7, Cas5, and Cas6 as indicated) and one plasmid encoding paired gRNAs under the control of CMV and human U6 (hU6) promoters ( Figure 36C , gRNA), followed by NGS analysis of the editing site. The diagram of the FokI-Cascade RNP complex is below the dotted line. Figure 36A Editing efficiency at target 7 (n=2) ( Figure 36A , black bars in the figure), and including plasmid mixtures lacking a single component ( Figure 36C , below the horizontal axis, gray boxes containing - / +) served as controls ( Figure 36C , hollow bars in the figure).

[0277] like Figure 36D As shown in (2-Plasmid Transfection System), paired gRNA expression plasmids ( Figure 36D , gRNA plasmid) and a polycistronic expression plasmid encoding all five proteins separated by T2A "ribosome skipping" sequence peptides ( Figure 36D HEK293 cells were transfected with CMV-Cas7-2A-Cas11-2A-Cas5-2A-Cas6-2A-FokI-Cas8 ( Figure 36D , star-shaped, gray, left side of the figure) and then NGS analysis of the editing site. The FokI-Cascade RNP complex is shown below the dotted line. Figure 37C 2 plasmid system transfection ( Figure 36D , open boxes) and 6 plasmid system transfection (n=3) ( Figure 36D , black bars), calculated Figure 36A Editing efficiencies at the 16 targets shown in . Figure 36D In the figure, the vertical axis is the percent editing efficiency ("Editing Efficiency (%)", the horizontal axis represents targets 1-16, and below the horizontal axis are brackets indicating the length of the interval in base (bp) pairs ( Figure 36D , from left to right, 25, 30, 35, 40, 45 bp).

[0278] The experiments were performed by nucleofecting HEK293 cells with purified Cascade-RNPs containing nuclear localization signal sequences on FokI and Cas6. As demonstrated by next-generation sequencing of PCR amplicons obtained from gDNA, editing efficiencies as high as ~4% were observed, and edits were typically located at sites containing 30-bp spacer lengths among the 16 target sites tested ( Figure 36A Close inspection of the spectra of the repair results revealed that the indels were clustered in the middle of the spacer region ( Figure 36B ), which is consistent with the design of type I CRISPR-Cas complex. Therefore, in one embodiment of the present invention, the modified type I CRISPR-Cas complex is directly introduced into the cell. For the 6 plasmid delivery experiment ( Figure 36C ), the assembled plasmid mixture contained 420 ng of each plasmid except one, and then water was added as a negative control or 700 ng of the deleted plasmid was added after nucleofection. For the initial two-plasmid delivery experiment of the FokI-EcoCascade polycistronic ( Figure 36D ), cells were electroporated with 500 ng of each plasmid or 500 ng of paired gRNA expression plasmids and 2.5 μg of polycistronic plasmid (3 μg total for each condition). In one embodiment, all five cas genes are constructed in a single polycistronic expression vector connected in series by a T2A "ribosome skipping" sequence (see, e.g., Kim, J., et al., PLoS ONE 6, e18556 (2011); Liu, Z., et al., Sci. Rep. 7: 2193 (2017)). Figure 36D ). Surprisingly, the editing efficiency and DNA repair results produced by co-transfection with a polycistronic plasmid and a paired gRNA expression plasmid were similar to those observed using the 6-plasmid method (Example 9A) and the direct RNP delivery method (Example 8A, Example 8B, Example 8C, Example 8D), supporting the conclusion that the biochemically active type I CRISPR-Cas effector complex is assembled and transported to the nucleus of human cells. In summary, these experiments validate a greatly simplified expression system that can reconstruct an exquisite 11-subunit RNA-guided nuclease in eukaryotic cells using only two molecular components similar in size to the widely used Cas9 and sgRNA plasmids.

[0279] Data from engineered type I CRISPR-Cas complexes (Escherichia coli (EcoCascade), Pseudomonas sp. S-6-2 (PseCascade), and Pseudomonas aeruginosa (SthCascade)) indicated that most target sites would be unique, as they must include two half-sites, the necessary spacer spacing, and a permissive PAM. Engineered Cascade homologs from EcoCascade, PseCascade, and SthCascade were selected for more detailed characterization.

[0280] Figure 37A 、 Figure 37B 、 Figure 37C and Figure 37D Editing efficiency is shown in relation to the FokI linker, spacer length, and Cascade homolog. Figure 37A , showing FokI-EcoCascade editing efficiency as a function of FokI-Cas8 linker length ( Figure 37A , open circles, lower line 10aa; open circles upper line, 20aa; black circles, 17aa; and grey circles, 30aa as a function of linker length) and spacer spacing. Figure 37A In the figure, the vertical axis is editing efficiency (%), and the horizontal axis is spacer spacing in bp. Each data point represents an average of 3–4 unique target sites.

[0281] Figure 37B FokI-Cascade nucleases with 30-aa linkers were provided. Twelve FokI-Cas8 linkers of type IE Cascade variants were generated and tested for genome editing at 4-7 target sites. Each data point represents a single genomic site, and the bars show the mean and standard deviation (SD) across sites. Targets containing AAG ( Figure 37B , gray bars) or GAA ( Figure 37B , white bars) PAM sequences and 30 bp spacer spacing, where the species on the horizontal axis are as follows: Eco, Escherichia coli; Pse, Pseudomonas sp. S-6-2; Sen, Salmonella enterica; Geo, Geobacterium geothermoides EPR-M; Mar, Methanosarcina oryzae; Ahe, Aranthibaea; Oce, Oceanus marinus HL-35; Pae, Pseudomonas aeruginosa; Sth, Streptococcus thermophilus; Str, Streptomyces sp. S4; Kpn, Klebsiella pneumoniae; Lba, Lachnospiraceae.

[0282] exist Figure 37C , FokI-PseCascade data are shown, where the vertical axis is the percent editing efficiency ( Figure 37C, editing efficiency (%), and the horizontal axis represents the intergenic interval length in base pairs (bp). The FokI-Cas8 linker is 17 amino acids long. Each data point represents a single genomic locus, and the bars show the mean and SD across 7–8 loci.

[0283] Figure 37D Data on FokI-PseCascade editing efficiency as a function of PAM sequence are provided, with the vertical axis being percent editing efficiency ( Figure 37D , editing efficiency (%)), and the horizontal axis corresponds to the PAM sequence ( Figure 37D , from left to right, CCG, CGC, AAG, AAA, ATG, AAC, AGG, ATA, GAG, and AAT). Genomic loci contain an AAG PAM and variable PAMs at the second half-site, as indicated on the horizontal axis. Each data point represents a single genomic locus, and bars show the mean and SD across 6–15 loci.

[0284] Figure 37E Provides FokI-EcoCascade editing efficiency ( Figure 37E , vertical axis, editing efficiency (%)) as a function of PAM sequence. The target site contains a fixed AAG PAM and a variable PAM at the second half site, as shown on the horizontal axis ( Figure 37E , from left to right, CCG, CGC, AAG, AGG, ATG, GAG, AAA, AAC, ATA, and AAT). Each dot represents a single target site in HEK293 cells, and 6-15 sites were tested for each PAM (n=1 / site). The bar graph shows the mean and SD.

[0285] Figure 37F Provides FokI-SthCascade efficiency ( Figure 37F , vertical axis, editing efficiency (%)) as a function of PAM sequence. The target site contains a fixed GAA PAM and a variable PAM at the second half site, as shown on the horizontal axis ( Figure 37F , from left to right, CC, AA, GA, TA, and CA). Each dot represents a single target site in HEK293 cells, and 18-33 sites were tested per PAM (n=1 / site). Bar graphs show mean and sd.

[0286] Figure 37G The provided heatmap shows the Figure 37C and Figure 37DFrequency of indel classes for 40 genomic sites that showed higher editing efficiency (10–53%). The bar chart in the top graph shows the percentage editing efficiency from 0–60. The heat map shown in the middle graph shows the insertion lengths of 1–8 bp, and the heat map in the bottom graph shows the deletion lengths of 1–50 bp. The 40 genomic target sites ( Figure 37G , target). Single bp insertions are separated by nucleotide identity, and the gray intensity scale at the bottom of the figure corresponds to the insertion frequency percentage ( Figure 37G , Ins Freq (%), scale is 0 to greater than or equal to 20) and missing frequency percentage ( Figure 37G , Del Freq (%), scale is 0 to greater than or equal to 20). The bar chart on the right shows the average frequency of each indel category ( Figure 37G , scale 0 to 20). The pie chart on the right shows the fraction of 2–4 bp insertions resulting from putative templated repair ( Figure 37G , the black area of ​​the pie chart), which is defined here as containing the repeat sequence adjacent to the cleavage site. "Other" is represented by the gray area of ​​the pie chart.

[0287] The most closely related sites of the five most highly edited FokI-PseCascade target sites in the human genome (~20-48% edited) were investigated, constrained only by the 30-33 bp spacer requirement. No sites with <22 mismatches in either half-site were identified in all five targets. Figure 37A ), cells were nucleofected with 2.4 μg of FokI-EcoCascade polycistronic plasmid and ∼0.5–3.5 μg of paired gRNA expression plasmids.

[0288] For FokI-Cascade homolog screening ( Figure 37B ), cells were nucleofected with 1.5 μg of FokI-Cascade polycistronic plasmid and ~0.4-2.2 μg of paired gRNA expression plasmids. Across the homologs, 4-7 sites were targeted, and sites with high editing efficiency of FokI-EcoCascade were selected. For homolog variants, FokI-Cas8 linker type and spacer spacing editing experiments ( Figure 37C and Figures 41A to 41C ), cells were nucleofected with 5 μg of polycistronic plasmid and 100-400 ng of oligo-templated paired gRNA expression amplicons. For this experiment, gRNA concentrations were not normalized across wells or homolog variants. Figures 41A to 41C, cells were nucleofected with an average of ∼1.5x or more FokI-PseCascade gRNA compared to FokI-EcoCascade or FokI-SthCascade gRNA.

[0289] Oligo-templated PCR amplification is described herein (eg, Example 20A). Figure 42A and Figure 42B The expression of the human U6 (hU6) promoter in mammalian cells is shown in Figure 42A , 420) generate oligo-templated PCR strategies for paired gRNA-expressed amplicons. Briefly, the reverse internal oligonucleotide ( Figure 42A , 424) encoding two gRNA sequences and modified for a new target site (also known as a unique primer encoding a "repeat-spacer-repeat-spacer-repeat" sequence ( Figure 42A , 421: repeat region, open rectangle; spacer 1, gray rectangle; repeat region, open rectangle; spacer 2, gray rectangle; repeat region, open rectangle), while the remaining primers are unchanged ( Figure 42A : forward outer primer, 422; forward inner primer, 423; reverse outer primer, 425). Figure 42B Figure 2 shows the editing efficiency at target 7 after co-transfection of HEK293 cells with a polycistronic plasmid encoding the FokI EcoCascade RNP complex and paired gRNA expression plasmids or paired gRNA expression amplicons (see Figure 36B ).exist Figure 42B In the figure, the vertical axis is editing efficiency (%) and the horizontal axis is paired gRNA cassette (ng). The data points are as follows: FokI-EcoCascade RNP complex (ng), paired gRNA plasmid, paired gRNA amplicon; 375, open triangle, open circle; 750, black triangle, black circle; 1,500, gray triangle, gray circle; 3,000, black triangle with white line, black circle with white line. Figure 42B The data in demonstrate comparable, if not higher, editing efficiencies of paired gRNA-expressing amplicons relative to paired gRNA-expressing plasmids.

[0290] For PAM screening ( Figure 37D 、 Figure 37E 、 Figure 37F 、 Figures 39A-39D 、 Figure 40C and Figure 40F), typically, cells were nucleofected with 3 μg of FokI-Cascade polycistronic plasmid and 150 ng (FokI-PseCascade and FokI-EcoCascade) or ∼80–120 ng (FokI-SthCascade) of oligo-templated paired gRNA expression amplicons (unless otherwise indicated).

[0291] To perform specificity analysis ( Figures 38A to 38C ), cells were nucleofected with 3 μg of polycistronic Cascade and 150 ng of oligo-templated paired gRNA expression amplicons and harvested 5 days after nucleofection. Figure 38A At the top of the , horizontal lines indicate spacer spacing, scissors indicate expected cleavage sites, and the half-sites of the genomic target are shown with their corresponding PAM regions ( Figure 38A , rectangular boxes with contrasting ends). The relationship of the indicated half sites to the target is shown by dotted lines. For each target, 32 base pairs are shown, and the PAM region is shown adjacent to the seed sequence. Figure 38A Pairs of gRNAs are provided that are designed to contain mismatches with one or both half-sites in the genomic target, as indicated by the filled boxes in the grid (excluding the PAM site). Note that for simplicity, both half-sites are shown in the same orientation. Figure 38B The relative editing efficiency at genomic target 70 for each combination of mismatched gRNA pairs is provided, plotted as a percentage of the editing efficiency of perfectly matched gRNAs. Figure 38B In the top row, the target ( Figure 38B , target 70), the next line represents the guide ( Figure 38B , gRNA1 and gRNA2), the next line identifies the unmatched set ( Figure 38B , mm set 1 and mm set 2), the bottom row shows the FokI-Cascade RNP complex. The left column shows the data of relative editing Guide 1-mm set 1 / Guide 2-mm set 2, the left column shows the data of Guide 1-mm set 2 / Guide 2-mm set 1, and both columns show the data of relative editing efficiency percentage ( Figure 38B Relative editing eff (%); scale 0-100), i.e., the left column shows data for gRNA1 and gRNA2 with mismatch (mm) sets 1 and 2, and the right column shows data for the same target but with mismatch (mm) sets swapped between gRNA1 and gRNA2 (n=1). Figure 38C The editing efficiency at target 73 (n=1) is provided as Figure 38B As shown in .

[0292] After developing a scalable method to generate paired gRNA expression cassettes by oligo-templated PCR amplification (as described herein), eliminating the need for labor-intensive cloning steps, a panel of 96 genomic targets was rescreened for each homolog variant with varying FokI linker and DNA spacer lengths. Using a 17-aa linker, FokI-PseCascade consistently produced an average editing efficiency of ∼15-25% within a ~30–33 bp spacer window, with some targets showing up to ∼40-50% indels ( Figure 37C Similar trends were observed with other homologs. PAM requirements were investigated by targeting genomic sites containing one homologous PAM and a second mutant PAM. In vitro, it has been shown that PAM recognition is much more promiscuous than the rigid 5'-GG-3' S. pyogenes PAM requirement (see, e.g., Szczelkun, M., et al., Proc. Natl. Acad. Sci. USA 111:9798–9803 (2014); Hayes, R., et al., Nature 530:499–503 (2016); Westra, E., et al., Mol. Cell. 46:595–605 (2012); Fineran, P., et al., Proc. Natl. Acad. Sci. USA 111:E1629–E1638 (2014); Leenay, R., et al., Mol. Cell. 62:137–147 (2016)). Surprisingly, in vitro data showed that a large number of PAMs indeed allowed activity, with a clear rank order bias ( Figure 37D ; Figures 39A to 39D In contrast, when the mutated PAM represented a “self” target from the CRISPR array, editing was completely abolished.

[0293] exist Figures 39A-39D In each of , the vertical axis corresponds to the editing efficiency (Editing Efficiency (%)), and the horizontal axis corresponds to the PAM sequence associated with the target. Figure 39A The FokI-PseCascade editing efficiency as a function of PAM sequence is provided. The genomic site contains a fixed ATG PAM and a variable PAM at the second half site, as shown on the horizontal axis. The bars show the mean and sd (6-14 sites per variable PAM, n=1 / target site). Note that Figure 37D Data describing the FokI-PseCascade, where one PAM is fixed at AAG and the other PAM is variable within a set of PAMs that includes ATG. Thus, a subset of those PAMs is AAG-ATG. Figure 39A describes data for a FokI-PseCascade in which one PAM is fixed at ATG and the other PAM is variable among a set of PAMs including AAG ( Figure 39A , horizontal axis, from left to right, AAG, AAC, AAA, ATG, GAG, ATA, AAT, and AGG). Therefore, a subset of those PAMs is also AAG-ATG and is Figure 37D The same AAG-ATG site in .

[0294] Figure 39B FokI-EcoCascade editing as a function of PAM sequence is provided ( Figure 39B , horizontal axis, from left to right, CCG, CGC, AAG, AGG, ATG, GAG, AAA, AAC, ATA, and AAT). The fixed PAM is AAG, and the bars show the mean and SD (6-15 sites per variable PAM, n=1 / target site). Figure 39C ( Figure 39C , horizontal axis, from left to right, AAG, ATG, AAC, AAA, AGG, GAG, AAT, and ATA) provide Figure 39B Similar analysis as shown, but with the first PAM fixed to the ATG (6-14 sites per variable PAM, n=1 / target site. Figure 39B The ATG column in the table corresponds to AAG-ATG pairs (mean value is ∼3) and Figure 39C The AA column in the corresponding AAG-ATG pair (mean is also ~3) is the same. Note that the vertical axes have different scales. Figure 39D FokI-SthCascade editing as a function of PAM sequence is provided ( Figure 39D , horizontal axis, from left to right, CC, AA, GA, TA, and CA). The fixed PAM is GAA, and the bars show the mean and sd (18-33 sites per variable PAM; n=1 / target site).

[0295] Figure 40A 、 Figure 40B 、 Figure 40C 、 Figure 40D 、 Figure 40E and Figure 40F Data are shown relating to exemplary changes in editing efficiency of engineered type I CRISPR-Cas complexes. Figure 40A (FokI-PseCascade) and Figure 40D The data shown for percent editing efficiency (vertical axis) versus spacer spacing in bps (horizontal axis) for (FokI-SthCascade) are essentially as described for Example 20C. Figure 41A and Figure 41C The data shown in Figure 40A and Figure 40D In the figure, the horizontal axis represents the 23-34 bp spacer distance, and the bars from left to right are the FokI-Cas8 polypeptide linker lengths of 17 aa (light gray bars), 20 amino acids (dark gray bars), and 30 aa (white bars). Figure 39B The acquisition Figure 40C and Figure 40F The data shown in . Figure 40C and Figure 40F Provides FokI-PseCascade and FokI-SthCascade editing ( Figure 40C , Figure 40F , vertical axis, editing efficiency (%)) as the PAM sequence ( Figure 40C , from left to right, CCG, CGC, AAG, AAA, ATG, AAC, AGG, ATA, GAG, and AAT; Figure 40F , from left to right, CC, AA, GA, TA and CA) functions. Figure 40B The FokI-PseCascade RNP complex is shown. The fixed PAM of FokI-PseCascade is AAG ( Figure 40B , AAG PAM), and another PAM in a group of PAMs ( Figure 40B , is variable in a variable PAM). Figure 40E The FokI-SthCascade RNP complex is shown. The fixed PAM of FokI-SthCascade is GAA ( Figure 40B , GAAPAM), and another PAM in a group of PAMs ( Figure 40E , variable PAM). The linker and spacer interval preferences of FokI-PseCascade were rescreened, and the data showed nearly 50% editing. PAM preference was also examined. From the data, the in vitro rank order preference of PAMs was determined. Basically, the same analysis was performed on variants of Streptococcus thermophilus. In the Streptococcus thermophilus system, editing was low. However, the data shown in this article show that in vivo, in human cells, the Streptococcus thermophilus system is very mixed in its preference for PAM. The fact that a single A upstream of the pre-spacer (i.e., target sequence) allows editing generally provides an increased number of potential target sequences in a gene (e.g., relative to the number of target sites associated with potential second class II type CRISPR-Cas9 PAMs within the same gene). In addition, the in vivo data presented herein are correlated with the in vitro PAM preference demonstrated by Sinkunas, T., et al., EMBO J. 32: 385-394 (2013).

[0296] Accumulated NGS data from hundreds of edited genomic sites provide the ability to characterize the DNA repair outcomes of DSBs introduced by FokI-PseCascade. Focusing on 40 unique sites with indel frequencies >10%, we analyzed the frequencies of deletions and insertions as a function of total mutant read length within a 50 bp window surrounding the predicted cut site. Insertions of 2–4 bp were highly enriched and present in the vast majority of sites examined ( Figure 37E Detailed inspection revealed that ~90% of these insertions contained perfect duplications of the sequence adjacent to the cleavage site. While not wishing to be bound by any particular theory, such duplications may be the result of templated repair of staggered cleavages introduced by dimeric FokI.

[0297] The specificity of FokI-PseCascade was assessed by editing two high-efficiency target sites using paired gRNAs with a large number of mismatches ( Figure 38A Previous studies at Cascade have highlighted mismatches at every sixth position in the ~8-nt PAM-proximal seed sequence and the 32-nt guide gRNA, as these bases are flipped out of the RNA-DNA heteroduplex formed upon target binding (see, e.g., Jung, C., et al., Cell 170:35–47 (2017); Mulepati, S., et al., Science 345:1479–1484 (2014); Fineran, P., et al., Proc. Natl. Acad. Sci. USA 111:E1629–E1638 (2014); Semenova, E., et al., Proc. Natl. Acad. Sci. USA 108:10098–10103 (2011)). Mismatches within the PAM-proximal seed region are highly deleterious for genome editing, whereas mismatches distal to the PAM are well tolerated, resulting in near-wild-state editing efficiencies ( Figure 38B ; Figure 38C However, when mismatched blocks were present at both half-sites, editing dropped dramatically across the entire set of gRNA pairs tested ( Figure 38B , Figure 38C ). Based on the PAM data and FokI-PseCascade-mediated genome editing and spacer interval data ( Figure 38C ; Figure 37D ), one advantage of the modified type I CRISPR-Cas complex of the present invention is that targetable sites can occur once every ∼20 to ∼30 bp in the human genome, while editing at potential off-target sites is impossible.

[0298] Thus, in one embodiment of the invention, the potential targetable sites or "target density" of a given engineered FokI-Cascade system is a function of its effective spacing and PAM preference, and will have some variability among homologs. In some embodiments, the target density of FokI-PseCascade, FokI-EcoCascade, and FokI-SthCascade in the human genome can be calculated using the following criteria (data are extrapolated to calculate predicted target density).

[0299] The following motif can be used to calculate FokI-PseCascade target density:

[0300] 5'–[half site 1–PAM1]–[spacer]–[PAM2–half site 2]–3'.

[0301] Here, [half site 1–PAM1] represents the reverse complement of the target sequence and PAM of the target strand of half site 1 gRNA 1, and [half site 2–PAM2] represents the PAM and target sequence of the non-target strand of half site 2 gRNA 2. Based on the distribution of spacer lengths that support editing with FokI-PseCascade (see, e.g., Figure 37D ), with an effective intergenic interval length of approximately 30-33 bp. PAMs were defined as belonging to set 1 (AAG, AAA, ATG, AAC), which gave the highest editing, or to set 2 (AAG, AGG, ATG, GAG, AAA, AAC, AAT, ATA) if they contained any of the tested PAMs that showed activity (see, e.g., Figure 39A ; Figure 40B ). Accordingly, potential target sites that meet the preferred interval lengths of two PAMs belonging to Set 1 or Set 2 will appear on average once every 33.4 bp or 9.2 bp, respectively.

[0302] The target density of FokI-EcoCascade was determined similarly, except that the intergenic interval length was defined as 31–33, and PAMs were defined as belonging to the highest editing set 1 (AAG, AGG, ATG, GAG, AAA), or to set 2 (AAG, AGG, ATG, GAG, AAA, AAC, AAT, ATA) if they contained any of the PAMs that showed activity (see, e.g. Figure 39C ; Figure 39D Based on this, potential target sites were calculated using Set 1 PAMs or Set 2 PAMs, which appeared once every 30.4 bp or 12.2 bp, respectively.

[0303] The human genomic target density of FokI-SthCascade was determined similarly, except that the intergenic interval length was defined as 29-31 bp and PAMs were defined as NNAs (see, e.g., Figure 39D ). Based on this, it was calculated that potential target sites appeared once every 4 bp on average.

[0304] Thus, as described herein, by providing a number of PAM adjacent target sequences that can be used for genome editing, the modified type I CRISPR-Cas complex provides a method for providing a variety of potential target sites. Thus, one embodiment of the present invention relates to a method for providing an increased number of available target sequences within a gene using a PAM sequence associated with a modified type I CRISPR-Cas complex (e.g., relative to the number of available target sequences associated with a PAM sequence of a second type CRISPR-Cas type II or type V system). Applications of this method involve the use of a modified type I CRISPR-Cas complex, which may include, but is not limited to, binding and / or cleavage of a target sequence, mutation of a target sequence, transcriptional regulation associated with a target sequence or its regulatory elements, and target sequences mediated by the use of the modified type I CRISPR-Cas complex described herein, as well as intentional modifications, changes, and / or significantly different structural changes (e.g., in a gene product) mediated by the use of the modified type I CRISPR-Cas complex described herein.

[0305] In some embodiments, the modified type I CRISPR-Cas effector complexes described herein can be used to generate non-human transgenic organisms by site-specifically introducing a selected polynucleotide sequence (e.g., a portion of a donor polynucleotide) at a DNA target site in the genome to generate modifications, alterations, and / or mutations of gDNA. The transgenic organism can be an animal or a plant.

[0306] Transgenic animals are typically produced by introducing a modified type I CRISPR-Cas effector complex into a fertilized egg cell. The basic technique described with reference to the preparation of transgenic mice (see, e.g., Cho, A., et al., "Generation of Transgenic Mice," Current Protocols in Cell Biology, CHAPTER. Unit-19.11 (2009)) involves five basic steps: first, preparing a system as described herein, which includes a suitable donor polynucleotide; second, harvesting donor fertilized eggs; third, microinjecting the system into mouse fertilized eggs; fourth, implanting the microinjected fertilized eggs into pseudopregnant recipient mice; and fifth, genotyping and analyzing the modifications of the gDNA established in the first-built mice. The first-built mice will pass the genetic modification to any offspring. The first-built mice are typically heterozygous for the transgene. Mating between these mice will produce mice that are homozygous for the transgene 25% of the time.

[0307] Methods for producing transgenic plants are also well known and can be applied using modified type II CRISPR-Cas effector complexes. For example, transgenic plants produced using Agrobacterium-mediated transformation typically contain a transgene inserted into one chromosome. By mating an independent, isolated transgenic plant containing a single transgene with its own sex (i.e., self-fertilization), it is possible to produce a transgenic plant that is homozygous for the transgene. Typical zygosity assays include, but are not limited to, single nucleotide polymorphism assays and thermal amplification assays that distinguish homozygotes from heterozygotes.

[0308] In the sixth aspect, the present invention relates to using the transformed type I CRISPR-Cas effector complex to produce substrate channel. In some embodiments, a fusion protein comprising a substrate channel element and a Cas7 subunit protein is constructed. These Cas7 fusion proteins are then assembled into the transformed type I CRISPR-Cas effector complex (for example, comprising Cse2, Cas5, Cas6, Cas7 substrate channel element fusion and Cas8). In some embodiments, the crRNA of the transformed type I CRISPR-Cas effector complex can be extended to accommodate other Cas7 subunits (see, for example, Luo, M., et al., Nucleic Acids Res. 44: 7385-7394 (2016)). Different substrate elements can be fused to Cas7 and then mixed with the desired stoichiometry. When these various Cas7 subunits are assembled into a complete type I CRISPR-Cas effector complex, the co-localization of substrate elements can enhance the efficacy of substrate channel action.

[0309] In some embodiments, an RNA scaffold is constructed such that multiple Cas7 substrate channel element fusions can bind to it in the absence of other type I CRISPR-Cas effector complex components.

[0310] The substrate access element can be fused to the N-terminus of Cas7 and / or the C-terminus of Cas7. In addition, a circular arrangement of Cas7 can be fused to the substrate access element.

[0311] Figure 11A and Figure 11B A schematic diagram of a substrate channel consisting of three consecutive enzymes in a pathway is shown. The substrate channel facilitates the direct delivery of intermediate metabolites to the active sites of consecutive enzymes in the metabolic pathway chain without being released into additional channel space. Figure 11A A typical arrangement of a modified substrate channel is shown. Enzymes E1, E2, and E3 interact covalently or non-covalently with the scaffold protein (S1, S2, S3) matrix. Double-headed arrows represent interactions (e.g., affinity interactions) between the enzyme and the scaffold protein. The substrate (X) is then processed into product (Y) without being released into additional channel space. Figure 11B An embodiment of the invention is shown, which includes a modified type I CRISPR-Cas effector complex carrying enzymes E1, E2, and E3 as fusion proteins (i.e., covalent interactions) with Cas7 subunit proteins, thereby generating a substrate channel. cpCas7 proteins and backbones formed by cpCas7 proteins may also be useful in practicing this aspect of the invention.

[0312] In other embodiments, substrate access elements can be fused to Cas6. The Cas6 subunit of the Cascade complex can recognize a specific RNA hairpin structure. An RNA scaffold can be constructed consisting of multiple Cas6 RNA hairpin structures connected in series. Cas6 peptides from different Cascade complexes have different recognition sequences. Therefore, an RNA scaffold can be constructed from multiple orthogonal Cas6 RNA hairpins. By fusing different substrate access elements to orthogonal Cas6 peptides, substrate access complexes can be assembled with specific stoichiometry.

[0313] The substrate access element can be fused to the N-terminus of Cas6 and / or the C-terminus of Cas6. In addition, the circularly arranged Cas6 can be fused to the substrate access element.

[0314] In some embodiments, the target heterologous metabolic pathway can be expressed in a model organism, such as E. coli. When a gene is heterologously expressed, the gene can be codon optimized to more efficiently express the gene.

[0315] In one embodiment, the target metabolic pathway is the mevalonate pathway from Saccharomyces cerevisiae. The substrate access elements of this pathway include, but are not limited to, acetoacetyl-CoA-sulfase (AtoB), hydroxy-methylglutaryl-CoA synthase (HMGS), and hydroxy-methylglutaryl-CoA reductase (HMGR).

[0316] In another embodiment, the target metabolic pathway is the glycerol synthesis pathway from Saccharomyces cerevisiae. The substrate access elements of this pathway include, but are not limited to, glycerol 3-phosphate dehydrogenase (GPD1) and glycerol-3-phosphate phosphatase (GPP2).

[0317] In yet another embodiment, the target metabolic pathway is the starch hydrolysis pathway from Clostridium stercorarium. The substrate access elements of this pathway include, but are not limited to, CelY and CelZ.

[0318] In another embodiment, the target metabolic pathway is the glucose phosphotransferase pathway from Escherichia coli. The substrate access elements of this pathway include, but are not limited to, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).

[0319] In a seventh aspect, the present invention relates to site-directed recruitment of functional domains fused to Cascade subunit proteins by a complex comprising a second type II Cas9 protein and a nucleic acid targeting nucleic acid (NATNA). Functional domains are disclosed herein and include, but are not limited to, protein domains having enzymatic functions capable of transcriptional activation or transcriptional repression. Examples 13A and 13B describe methods for modifying a second type II CRISPRsgRNA, crRNA, tracrRNA, or crRNA and tracrRNA sequence with a first type I CRISPR repeat stem sequence to allow recruitment of one or more Cascade subunit proteins to a type II CRISPRCas protein / guide RNA complex binding site.

[0320] Figure 12A 、 Figure 12B and Figure 12C A generalized view of the site-directed recruitment of functional protein domains fused to Cascade subunit proteins to target sites by the dCas9:NATNA complex is shown. Figure 12A , 101) of the second type II CRISPRNATNA ( Figure 12A , 102) through the linker nucleic acid sequence ( Figure 12A , 103) covalently linked to the first type I CRISPR repeat stem sequence ( Figure 12A , 104). Covalently linked to a type I CRISPR repeat stem sequence ( Figure 12A, 105) type II CRISRP NATNA is able to bind to type II dCas9 ( Figure 12A , 106 ) and type I Cascade subunit proteins (e.g., Cas6; Figure 12A , 107), which is connected by a subsequence ( Figure 12A , 108 ) fused to a functional protein domain (e.g., an enzymatic domain, a transcriptional activation or repression domain; Figure 12A , 109), thereby forming an RNP complex. The RNP complex ( Figure 12B , 110) can target sequences containing type II CRISPRNATNA spacer sequences ( Figure 12A , 101) complementary target sequence ( Figure 12B , 112) double-stranded DNA ( Figure 12B , 111). Target recognition by the RNP complex leads to the spacer sequence ( Figure 12A , 101) and target sequence ( Figure 12B , 112) between the hybrid ( Figure 12B , 113). Targeting Cascade subunit domain fusion proteins to DNA allows modification of DNA by the functional protein domains of adjacent genes or transcriptional regulation ( Figure 12C , 114).

[0321] In an eighth aspect, the present invention relates to compositions comprising a modified type I CRISPR-Cas effector complex, a modified guide polynucleotide and a combination thereof. In some embodiments, the modified type I CRISPR-Cas effector complex comprises a related Cas3 fusion protein. The wild-type type I CRISPR-Cas system requires the synergistic effect of the Cascade effector complex for DNA targeting and the Cas3 helicase-nuclease for progressive DNA degradation. In one embodiment of the invention, the type I CRISPR-Cas effector complex is modified to prepare precise DSBs by fusing the complex to a nuclease domain (e.g., a non-specific FokI nuclease domain). The method uses paired guide polynucleotides targeting two half-site DNA sequences separated by an intermediate sequence (i.e., a spacer).

[0322] Embodiments of this aspect of the invention relate to compositions comprising two engineered type I CRISPR-Cas effector complexes, each of which comprises a spacer and an endonuclease (e.g., FokI; see, e.g., Figure 2A 、 Figure 2B and Figure 2C The invention relates to a fusion protein of a Cascade complex (of a gene encoding a Cascade protein) wherein at least two parameters are varied to modulate genome editing efficiency. Such parameters include:

[0323] The length of the linker polypeptide used to generate a fusion protein comprising a Cas subunit protein and an endonuclease (e.g., FokI); and

[0324] The length of the spacer spacing between nucleic acid target sequences to which the spacer is capable of binding.

[0325] Guidance regarding amino acid composition and sequence linker polypeptides is provided herein.

[0326] One embodiment of this aspect of the invention is a composition comprising:

[0327] A first engineered type I CRISPR-Cas effector complex comprising:

[0328] a first Cse2 subunit protein, a first Cas5 subunit protein, a first Cas6 subunit protein, and a first Cas7 subunit protein,

[0329] a first fusion protein comprising a first Cas8 subunit protein and a first FokI, wherein the N-terminus of the first Cas8 subunit protein or the C-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus or N-terminus of the first FokI, respectively, via a first linker polypeptide, and wherein the first linker polypeptide has a length of about 10 amino acids to about 40 amino acids, and

[0330] a first guide polynucleotide comprising a first spacer capable of binding to a first nucleic acid target sequence; and

[0331] A second engineered type I CRISPR-Cas effector complex comprising:

[0332] a second Cse2 subunit protein, a second Cas5 subunit protein, a second Cas6 subunit protein, and a second Cas7 subunit protein,

[0333] a second fusion protein comprising a second Cas8 subunit protein and a second FokI, wherein the N-terminus of the second Cas8 subunit protein or the C-terminus of the second Cas8 protein is covalently linked to the C-terminus or N-terminus of the second FokI, respectively, via a second linker polypeptide, and wherein the second linker polypeptide has a length of about 10 amino acids to about 40 amino acids, and

[0334] A second guide polynucleotide comprising a second spacer capable of binding to a second nucleic acid target sequence, wherein the protospacer adjacent motif (PAM) of the second nucleic acid target sequence and the PAM of the first nucleic acid target sequence have a spacer spacing of about 20 base pairs to about 42 base pairs.

[0335] Examples of such a first engineered type I CRISPR-Cas effector complex that binds to a first nucleic acid target sequence and a second engineered type I CRISPR-Cas effector complex that binds to a second nucleic acid target sequence are shown in Figure 2A 、 Figure 2B and Figure 2C middle.

[0336] In some embodiments, the length of the first linker polypeptide and / or the second linker polypeptide is about 15 amino acids to about 30 amino acids, or about 17 amino acids to about 20 amino acids in length. In one embodiment, the length of the first linker polypeptide and the second linker polypeptide is the same.

[0337] The first Cas8 subunit protein and the second Cas8 subunit protein may each comprise the same amino acid sequence of the Cas8 subunit protein.

[0338] Similarly, the first Cse2 subunit protein and the second Cse2 subunit protein can each comprise the same amino acid sequence of the Cse2 subunit protein, the first Cas5 subunit protein and the second Cas5 subunit protein can each comprise the same amino acid sequence of the Cas5 subunit protein, the first Cas6 subunit protein and the second Cas6 subunit protein can each comprise the same amino acid sequence of the Cas6 subunit protein, the first Cas7 subunit protein and the second Cas7 subunit protein can each comprise the same amino acid sequence of the Cas7 subunit protein, and combinations of the above.

[0339] Typically, the N-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus of the first FokI through a first linker polypeptide, the C-terminus of the first Cas8 subunit protein is covalently linked to the N-terminus of the first FokI through a first linker polypeptide, the N-terminus of the second Cas8 subunit protein is covalently linked to the C-terminus of the second FokI through a second linker polypeptide, the C-terminus of the second Cas8 subunit protein is covalently linked to the N-terminus of the second FokI through a second linker polypeptide, and combinations thereof.

[0340] Embodiments of this aspect of the invention include embodiments wherein the length of the spacer distance between the second nucleic acid target sequence and the first nucleic acid target sequence is from about 22 base pairs to about 40 base pairs, from about 26 base pairs to about 36 base pairs, from about 29 base pairs to about 35 base pairs, or from about 30 base pairs to about 34 base pairs.

[0341] The first FokI and the second FokI can be monomeric subunits capable of combining to form a homodimer, or different subunits capable of combining to form a heterodimer.

[0342] In preferred embodiments, the guide polynucleotide comprises RNA.

[0343] In some embodiments, the gDNA comprises a PAM of the second nucleic acid target sequence and a PAM of the first nucleic acid target sequence.

[0344] In some embodiments, the engineered type I CRISPR-Cas effector complex is based on a type I CRISPR-Cas effector complex of one or more organisms selected from: Salmonella enterica, Bacillus geothermophilus (strain EPR-M), Methanospora oryzae MRE50, Pseudomonas aeruginosa (e.g., Pseudomonas aeruginosa (strain ND07), Pseudomonas S-6-2, and Escherichia coli. In preferred embodiments, the engineered type I CRISPR-Cas effector complex is based on a type I CRISPR-Cas effector complex of Pseudomonas aeruginosa (e.g., Pseudomonas aeruginosa (strain ND07), Pseudomonas S-6-2, and / or Escherichia coli. Pseudomonas S-6-2 induced ~10-fold higher editing efficiency than the E. coli homolog, and approximately the same level of activity as E. coli was shown in the other homologs tested, demonstrating that engineered type I CRISPR-Cas effector complexes from different type I systems can be functionally used for genome editing in human cells.

[0345] The data shown in Example 18A, Example 18B, Example 18C, Example 18D, Example 20A, Example 20B and Example 20C demonstrate that changing the length of the linker polypeptide used to generate a fusion protein comprising a Cas subunit protein and FokI and / or changing the length of the spacer spacing between nucleic acid target sequences to which the spacer can bind promotes the regulation of genome editing efficiency in cells.

[0346] In another embodiment, the present invention relates to an engineered type I CRISPR-Cas effector complex comprising a first fusion protein comprising a Cascade subunit protein (e.g., a Cas8 subunit protein) and a first functional domain (e.g., FokI), and a second fusion protein comprising a dCas3* protein and a second functional domain (e.g., FokI). Figure 13A : Cas7, Cas5, Cas8, Cse2 and Cas6, the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin). Contains a first functional domain (e.g., FokI) ( Figure 13A , Cas8-linker 1-FP1 fusion) can bind DNA and then recruit dCas3*-second functional domain (e.g., FokI) fusion protein ( Figure 13A , dCas3*-linker 2-FP2). In which the first functional domain ( Figure 13A, Cas8-linker 1-FP1 fusion) and the second functional domain ( Figure 13A In the case where the dCas3*-linker 2-FP2) subunits comprise a dimeric protein, the dCas3*-second functional domain (e.g., FokI) fusion protein binds to the engineered type I CRISPR-Cas effector complex comprising the first functional domain (e.g., FokI) to promote dimerization of the first functional domain and the second functional domain ( Figure 13A ). Figure 14A Shown are polypeptides comprising a linker ( Figure 14A , linker 1) connected to the Cas subunit protein ( Figure 14A , striped box) of the first functional domain ( Figure 14A , FD1) and a linker polypeptide associated with the Cascade complex ( Figure 14A , linker 2) is connected to the second functional domain ( Figure 14A , FD2) of the modified type I CRISPR-Cas effector complex ( Figure 14A , Cascade) dsDNA; thereby bringing FD1 and FD2 into proximity and promoting the interaction between FD1 and FD2. The binding of the Cascade complex involves a single PAM sequence ( Figure 14A , PAM, open box). Figure 14A In the figure, dsDNA is shown as a pair of horizontal dashed lines. In the case where the functional domain is a dimeric endonuclease (eg, FokI), the proximity of FD1 and FD2 favors the formation of a functional dimer.

[0347] One advantage of embodiments of the present invention is that, compared to using two FokI-Cascade complexes ( Figure 14A and Figure 2A 、 Figure 2B and Figure 2C For comparison), a single Cascade complex (recognizing a single PAM sequence) can be used to cleave a double-stranded nucleic acid target sequence. Using two FokI-Cascade complexes requires two PAM sequences ( Figure 2A 、 Figure 2B and Figure 2C ), which may limit the selection of proximal nucleic acid target sequences.

[0348] The length and / or composition of the linker polypeptides used to generate fusion proteins comprising Cas subunit proteins and endonucleases (e.g., FokI), as well as the length and / or composition of the linker polypeptides used to generate fusion proteins comprising dCas3* proteins and endonucleases, can be varied to modulate genome editing efficiency. Examples 21A, 21B, 21C, and 21D describe the design and testing of various Cas3-FokI linker compositions and lengths, as well as FokI-Cas8 linker compositions and lengths for modulating genome editing efficiency.

[0349] Another embodiment of this aspect of the invention comprises an engineered type I CRISPR-Cas effector complex ( Figure 13B : Cas7, Cas5, Cas8, Cse2 and Cas6; the dashed box around Cas6 indicates its interaction with the crRNA hairpin; cRNA is shown as a black line including the hairpin), and the polypeptide comprising a linker ( Figure 13B , linker) connected dCas3* protein ( Figure 13B , dCas3*) and functional domains ( Figure 13B , FP) (e.g., cytidine deaminase) fusion protein. The modified type I CRISPR-Cas effector complex can bind to DNA and recruit dCas3*-functional domain (e.g., cytidine deaminase) fusion protein. This embodiment can promote site-specific targeting of nucleic acid target sequences for modification by functional domains or interaction with functional domains. In the case of cytidine deaminase, the modified type I CRISPR-Cas effector complex and a fusion protein comprising dCas3* protein and cytidine deaminase can be used for site-specific base editing in nucleic acid target sequences. Figure 14B The engineered type I CRISPR-Cas effector complex ( Figure 14B , Cascade), which comprises a fusion protein comprising a linker polypeptide ( Figure 14B , linker) and functional domain ( Figure 14B , FD) linked to the dCas3* protein ( Figure 14B , dCas3*), where the complex binds to dsDNA ( Figure 14B , paired horizontal dashed lines). Figure 14B In the Cascade complex, the contact between the functional domain and the dsDNA is promoted. The binding of the Cascade complex involves a single PAM sequence ( Figure 14B , PAM, open box). Figure 14C The engineered type I CRISPR-Cas effector complex ( Figure 14C , Cascade), which comprises a fusion protein comprising a linker polypeptide ( Figure 14C, linker) and functional domain ( Figure 14C , FD) linked to the dCas3* protein ( Figure 14C , dCas3*), where the complex binds to dsDNA ( Figure 14C , paired horizontal dashed lines). Cascade complex binding involves a single PAM sequence ( Figure 14C , PAM, open box). Figure 14C In the mitochondrial domain, the contact between the functional domain and ssDNA is promoted.

[0350] Additional functional domains and proteins that can be used to construct fusion proteins with type I CRISPR-Cas subunit proteins are described in this specification and in the Examples. The length of the linker polypeptide composition and Cas3-linker polypeptide-functional domain fusion protein can be evaluated following the guidance of Examples 21A to 21D and this specification to assess the impact on the performance of the functional domain.

[0351] Some embodiments of the present invention can use a modified type I CRISPR-Cas effector complex and an mCas3 protein, wherein the mCas3 protein comprises a downregulated helicase activity (e.g., an mCas3 protein—a Cas3 processive mutant protein that has reduced movement along DNA relative to a wild-type type I CRISPRCas3 protein), or the mCas3 protein lacks helicase activity (e.g., the mCas3 protein is no longer a processive nuclease such as the wtCas3 protein, but the mCas3 protein retains nicking activity). The modified type I CRISPR-Cas effector complex can bind to DNA and then recruit the mCas3 protein. This embodiment can promote site-specific cutting of genomic DNA.

[0352] Table 48 describes a number of mCas3 proteins in which mutations in the Cas3 protein affect the ATP binding / hydrolysis region of the helicase domain or the conserved ssDNA pathway region of the helicase domain. Figure 44 A linear representation of the functional domains of the EcoCas3 protein and the relative positions of mutations made in the Cas3 coding sequence are shown. Figure 44In the figure, the HD nuclease domain (amino acids 1-272), helicase domain (RecA1 region, amino acids 273-521; RecA2 region, amino acids 522-737), linker (amino acids 738-794), and C-terminal domain (CTD, amino acids 795-888) are indicated. Huo, Y., et.al., Nat. Struct. Mol. Biol. 9:771-777 (2014) provided a method for synthesizing a novel thermophilic spore fragment using a novel spore fragment from Thermobifida fusca (accession code: Q47PJ0; SEQ ID NO: 1869), Saccharomonospora viridis (C7MTA6; SEQ ID NO: 1870), Thermomonospora curvata (D1A6Q2; SEQ ID NO: 1922), Streptomyces avermitilis (Q825B5; SEQ ID NO: 1925), Streptomyces botropensis (M3DI13; SEQ ID NO: 1923), Thermus thermophiles strain HD8 (Q53VY2; SEQ ID NO: 1930), Streptomyces avermitilis (Q825B5; SEQ ID NO: 1931), Streptomyces botropensis (M3DI13; SEQ ID NO: 1932), Thermus thermophiles strain HD8 (Q53VY2; SEQ ID NO: 1933), Streptomyces avermitilis (Q825B5; SEQ ID NO: 1934), Streptomyces bottropensis (M3DI13; SEQ ID NO: 1935), Streptomyces avermitilis (Q825B5; SEQ ID NO: 1936), Streptomyces bottropensis (M3DI13; SEQ ID NO: 1937), Thermus thermophiles strain HD8 (Q53VY2; SEQ ID NO: 1938), Streptomyces avermitilis (Q825B5; SEQ ID NO: 1939), Streptomyces bottropensis (M3DI13; SEQ ID NO: Sequence conservation analysis was performed by comparing the sequence alignment of the Cas3 family of proteins from Escherichia coli (P38036; SEQ ID NO: 1924) and Escherichia coli (P38036; SEQ ID NO: 1844). 24 different EcoCas3 protein variants with mutations in the ATP binding portion of the helicase domain or the ssDNA loop binding domain were screened (Examples 23A to 23C). Seven mutants showed significantly more and / or positionally shifted deletion categories in the amplicon window; this finding supports that those mCas3 proteins have reduced processivity relative to wtCas3.

[0353] Examples 23A to 23C describe mCas3 proteins in which the average mCas3 protein-induced deletion is shorter relative to the average deletion produced by the corresponding wtCas3 protein. Such mCas3 proteins can be used for genome editing (e.g., in human cells). Figure 45A 、 Figure 45B 、 Figure 45C and Figure 45DData indicating that mCas3 proteins, when introduced and expressed in human cells, produce shorter average deletion lengths when associated with the Cascade RNP complex relative to wtCas3 proteins associated with the Cascade RNP complex are shown. With reference to the teachings of this specification, one of ordinary skill in the art can perform similar mutations in corresponding regions of Cas3 proteins obtained from bacterial species other than E. coli.

[0354] Examples 26A to 26C provide additional examples of mCas3 proteins that can be used to generate genomic deletions, wherein the average mCas3 protein-induced deletions are shorter relative to the average deletions generated using the corresponding wtCas3 protein. The data shown in the Examples support that an ATPase / helicase-deficient variant of Cas3 from Pseudomonas sp. S-6-2 (mPseCas3 protein) can be used with the PseCascade RNP complex to generate deletions at the intended cleavage site (i.e., cleavage site-localized deletions).

[0355] The wtPseCas3 protein / PseCascade activity was further characterized. Additional experiments were performed using target enrichment probes that enable detection of large genomic deletions. Specifically, HEK293 cells were transfected with DNA templates encoding the PseCascade RNP complex, the wtPseCas3 protein, and a minimal CRISPR array for the TRAC site, essentially as described in Examples 26A to 26C. Target enrichment probes were used to isolate and sequence genomic fragments; whereas in Example 26C, an amplicon window was used to identify the presence of deletions. The target enrichment / sequencing method provides an unbiased view of larger deletions, which cannot be provided by using an amplicon window to identify deletions. Overall, it was found that deletions assessed using target enrichment and genomic fragment sequencing were largely unidirectional, starting upstream of the wtPseCas3 protein start site. Deletions ranged from 1 bp to nearly 250 kb. In addition to providing a method for cutting genomic DNA and providing deletions of a given length, this method can also be used to generate larger random subsets of deletions at a determined position to probe the regulatory / promoter region of a gene.

[0356] The mCas3 protein can comprise one or more mutations (e.g., a combination of mutations as described in Table 48).

[0357] Control of deletion length for several mCas3 proteins was demonstrated. In some embodiments, the mCas3 protein associated with the Cascade complex comprising a guide polynucleotide of the present invention can provide an average deletion length of about 1 to about 600 base pairs, about 1 to about 500 base pairs, about 1 to about 400 base pairs, about 1 to about 300 base pairs, preferably about 1 to about 250 base pairs, about 1 to about 200 base pairs, or about 1 to about 100 base pairs.

[0358] In some embodiments, wtCas3 protein or mCas3 protein can be fused to the various subunits of the Cascade complex to further control the Cas3 average deletion length. Constraining the Cascade complex can limit or prevent Cas3 protein or mCas3 protein from moving along DNA because it will be fixed to the site bound by the Cascade complex. Generally, a linker polypeptide can be utilized to fuse wtCas3 protein or mCas3 protein to the N-terminal or C-terminal domain (for example, for the EcoCascade complex, the fusion can be with EcoCas8, EcoCas6 or EcoCas5) of the protein component of the Cascade complex. NLS sequences can also be attached to the N-terminal of the fusion protein. Examples of such constructs of E. coli Cascade protein components are shown in Table 12. These EcoCas3 fusion proteins also have an NLS sequence attached to their N-terminal.

[0359]

[0360]

[0361] *Protein sequence is the protein sequence encoded by the polycistronic

[0362] Embodiments of the present invention include engineered type I CRISPR mCas3 proteins that can reduce movement along DNA relative to wild-type type I CRISPR Cas3 proteins (wtCas3 proteins). In some embodiments, the mCas3 protein comprises about 90% or more, preferably about 95% or more, more preferably about 98% or more sequence identity to the corresponding wtCas3 protein. The coding sequence of the mCas3 protein may comprise a covalently linked nuclear localization signal at the amino terminus, the carboxyl terminus, or both the amino terminus and the carboxyl terminus. The mCas3 protein may comprise one or more mutations that downregulate helicase activity, wherein the engineered mCas3 protein retains nuclease activity (or at least a portion thereof) relative to the corresponding wtCas3 protein. Typically, the DNA is dsDNA comprising a target region containing a nucleic acid target sequence. When the wtCas3 protein is associated with the corresponding Cascade nucleoprotein complex ("Cascade NP complex / wtCas3 protein"; e.g., Cascade RNP complex), and the Cascade NP complex comprises a guide comprising a spacer complementary to a nucleic acid target sequence, binding of the Cascade NP complex / wtCas3 protein to the nucleic acid target sequence facilitates cleavage in the DNA target region, typically resulting in a deletion in the target region; and the mCas3 protein, when associated with the Cascade NP complex ("Cascade NP complex / mCas3 protein"; e.g., Cascade RNP complex / mCas3 protein) and bound to a nucleic acid target sequence, facilitates cleavage in the DNA target region and results in an average deletion length that is shorter than the average deletion length of wtCas3.

[0363] In some embodiments, the one or more mutations in the mCas3 protein are amino acid substitutions relative to the wtCas3 protein. In other embodiments, one or more deletions include amino acid deletions or insertions relative to the wtCas3 protein in the mCas3 protein coding sequence. One or more mutations may be located in the RecA1 region or the RecA2 region of the helicase domain. In one embodiment, relative to the wtCas3 protein (e.g., mutations that affect ssDNA ring binding and / or mutations in the conserved region of the ssDNA pathway of the helicase domain), one or more mutations downregulate the binding of the mCas3 protein to ssDNA. In another embodiment, relative to the wtCas3 protein, one or more mutations downregulate ATP hydrolysis by the mCas3 protein, or relative to the wtCas3 protein, downregulate the binding of ATP to the mCas3 protein. In other embodiments, the mCas3 protein comprises a combination of one or more mutations that downregulate the binding of the mCas3 protein to ssDNA, downregulate ATP hydrolysis by the mCas3 protein relative to the wtCas3 protein, or downregulate the binding of ATP to the mCas3 protein.

[0364] Other embodiments include the coding sequence of the mCas3 protein covalently linked to the amino-terminal or carboxyl-terminal coding sequence of the Cas protein of the Cas nucleoprotein complex (e.g., Cascade RNP complex). Such Cas protein can be selected from Cse2, Cas8 protein, Cas7 protein, Cas6 and Cas5 protein.

[0365] In some embodiments, the wtCas3 protein is an Escherichia coli type 1 CRISPRCas3 protein. In other embodiments, the wtCas3 protein is a wtCas3 protein selected from Pseudomonas S-6-2, Thermospora fusca, Saccharomonas viridis, Thermospora curvatum, Streptomyces avermannii, Streptomyces podophylla, Thermus thermophilus, Vibrio cholerae, Salmonella enterica, Thermobacillus geothermophilus EPR-M, Methanospora oryzae MRE50, and Pseudomonas aeruginosa (strain ND07).

[0366] For the E. coli type 1 CRISPR wtCas3 protein, the one or more mutations may include, but are not limited to, D452H, A602V, or D452H and A602V.

[0367] In other embodiments, the cell comprises DNA, wherein the cell can be a eukaryotic cell (eg, a human cell).

[0368] In additional embodiments, the present invention includes a polynucleotide comprising a coding sequence for an mCas3 protein, an expression cassette comprising a coding sequence for an mCas3 protein, a plasmid comprising a coding sequence for an mCas3 protein, and a Cascade nucleoprotein complex comprising an mCas3 protein.

[0369] In a ninth aspect, the present invention relates to methods of using engineered type I CRISPR-Cas effector complexes.

[0370] In some embodiments, the present invention includes a method of binding a nucleic acid target sequence in a polynucleotide (e.g., dsDNA), comprising providing one or more engineered type I CRISPR-Cas effector complexes for induction into a cell or biochemical reaction, and introducing the engineered type I CRISPR-Cas effector complex into the cell or biochemical reaction, thereby promoting contact of the engineered type I CRISPR-Cas effector complex with the polynucleotide. Contact of the complex with the polynucleotide results in binding of the engineered type I CRISPR-Cas effector complex to the nucleic acid target sequence in the polynucleotide.

[0371] In one embodiment, the engineered type I CRISPR-Cas effector complex comprises a guide that is complementary to a nucleic acid target sequence in a polynucleotide. The engineered type I CRISPR-Cas effector complex binds to a nucleic acid target sequence in a polynucleotide.

[0372] In other embodiments, a first engineered Type I CRISPR-Cas effector complex comprises a guide that is complementary to a first nucleic acid target sequence in a polynucleotide, and a second engineered Type I CRISPR-Cas effector complex comprises a guide that is complementary to a second nucleic acid target sequence in a polynucleotide. The first engineered Type I CRISPR-Cas effector complex binds the first nucleic acid target sequence, and the second engineered Type I CRISPR-Cas effector complex binds the second nucleic acid target sequence in the polynucleotide.

[0373] In yet another embodiment, the engineered type I CRISPR-Cas effector complex comprises a guide that is complementary to a nucleic acid target sequence in a polynucleotide and further comprises a dCas3* fusion protein capable of binding to the complex. The engineered type I CRISPR-Cas effector complex binds to a nucleic acid target sequence in a polynucleotide, and the effector complex comprises a dCas3* fusion protein that binds to the complex.

[0374] Such methods of binding a nucleic acid target sequence can be performed in vitro (e.g., in a biochemical reaction or in cultured cells; in some embodiments, the cultured cells are cultured human cells that remain in culture and are not introduced into a human body); in vivo (e.g., in cells of a living organism, with the proviso that, in some embodiments, the organism is a non-human organism); or ex vivo (e.g., in cells removed from a subject, with the proviso that, in some embodiments, the subject includes a human subject, and in other embodiments, the subject is a non-human subject).

[0375] The various methods of interaction between assessment and / or quantitative nucleic acid sequence and nucleotide known in the art include but are not limited to the following: immunoprecipitation (ChIP) assay, DNA electrophoretic mobility shift assay (EMSA), DNA pull-down assay, and microplate capture and detection assay. Commercial kits, materials and reagents can be used for implementing many such methods, and for example, can be obtained from the following suppliers: Thermo Scientific (Wilmington, DE), Signosis (Santa Clara, CA), Bio-Rad (Hercules, CA) and Promega (Madison, WI). A common method for detecting the interaction between a polypeptide and a nucleic acid sequence is EMSA (see, for example, Hellman LM, et al., Nature Protocols 2: 1849-1861 (2007)).

[0376] In another embodiment, the invention includes a method of cleaving a nucleic acid target sequence in a polynucleotide (e.g., single-stranded cleavage in dsDNA or double-stranded cleavage in dsDNA) comprising providing one or more engineered Type I CRISPR-Cas effector complexes for use in a cell or biochemical reaction, and introducing the engineered Type I CRISPR-Cas effector complex into the cell or biochemical reaction, thereby facilitating contact of the engineered Type I CRISPR-Cas effector complex with the polynucleotide.

[0377] In one embodiment, a first engineered type I CRISPR-Cas effector complex (CRISPR-Cas) comprising a guide complementary to a first nucleic acid target sequence in a polynucleotide and a first nuclease domain (e.g., FokI) is introduced into the Figure 15A , Cascade1, solid outline box, connected via a linker polypeptide, black curve, to a first nuclease domain, represented as a circular sector), and a second engineered type I CRISPR-Cas effector complex ( Figure 15A , Cascade 2, dotted outline box, connected via a linker polypeptide, black curve, to the second nuclease domain, represented as a circular sector) is introduced into a cell or biochemical reaction. The first modified type I CRISPR-Cas effector complex ( Figure 15B , Cascade1) binds to dsDNA ( Figure 15B , a first nucleic acid target sequence in the dsDNA represented by paired horizontal black lines), and the first nuclease domain cleaves the first strand of the dsDNA ( Figure 15C , Cascade1), and a second engineered type I CRISPR-Cas effector complex ( Figure 15B, Cascade2) binds to a second nucleic acid target sequence in dsDNA, and the second nuclease domain cuts the second strand of dsDNA. The binding of the modified type I CRISPR-Cas effector complex causes the nucleic acid target sequence in the polynucleotide (e.g., dsDNA) to be cut by the modified type I CRISPR-Cas effector complex.

[0378] In another embodiment, a first modified type I CRISPR-Cas effector complex comprises a guide complementary to a first nucleic acid target sequence in a polynucleotide, a second modified type I CRISPR-Cas effector complex comprises a guide complementary to a second nucleic acid target sequence in a polynucleotide, and a Cas3 nickase (e.g., an ATPase-deficient Cas3 variant having only nickase activity) is introduced into a cell or biochemical reaction. The first modified type I CRISPR-Cas effector complex binds to a first nucleic acid target sequence in dsDNA, the Cas3 nickase protein is associated with the first complex, and cuts the first strand of the dsDNA, and the second modified type I CRISPR-Cas effector complex binds to a second nucleic acid target sequence in dsDNA, the Cas3 nickase protein is associated with the second complex, and cuts the second strand of the dsDNA. Binding of the modified type I CRISPR-Cas effector complex to the associated Cas3 nickase protein results in cleavage of the nucleic acid target sequence in the polynucleotide (e.g., dsDNA) by the modified type I CRISPR-Cas effector complex. The data shown in Example 25A, Example 25B, and Example 25C demonstrate that the Cascade RNP complex comprising a Cas3 ATPase-deficient mutant protein can induce targeted genomic deletions by paired nicking. The paired nicking can promote targeted deletions in the genome of a host cell (e.g., a human cell).

[0379] In another embodiment, an engineered type I CRISPR-Cas effector complex (CRISPR-Cas) comprising a guide complementary to a nucleic acid target sequence in a polynucleotide and a first nuclease domain (e.g., FokI) is inserted into the CRISPR-Cas effector complex. Figure 16A , Cascade; dashed outline box, connected via a linker polypeptide, black curve, to the first nuclease domain, represented as a circular sector), and a dCas3*-second nuclease domain (e.g., FokI) fusion protein capable of binding to the complex ( Figure 16A , dCas3; solid outline box, connected via a linker polypeptide, black curve, to the second nuclease domain, represented as a circular sector) is introduced into cells or biochemical reactions. Figure 16B , Cascade) binds to nucleic acid target sequences in dsDNA ( Figure 16B , paired horizontal black lines) and cut the first strand of dsDNA ( Figure 16C, Cascade), and the dCas3* fusion protein is associated with the Cascade RNP complex ( Figure 16B ,dCas3*), and cuts the second strand of dsDNA ( Figure 16C ,dCas3*).

[0380] In other embodiments, a modified type I CRISPR-Cas effector complex comprising a guide complementary to a target region comprising a nucleic acid target sequence in a polynucleotide and a Cas3 protein (e.g., Cas3 protein or mCas3 protein) capable of binding to the complex is introduced into a cell or biochemical reaction. The modified type I CRISPR-Cas effector complex binds to the nucleic acid target sequence in the dsDNA, and the Cas3 protein (e.g., Cas3 protein or mCas3 protein) is associated with the complex and cuts at least one chain of the dsDNA in the target region. In some embodiments, dsDNA is cut by the mCas3 protein, resulting in a deletion in the dsDNA target region. This method can be used to make long-range deletions of a specific length and can be used to generate gene knock-outs or knock-ins. In some embodiments, the Cas3 protein (e.g., Cas3 protein or mCas3 protein) can be fused to a Cascade complex subunit protein (e.g., Cas7 protein, Cas8 protein, Cas5 protein, Cse2 protein). Examples 23A to 23C describe embodiments of the mCas3 protein.

[0381] In another embodiment, the present invention relates to the use of type I CRISPR-Cas effector complexes, wherein the nuclease domain is fused to a Cascade complex protein (see, e.g., Example 11A, Table 38) or a dCas3* protein (e.g., a dCas3* protein fused to a DNA enzyme) to delete a nucleic acid target sequence. This method can be used to make cuts and deletions in a dsDNA target region and can be used to generate gene knockouts. In some embodiments, the nuclease domain can be fused to a Cascade complex subunit protein such as a Cas7 protein, a Cas8 protein, a Cas5 protein, or a Cse2 protein.

[0382] The method of cleaving a nucleic acid target sequence in a polynucleotide can further comprise introducing a donor polynucleotide into a cell to facilitate incorporation of at least a portion of the donor polynucleotide into gDNA of the cell.

[0383] Figure 17A A guide complementary to a first nucleic acid target sequence in a polynucleotide is shown. Figure 17A , Cascade1) and a first nuclease domain (e.g., FokI) of the first engineered type I CRISPR-Cas effector complex ( Figure 17A, a linker polypeptide, shown as a curved line connecting Cascade 1, and a gray circular sector), and a guide comprising a second nucleic acid target sequence complementary to the polynucleotide ( Figure 17A , Cascade 2) and a second engineered type I CRISPR-Cas effector complex ( Figure 17A , linker polypeptide, shown as a curved line connecting Cascade2, and a gray circular sector) cleaved two strands of dsDNA ( Figure 17A , paired black horizontal lines). Figure 17B Shown are homology arms comprising complementary DNA sequences adjacent to the double-stranded cleavage site ( Figure 18B , donor, dotted line) of the donor polynucleotide ( Figure 17B , paired dashed lines, shown above Cascade2). Figure 17C A portion of the donor polynucleotide incorporated into the double-stranded cleavage site region is shown ( Figure 17C , pairs of dashed lines connecting pairs of black horizontal lines representing dsDNA). Incorporation of the donor polynucleotide is mediated by cellular DNA repair mechanisms (e.g., HDR) ( Figures 17B to 17C , pointing downward, vertical arrows represent cellular DNA repair mechanisms).

[0384] In other embodiments, a modified type I CRISPR-Cas effector complex comprising a guide complementary to a first nucleic acid target sequence in a polynucleotide and a first nuclease domain can be paired with a second component comprising a second nuclease domain, wherein the second component is capable of binding to a second nucleic acid target sequence of the polynucleotide. Examples of such second components include transcription activator-like effector nucleases (TALENs) comprising a second nuclease domain, zinc finger nucleases (ZFNs) comprising a second nuclease domain, or dCas9 / NATNA complexes comprising a second nuclease domain.

[0385] In one embodiment, a combination of a Cascade complex comprising a guide complementary to a first nucleic acid target sequence in a target polynucleotide and a dCas9 / NATNA complex can be used to delete a region of a target polynucleotide (e.g., gDNA), wherein NATNA comprises a spacer sequence complementary to a second nucleic acid target sequence in a target polynucleotide. The first and second nucleic acid target sequences are selected to be located on both sides of the nucleic acid target sequence targeted for deletion. The Cas3 protein comprising active endonuclease activity binds to the Cascade complex and then progressively deletes a single strand of dsDNA comprising a nucleic acid target sequence targeted for deletion. When the Cas3 protein collides with the dCas9 / NATNA complex (i.e., a "roadblock"), the Cas3 nuclease activity can be terminated at the second nucleic acid target sequence by the dCas9 / NATNA complex. Figures 21A-21DExamples of Cas3 deletions of nucleic acid target sequences are shown. Figure 21A A nucleic acid target sequence 1 ( Figure 21A , NATS1) and nucleic acid target sequence 2 ( Figure 21A , NATS2) dsDNA ( Figure 21A , paired horizontal black lines). Figure 21A The Cascade complex is shown, which contains a guide that complements NATS1 ( Figure 21A , Cascade; rectangle with black frame), Cas3 protein ( Figure 21A , Cas3; gray circle sector) and the dCas9 / NATNA complex containing a spacer complementary to NATS2 ( Figure 21A , dCas9; dashed frame rectangle). Figure 21B The binding of the Cascade complex to NATS1, the association of the Cas3 protein with the Cascade complex, and the binding of the dCas9 / NATNA complex to NATS2 are shown. Figure 21C Shown is progressive deletion by Cas3 targeting a single-stranded nucleic acid target sequence for deletion. Figure 21D The dissociation of Cas3 protein from dsDNA at the position of the dCas9 / NATNA complex bound to NATS2 is shown. The data shown in Examples 24A-24D support the use of protein roadblocks to control the length of deletions mediated by Cas3 proteins associated with the Cascade nucleoprotein complex; thereby providing a method for using Cas3 proteins associated with the Cascade nucleoprotein complex to promote the formation of deletions of defined length in gDNA of cells (e.g., human cells).

[0386] In another embodiment, a region of a target polynucleotide (e.g., gDNA) can be deleted using a combination of a first Cascade complex comprising a guide complementary to a first nucleic acid target sequence in a target polynucleotide and a second Cascade complex comprising a guide complementary to a second nucleic acid target sequence in a target polynucleotide. The first and second nucleic acid target sequences are selected to be located on both sides of the nucleic acid target sequence targeted for deletion. Each Cascade complex of a Cas3 protein containing active endonuclease activity binds and then progressively deletes the two chains of the nucleic acid target sequence targeted for deletion. When each Cas3 protein collides with a Cascade complex, the Cas3 nuclease activity can be terminated at the first and second nucleic acid target sequences of the Cascade complex. Figures 22A-22D Examples of Cas3 deletions of both strands of a nucleic acid target sequence are shown. Figure 22A A nucleic acid target sequence 1 ( Figure 22A, NATS1) and nucleic acid target sequence 2 ( Figure 22A , NATS2) dsDNA ( Figure 22A ; paired horizontal black lines). Figure 22A The first Cascade complex is shown, which contains a guide complementary to NATS1 ( Figure 22A , Cascade1; rectangle with black frame), Cas3 protein ( Figure 22A , Cas3; grey circle sector), and a second Cascade complex containing a guide complementary to NATS2 ( Figure 22A , Cascade2; rectangle with dashed frame). Figure 22B The binding of the Cascade complex to NATS1 and NATS2, as well as the association of the Cas3 protein with the Cascade complex, are shown. Figure 22C Progressive deletions resulting from movement along the DNA and nuclease degradation by Cas3 targeted to both strands of the nucleic acid target sequence for deletion are shown. Figure 22D The dissociation of the Cas3 protein from dsDNA is shown at the site of the Cascade complex bound to NATS1 and NATS2.

[0387] In other embodiments, the Cascade complex can be modified so that it cannot bind to the Cas3 protein, and such a modified Cascade complex can be substantially the same as Figures 21A-21D The same approach shown is used as a roadblock to prevent the progressive degradation of DNA by catalytically activating Cas3 associated with the Cascade RNP complex. Additional site-specific binding proteins (e.g., transcription activator-like effector (TAL) or zinc finger (ZnF) DNA binding proteins) can be used as roadblocks in a similar manner.

[0388] In some embodiments, the nucleic acid target sequence is dsDNA (e.g., genomic) DNA. In some embodiments, the nucleic acid target sequence is double-stranded, and one or both strands are cleaved. Such methods of cleaving nucleic acid target sequences can be performed in vitro, in vivo, or ex vivo.

[0389] As described above, in some embodiments, the present invention relates to introducing one or more modified type I CRISPR-Cas effector complexes into a host cell to promote the cleavage of a nucleic acid target sequence in dsDNA in the presence of a donor polynucleotide, wherein the one or more modified type I CRISPR-Cas effector complexes generate a cleavage site (or a cleavage site and an associated deletion) in a target region of a nucleic acid target sequence comprising host cell DNA, thereby facilitating the insertion of at least a portion of the donor polynucleotide into the target region. In some embodiments, the cleavage site is a double-strand break in the target region (e.g., when using two modified type I CRISPR-Cas effector complexes each comprising a spacer and a fusion protein comprising a Cas protein and an endonuclease (e.g., FokI) or two modified type I CRISPR-Cas effector complexes each comprising a spacer associated with a Cas3 protein or an mCas3 protein). In some embodiments, the cleavage site is a single-strand break in the target region (e.g., when using a type I CRISPR-Cas effector complex associated with an mCas3 protein). In other embodiments, the cleavage site is a deletion in the target region (e.g., when using a type I CRISPR-Cas effector complex associated with a Cas3 or mCas3 protein).

[0390] To demonstrate homology-directed repair (HDR), a minimal CRISPR array was designed to target the FokI-PseCascadeRNP complex to four sites in the human genome (WDR92, B2M, CCR5, and TRAC). Using three oligonucleotides (SEQ ID NO: 1513 to SEQ ID NO: 1515; Example 20A) and unique primers encoding a "repeat-spacer-repeat-spacer-repeat" sequence, a minimal CRISPR array was generated using PCR-based assembly, wherein the first and second spacers guide the FokI-PseCascadeRNP complex to adjacent nucleic acid target sequences to enable FokI dimerization and genomic cleavage (i.e., generating cleavage sites).

[0391] For each HDR insertion site in the target region containing the cleavage site—in this case overlapping the cleavage site—the cells were transfected with 3 μg of a vector encoding a FokI-PseCascade complex protein component comprising FokI fused to the N-terminus of Cas8 in which the NLS is linked to the N-terminus of FokI, 150 ng of a minimal CRISPR array, and 0-60 pmol of a single-stranded oligodeoxynucleotide (ssODN) template donor polynucleotide for HDR. The ssODN comprises homology arms, each of which is 75 nucleotides, and the two arms are symmetrically located around the cleavage site. The donor polynucleotide also comprises a phosphorothioate bond at the 3' end nucleotide of the homology arm to reduce or prevent cellular degradation of the donor polynucleotide. At the 5' end of the phosphorothioate bond, the donor polynucleotide also comprises an insertion sequence of "TAATAAT" to insert two stop codons and increase the spacing between the intergenic regions in the repaired chromosome, thereby hindering re-cutting by the FokI-PseCascade RNP complex.

[0392] Transfection was performed in HEK293 cells essentially as described in Example 20B, except that ssODN was included in the mixture to enable HDR. A few days after transfection, gDNA was purified from the cells, treated with an exonuclease to remove any residual ssODN that might contaminate subsequent PCRs, and then used as a template for amplification to measure donor insertions. Deep sequencing analysis was performed essentially as described in Example 20C. The percentage of mutant reads in the total reads from this experiment is shown in Table 13 (the first column is pmol of ssODN):

[0393]

[0394] The percentage of mutant reads indicates mutant reads containing indels resulting from nonhomologous end joining and insertion of the “TAATAAT” HDR sequence.

[0395] The percentage of HDR reads containing only the "TAATAAT" insertion sequence among the total mutant reads from this experiment is shown in Table 14 (first column is pmol of ssODN):

[0396]

[0397] As can be seen from the data, dsDNA cleavage by the Cascade RNP complex enables HDR and incorporation of donor polynucleotides at multiple loci in the human genome.

[0398] In yet another embodiment, the invention includes a method for modifying one or more nucleic acid target sequences in a polynucleotide (e.g., DNA) in a cell or biochemical reaction, comprising providing one or more engineered type I CRISPR-Cas effector complexes (e.g., comprising a Cas subunit protein-cytidine deaminase fusion protein) for introduction into the cell or biochemical reaction, and introducing the engineered type I CRISPR-Cas effector complex into the cell or biochemical reaction, thereby promoting contact of the engineered type I CRISPR-Cas effector complex with the polynucleotide, resulting in binding of the engineered type I CRISPR-Cas effector complex to the nucleic acid target sequence in a polynucleotide that favors mutations in the nucleic acid target sequence (e.g., C to T, G to A, A to G, and T to C). Figures 18A-18D The Cascade complex comprising a Cas subunit protein-linker polypeptide-cytidine deaminase fusion protein (Cascade / CD complex) is shown to mutate target nucleotides in cellular gDNA ( Figure 18A , paired black horizontal lines, "C" is cytosine and "G" is guanine). Figure 18A ; "Cascade" with a linker polypeptide is shown as a curved line connecting Cascade and cytidine deaminase, and "CD" is represented as a gray circular sector) is introduced into the cell. The Cascade / CD complex contains a guide ( Figure 18B , “C”). Figure 18B In the Cascade / CD complex, the Cascade / CD complex binds to the DNA target sequence, and cytidine deaminase converts cytosine ( Figure 18B , "C") into cytosine ( Figure 18C "U"). The cell's repair machinery can then repair the cytosine to thymine and change the mismatched guanidine to adenine ( Figure 18C-Figure 18D , pointing downward, vertical arrows represent cellular DNA repair mechanisms).

[0399] In another embodiment, the present invention includes methods for regulating in vitro or in vivo transcription, for example, transcription of a gene comprising a regulatory element sequence. Such methods include providing one or more modified type I CRISPR-Cas effector complexes (e.g., comprising Cas subunit protein-transcription factor fusion proteins) for introduction into a cell or biochemical reaction, and introducing the modified type I CRISPR-Cas effector complex into a cell or biochemical reaction, thereby promoting contact of the modified type I CRISPR-Cas effector complex with the regulatory element sequence, resulting in the modified type I CRISPR-Cas effector complex binding to the regulatory element sequence, thereby promoting regulation of the in vitro or in vivo transcription of a gene comprising a regulatory element sequence.

[0400] Figure 19A and Figure 19B A generalized diagram showing an example of transcriptional activation of a universal gene ("Gene 1"). Figure 19A Provides an overview of the transcriptional regulation of endogenous genes in eukaryotic cells. Figure 19A In the figure, two black parallel lines represent double-stranded DNA, indicating the location of gene 1 ( Figure 19A , gene 1), and the transcription start site associated with gene 1 ( Figure 19A , TSS). Figure 19A In the first figure, the transcription factor required for the transcriptional activation of gene 1 ( Figure 19A , TF) and polymerase II ( Figure 19A , Pol II) is shown as not yet associated with gene 1-TSS. The second figure shows the association of TF with its cognate TSS. TF then recruits transcriptional activator proteins ( Figure 19A , TP), which subsequently recruits RNA polymerase II ( Figure 19A , Pol II). Typically, in eukaryotes, TF factors and TP form a complex containing multiple proteins and possibly other molecules. The third figure shows the transcription of gene 1 by Pol II ( Figure 19A , the curved arrow at the end of gene 1 indicates the direction of transcription). This type of transcriptional activation often depends on TFs specific for gene expression. Figure 19B A schematic diagram of one embodiment of the present invention is shown, wherein the Cascade complex is engineered to include proteins or factors ( Figure 19B , CASCADEa), which attracts one or more components in the cell responsible for transcriptional activation (transcription activators; Figure 19B , TA). An example of such a protein or factor is the protein VP64. CASCADEa comprises a guide ( Figure 19B , TSS). Figure 19B In the figure, two black parallel lines represent double-stranded DNA, indicating the location of gene 1 ( Figure 19B , gene 1), and the transcription start site (TSS) associated with gene 1. Figure 19B In the first figure, CASCADEa and polymerase II ( Figure 19B , Pol II) is shown as not yet associated with gene 1-TSS. In the second figure, the association of CASCADEa with its target TSS is shown. CASCADEa then recruits transcriptional activator proteins ( Figure 19B , TA), which then recruits RNA polymerase II ( Figure 19B , Pol II). The third figure shows the transcription of gene 1 by Pol II ( Figure 19B, the curved arrow at the end of gene 1 indicates the direction of transcription). One advantage of this embodiment of the present invention is that transcriptional activation of a gene does not rely on endogenous transcription factors binding to the TSS of the gene, but rather can be targeted to the TSS of the gene by selecting an appropriate Cascade guide.

[0401] Figure 20A and Figure 20B The use of a protein containing a Cas subunit protein-KRAB domain fusion and a regulatory sequence associated with gene 1 ( Figure 20A , promoter) complementary guide ( Figure 20A , the Cascade complex with a linker polypeptide shown as a curved line connecting Cascade and a circular element representing the KRAB domain, for the universal gene ( Figure 20A , gene 1). Binding of CASCADEi to regulatory sequences ( Figure 20B ) leads to transcriptional repression of gene 1 ( Figure 20B , black lines ending in X represent transcriptional repression).

[0402] The modified type I CRISPR-Cas effector complexes as described herein can be incorporated into a kit. In some embodiments, the kit comprises packaging having one or more containers for holding the kit elements as one or more individual compositions or, optionally, as a mixture if the compatibility of the components permits. In some embodiments, the kit further comprises one or more of the following excipients: a buffer, a buffering agent, a salt, a sterile aqueous solution, a preservative, and combinations thereof. An exemplary kit may comprise one or more modified type I CRISPR-Cas effector complexes and one or more excipients, or one or more nucleic acid sequences encoding one or more components of the modified type I CRISPR-Cas effector complexes.

[0403] Additionally, the kit may include instructions for using the engineered type I CRISPR-Cas effector complex composition.

[0404] Another aspect of the present invention relates to a method for preparing or producing one or more modified type I CRISPR-Cas effector complexes or components thereof. In one embodiment, the preparation or production method comprises generating the modified type I CRISPR-Cas effector complex in a cell and purifying the modified type I CRISPR-Cas effector complex from a cell lysate.

[0405] The modified type I CRISPR-Cas effector complex composition may also include a detectable label, such as a portion that can provide a detectable signal. Examples of detectable labels include, but are not limited to, enzymes, radioisotopes, multiple specific binding pairs, fluorophores (FAM), fluorescent proteins (green fluorescent protein (GFP), red fluorescent protein, mCherry, tdTomato), DNA or RNA aptamers together with suitable fluorophores (enhanced GFP (eGFP), "Spinach"), quantum dots, antibodies, etc. A large number and variety of suitable detectable labels are well known to those of ordinary skill in the art.

[0406] In some embodiments, the engineered type I CRISPR-Cas effector complex (i.e., nucleoprotein particle) can be introduced into cells by methods including, but not limited to, nuclear transfection, gene gun delivery, sonication, cell squeezing, lipofection, or the use of other chemicals, cell-penetrating peptides, etc. In other embodiments, the coding sequences of one or more components of the engineered type I CRISPR-Cas effector complex and associated proteins can be introduced into cells using a vector system, an expression cassette comprising a DNA sequence encoding one or more components, and one or more RNA molecules (e.g., mRNA) comprising an expression cassette comprising an RNA sequence encoding one or more components.

[0407] One embodiment of the present invention relates to the use of a modified type I CRISPR-Cas effector complex to produce recombinant cells (e.g., modified lymphocytes). The method generally includes promoting the contact of dsDNA containing a target region containing a nucleic acid target sequence in a host cell with one or more modified first type I CRISPR-Cas effector complexes of the present invention. The contact of the first type I CRISPR-Cas effector complex of the modified nucleic acid target sequence causes the first type I CRISPR-Cas effector complex of the modified nucleic acid target sequence to bind to the target region containing the nucleic acid target sequence, cut the target region containing the nucleic acid target sequence, and modify the dsDNA in the target region, thereby producing a recombinant cell. In some embodiments, the dsDNA comprises more than one nucleic acid target sequence, and the first type I CRISPR-Cas effector complex of the modified spacer sequence comprising a complementary spacer sequence to each nucleic acid target sequence is used to bind, cut, and modify each nucleic acid target sequence. In some embodiments, the modification of the target region is an insertion, a deletion, or an insertion and a deletion. Methods of cleaving a nucleic acid target sequence in a polynucleotide (e.g., a single-stranded nick in dsDNA or a double-stranded nick in dsDNA) are described above, comprising providing one or more engineered type I CRISPR-Cas effector complexes for introduction into a cell.

[0408] Embodiments of the present invention include using one or more transformed first type I CRISPR-Cas effector complexes to produce recombinant cells, wherein the gDNA of recombinant cells includes knocking out mutations (for example, B2M genes and / or PDCD1 genes), knocking in (for example, the editor at TRAC sites and the integration of CAR from donor polynucleotides), or the above combination. In some embodiments, after the cutting at the nucleic acid target sequence in the TRAC gene of gDNA, at least a portion of the donor polynucleotides are incorporated at the nucleic acid target sequence. Donor polynucleotides can include a CAR construct, wherein CAR is inserted into the nucleic acid target sequence.

[0409] The recombinant cells prepared by the method of the present invention can be used for adoptive cell transfer (ACT). ACT is a rapidly emerging immunotherapy that uses transplanted immune cells to treat cancer. ACT is the transfer of cells into the patient. Most commonly, immune cells are derived from the immune system and are intended to improve immune function. In autologous cancer immunotherapy, immune cells or stem cells are harvested from the patient and amplified by ex vivo culture to a large number and then returned to the patient. Immune cells or stem cells can be modified in culture in various ways (for example, using genome editing to integrate CAR into the genome of T cells). In some embodiments, the lymphocytes used for modification are separated from the subject, modified, and then reintroduced into the same subject. This technology is called autologous lymphocyte therapy. In allogeneic cancer immunotherapy, immune cells or stem cells derived from a single donor's culture expansion can provide treatment for a large number of patients. Such immune cells or stem cells can also be modified in culture in various ways. In some embodiments, lymphocytes can be separated, modified, and introduced into different subjects. This technology is called allogeneic lymphocyte therapy.

[0410] In certain embodiments, this immunotherapy method can utilize lymphocytes, including but not limited to T cells, natural killer cells (NK cells), B cells, tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), T cell receptor modified T cells (TCR), TCR CAR-T cells, CAR TIL cells, CAR-NK cells, modified NK cells or hematopoietic stem cells that produce lymphocytes. In other embodiments, cells are stem cells, dendritic cells, etc. The genome of such cells can be modified (e.g., the generation of insertions and / or deletions in the lymphocyte genome) by using one or more modified class I type I Cascade effector complexes of the present invention.

[0411] Can be from object such as human subject, for example from blood or from solid tumor, as in the case of TILs, or from lymphoid organs such as thymus, bone marrow, lymph node and mucosa associated lymphoid tissue, separation of lymphocytes is used for modification.The technology of separation of lymphocytes is well known in the art.For example, can be from peripheral blood mononuclear cells (PBMCs) separation of lymphocytes, peripheral blood mononuclear cells can use for example ficoll, a kind of hydrophilic polysaccharide and density gradient centrifugation of separation blood layer to separate from whole blood.Usually, anticoagulation or defibrotized blood sample is layered on the top of ficoll solution, and centrifuged to form different cell layers.Bottom layer comprises red blood cells (erythrocytes), and they are collected or gathered by ficoll medium, and sink to bottom layer completely.Next layer mainly comprises granulocytes, and they also migrate downwards by ficoll-paque solution.Next layer comprises lymphocytes and monocytes and platelets, and lymphocytes are usually located at the interface between blood plasma and ficoll solution.In order to separate lymphocytes, reclaim this layer, wash with saline solution to remove platelets, ficoll and blood plasma, and then centrifuge again. Alternatively, the sample can be separated by centrifugation techniques (e.g., using Cells were isolated from donor blood using either a CAR T cell separation machine (Haemonetrics, Braintree, MA) or a Lovo automated cell processing system (Fresenius Kabi USA, LLC, Lake Zurich, IL).

[0412] Other techniques for isolating lymphocytes include biopanning, which separates a cell population from a solution by binding target cells to an antibody-coated plastic surface. Unwanted cells are then removed by treatment with specific antibodies and complement. Additionally, fluorescence-activated cell sorting (FACS) analysis can be used to detect and count lymphocytes. FACS analysis uses a flow cytometer that can separate labeled cells based on differences in light scattering and fluorescence.

[0413] For TILs, lymphocytes are isolated from the tumor and, for example, grown in high doses of IL-2 and selected using a cytokine release co-culture assay against autologous tumor or HLA-matched tumor cell lines. Cultures showing evidence of increased specific reactivity compared to allogeneic non-MHC-matched controls are selected for rapid expansion and then introduced into subjects to treat cancer (see, e.g., Rosenberg, S., et al., Clin. Cancer Res. 17: 4550-4557 (2011); Dudly, M., et al., Science 298: 850-854 (2002); Dudly, M., et al., J. Clin. Oncol. 26: 5233-5239 (2008); Dudley, M., et al., J. Immnother. 26: 332-342 (2003)).

[0414] After isolation, lymphocytes can be characterized based on specificity, frequency, and function. Commonly used assays include the ELISPOT assay, which measures the frequency of T cell responses.

[0415] In some embodiments, CD4+ and CD8+ T cells are isolated from donor peripheral blood mononuclear cells (PBMCs). One of ordinary skill in the art can isolate T cells or other lymphoid cells by various methods as described above. Such cells can also be isolated by differentiation from iPSC cells.

[0416] After separation, lymphocytes can be activated using techniques known in the art to promote proliferation and differentiation into specialized effector lymphocytes. Surface markers of activated T cells include, for example, CD3, CD4, CD8, PD1, IL2R, and others. Activated cytotoxic lymphocytes can kill target cells after binding to cognate receptors on the target cell surface. Surface markers of NK cells include, for example, CD16, CD56, and others.

[0417] After isolation and optional activation, the lymphocytes can be modified to provide the desired properties. One or more modified type I Cascade effector complexes of the present invention can be used to introduce genomic modifications, including but not limited to introducing coding sequences to be expressed and / or inactivating endogenous gene expression. In some embodiments, one or more modified type I Cascade effector complexes of the present invention can be used to edit the TRAC gene (encoding T cell receptor alpha constant), the B2M gene (encoding beta 2 microglobulin), and / or the PDCD1 gene (encoding programmed cell death protein 1; also known as PD-1).

[0418] T cells and NK cells are examples of lymphocytes that can be modified by the methods of the present invention. In some embodiments, one or more modified type I Cascade effector complexes of the present invention can introduce a cleavage site in the target region of a gene in the presence of a donor polynucleotide comprising a CAR, wherein the CAR is integrated into the target region of the lymphocyte genome. In other embodiments, one or more modified type I Cascade effector complexes of the present invention can be used to introduce a cleavage site in the target region of a gene to promote the generation of knock-out mutations to prevent the expression of a gene.

[0419] In another embodiment, the type I Cascade effector complexes of the transformation of the present invention can be used to introduce genome modification into human iPSCs. In some embodiments, the type I Cascade effector complexes of one or more transformations of the present invention can be used to edit TRAC genes, B2M genes and / or PDCD1 genes. In other embodiments, the type I Cascade effector complexes of the transformation can be used to introduce genome modification and coding sequences, such as CAR or cytokines (e.g., IL2, IL15, etc.), together with donor polynucleotides. The modified iPSC cells can then be further differentiated into mature cell types comprising T cells and NK cells or dendritic cells. In some embodiments, the modified iPSCs can be differentiated into CAR-T cells and CAR-NK cells.

[0420] In some embodiments of the method of the present invention, the donor polynucleotide includes a polynucleotide encoding CAR. CAR can be targeted to insert the target region of the gene (e.g., TRAC gene) comprising the cleavage site via homologous recombination ("knock-in"). An advantage of this method is that it can also provide the knock-out of the targeted TRAC gene; that is, to render the TRAC gene ineffective. The examples of the extracellular antigen recognition domains that can be incorporated into the CAR construct are described above (see Table 2). In one embodiment, the extracellular antigen recognition domain includes a CD19 binding portion (e.g., anti-CD19 scFv). In another embodiment, the extracellular antigen recognition domain includes a B cell maturation antigen (BCMA) binding portion (e.g., anti-BCMA scFv).

[0421] In embodiments of the present invention comprising methods for generating a cleavage site in a target region of DNA, the method may further comprise introducing a donor polynucleotide into the modified cell, thereby promoting insertion of at least a portion of the donor polynucleotide into the target region comprising the cleavage site of the modified cell. The donor polynucleotide may be introduced directly into the modified cell. In some embodiments, a vector is used to introduce the donor polynucleotide. General methods for constructing vectors are known in the art. Examples of viral vectors include, but are not limited to, lentiviruses, retroviruses, adenoviruses, herpes simplex virus I or II, parvoviruses, reticuloendotheliosis virus, and AAV vectors.

[0422] Other embodiments of the methods of the present invention include introducing a mutation in the B2M gene. In a preferred embodiment, the mutation is a knock-out mutation in the B2M gene.

[0423] Other embodiments of the methods of the present invention include introducing a mutation in the PDCD1 gene. In a preferred embodiment, the mutation is a knock-out mutation in the PDCD1 gene.

[0424] Genome modification facilitated by one or more engineered type I Cascade effector complexes of the invention can be performed by simultaneously or sequentially introducing an engineered Cascade complex, a polynucleotide (e.g., a plasmid or expression cassette), or a mixture thereof into a host cell (e.g., a lymphocyte).

[0425] After the modified lymphocytes are generated, the lymphocytes can be screened to select cells that express (e.g., express a desired cell surface receptor) or do not express (e.g., a cell surface protein whose expression has been inactivated by genome editing using one or more engineered type I Cascade effector complexes) using methods such as high-throughput screening techniques, including but not limited to FACS, microfluidics-based screening platforms, etc. These techniques are known in the art (see, e.g., Wojcik, M., et al., Int. J. Mol.. Sci. 16: 24918-24945 (2015)).

[0426] Once the modified lymphocytes are produced, they can be formulated into pharmaceutical compositions for delivery to the subject to be treated. The compositions of the present invention include modified lymphocytes and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobials, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerol, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, derivatized sugars such as sugar alcohols, aldonic acids, esterified sugars, and / or sugar polymers can be provided as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, etc. Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof. Refrigerants (e.g., (BioLife Solutions Inc, Bothell, WA) CS2, CS5, or CS10 freezing medium can be used to freeze cells for storage and shipping.

[0427] Pharmaceutical compositions of the present invention may also include an antimicrobial agent for preventing or stopping microbial growth. Non-limiting examples of antimicrobials suitable for use in the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0428] Antioxidants may also be present in the pharmaceutical composition. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of lymphocytes or other components in the formulation. Antioxidants suitable for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0429] Surfactants can be present as excipients. Exemplary surfactants include polysorbates such as TWEEN 20 and TWEEN 80, and pluronic compounds such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitol esters; lipids such as phospholipids, such as lecithin and other phosphatidylcholines, phosphatidylethanolamine (although preferably not in liposomal form), fatty acids, and fatty esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other such suitable cations.

[0430] Acid or alkali can exist as the excipient in the pharmaceutical composition. Non-limiting examples of operable acid include those selected from the following acids: hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid and above combinations. Examples of suitable alkalis include, but are not limited to, those selected from the following alkalis: sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate and above combinations.

[0431] The number of lymphocytes (or other recombinant cells) in the composition will vary depending on a variety of factors, but will optimally be a therapeutically effective dose when the composition is in unit dosage form or container (e.g., a bag). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition to determine which amount produces a clinically desired endpoint.

[0432] The amount of any single excipient in the composition will vary depending on the nature and function of the excipient and the specific needs of the composition. Generally, the optimal amount of any single excipient is determined by routine experimentation, i.e., by preparing compositions containing different amounts of the excipient (ranging from low to high), examining stability and other parameters, and then determining the range that achieves optimal performance without significant side effects. However, generally, the excipient is present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15% to about 95% by weight of the excipient, with concentrations of less than 30% by weight being most preferred. These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy," current edition, Williams & Williams; the "Physician's Desk Reference," current edition, Medical Economics, Montvale, NJ; and Kibbe, AH, Handbook of Pharmaceutical Excipients, current edition, American Pharmaceutical Association, Washington, DC.

[0433] The pharmaceutical composition may be contained in a syringe, implant device, or the like, depending on the intended mode of delivery and use. Preferably, the composition is present in an amount suitable for a single dose, in pre-measured or pre-packaged form.

[0434] The pharmaceutical composition herein may optionally include one or more additional agents, such as other drugs for treating the target cancer of the subject or treating the known side effects of the treatment. For example, T cells release cytokines into the blood, which may cause dangerous high fever and a sharp drop in blood pressure. This condition is called cytokine release syndrome (CRS). In many patients, CRS can be managed by standard supportive therapy, including steroids and immunotherapy, such as tocilizumab (ActemraTM, Genentech, South San Francisco, CA) that can block IL-6 activity.

[0435] At least one therapeutically effective cycle of treatment with the modified lymphocyte composition will be administered to the subject. By "a therapeutically effective cycle of treatment" is meant a treatment cycle that, when administered, results in a positive therapeutic response with respect to the treatment of the target disease in the individual. By "positive therapeutic response" is meant an individual treated according to the present invention exhibiting an improvement in one or more symptoms of the disease, including improvements such as tumor reduction and / or a reduced need for lymphocyte therapy.

[0436] In certain embodiments, the compositions comprising lymphocytes or other drugs of multiple therapeutic effective doses will be given. Compositions of the present invention, although not necessarily, are typically injected, such as subcutaneously, intradermally, intravenously, intra-arterially, intramuscularly, intraperitoneally, intramedullary, intratumorally, intranodally), by infusion or topical administration. Before being administered, pharmaceutical preparations can be in the form of liquid solutions or suspensions. The foregoing is intended to be exemplary, because other modes of administration are also contemplated. Pharmaceutical compositions can be given according to any medically acceptable method known in the art, using the same or different routes of administration.

[0437] The actual dosage administered will vary depending on the age, weight, and general condition of the subject and the severity of the condition being treated, the judgment of the healthcare professional, and the specific lymphocytes being administered. A therapeutically effective amount can be determined by one skilled in the art and will be adjusted to the specific requirements of each particular case.

[0438] Typically, a therapeutically effective amount of lymphocytes per patient will range from a total of about 1 x 10 5 to about 1x 10 10 lymphocytes or more, such as 1x 10 6 to about 1x 10 10 For example, 1x 10 7 to 1x 10 9 pcs, such as 5x 10 7 to 5x 10 8 Other dosage ranges may be 1 x 10 per kg / body weight.4 to 1x10 10 The total number of lymphocytes can be administered in a single bolus or in two or more divided doses, such as one or more days apart. The total number of lymphocytes can be administered in a single bolus or in two or more divided doses, such as one or more days apart. The amount of compound administered will depend on the potency of the particular lymphocyte composition, the disease being treated, and the route of administration.

[0439] Alternatively, the dose may include a mixture of lymphocytes, such as a mixture of CD8+ and CD4+ cells. If a mixture of CD8+ and CD4+ cells is provided, the ratio of CD8+ to CD4+ cells may be, for example, 1:1, 1:2, or 2:1, 1:3, or 3:1, 1:4, or 4:1, 1:5, or 5:1, etc.

[0440] The lymphocytes of modification can be administered before, at the same time or after other reagents. If provided simultaneously with other reagents, the lymphocytes of modification can be provided in the same or different compositions. Therefore, lymphocytes and other reagents can be provided to individuality by the mode of simultaneous treatment. By "simultaneous treatment", it is meant to give the object so that the therapeutic effect of the combination of substances is caused in the object being treated. For example, according to a specific dosing regimen, a pharmaceutical composition comprising a modified lymphocyte and a dose comprising at least one other reagent, such as a pharmaceutical composition of another chemotherapeutic agent, comprising a therapeutically effective dose can be given by giving a combination to achieve simultaneous treatment. Similarly, the lymphocytes of modification and therapeutic agents can be given in at least one therapeutic dose. The administration of a separate pharmaceutical composition can be carried out simultaneously or at different times (for example, continuously, in any order, on the same day or on different days), as long as the therapeutic effect of these combinations of substances is caused in the object being treated.

[0441] As described herein, the modified type I Cascade effector complex of the present invention provides a genome editing tool. Experiments demonstrating that the first type of CRISPR-Cas system has been functionally reconstructed in mammalian cells for genome editing show that such improved plasmid design can allow the use of other first type CRISPR-Cas systems, including those showing fewer protein components and unique PAM requirements, and possibly even RNA and DNA targeting effector complexes from type III CRISPR–Cas systems (see, e.g., Hille, F., et al., Cell 172: 1239-1259 (2018); Tamulaitis, G., et al., Trends Microbiol. 25: 49-61 (2017)). The multi-subunit nature of the Cascade complex provides the possibility of multivalent and / or spatially precise recruitment of effector fusions, such as synthetic transcription factors, epigenomic modifiers, and base editors. Alternatively, heterologous expression of the complete DNA interference pathway from the Type I system—i.e., Cascade-mediated recruitment of the Cas3 helicase-nuclease to the genomic target site—can be utilized to generate larger DNA deletions, to expose longer ssDNA strands for homology-directed repair, and / or to mechanically disrupt protein-DNA roadblocks at defined genomic sites. Thus, in one embodiment of the present invention, an engineered Type I CRISPR-Cas system can be used to generate a larger deletion region, and a donor polynucleotide (e.g., a more appropriate homology arm) can be introduced into the cell to facilitate insertion of at least a portion of the donor polynucleotide into the region.

[0442] Embodiments of the present invention include, but are not limited to, the following.

[0443] Embodiment 1. A composition comprising:

[0444] A first engineered first type I CRISPR-Cas effector complex comprising:

[0445] a first Cse2 subunit protein, a first Cas5 subunit protein, a first Cas6 subunit protein, and a first Cas7 subunit protein,

[0446] a first fusion protein comprising a first Cas8 subunit protein and a first FokI, wherein the N-terminus of the first Cas8 subunit protein or the C-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus or N-terminus of the first FokI, respectively, via a first linker polypeptide, and wherein the first linker polypeptide has a length of about 10 amino acids to about 40 amino acids, and

[0447] a first guide polynucleotide comprising a first spacer capable of binding to a first nucleic acid target sequence; and

[0448] A second engineered first type I CRISPR-Cas effector complex comprising:

[0449] a second Cse2 subunit protein, a second Cas5 subunit protein, a second Cas6 subunit protein, and a second Cas7 subunit protein,

[0450] a second fusion protein comprising a second Cas8 subunit protein and a second FokI, wherein the N-terminus of the second Cas8 subunit protein or the C-terminus of the second Cas8 subunit protein is covalently linked to the C-terminus or N-terminus of the second FokI, respectively, through a second linker polypeptide, and wherein the second linker polypeptide has a length of about 10 amino acids to about 40 amino acids, and

[0451] A second guide polynucleotide comprising a second spacer capable of binding to a second nucleic acid target sequence, wherein the protospacer adjacent motif (PAM) of the second nucleic acid target sequence and the PAM of the first nucleic acid target sequence have a spacer spacing of about 20 bp to about 42 bp.

[0452] Embodiment 2. The composition of embodiment 1, wherein the first linker polypeptide has a length of about 15 amino acids to about 30 amino acids.

[0453] Embodiment 3. The composition of embodiment 2, wherein the first linker polypeptide has a length of about 17 amino acids to about 20 amino acids.

[0454] Embodiment 4. The composition of any one of embodiments 1-3, wherein the second linker polypeptide has a length of about 15 amino acids to about 30 amino acids.

[0455] Embodiment 5. The composition of embodiment 4, wherein the second linker polypeptide has a length of about 17 amino acids to about 20 amino acids.

[0456] Embodiment 6. The composition of any preceding embodiment, wherein the lengths of the first linker polypeptide and the second linker polypeptide are the same.

[0457] Embodiment 7. The composition of any preceding embodiment, wherein each of the second nucleic acid target sequence and the first nucleic acid target sequence has a spacer spacing of about 22 bp to about 40 bp.

[0458] Embodiment 8. The composition of embodiment 7, wherein each of the second nucleic acid target sequence and the first nucleic acid target sequence has a spacer spacing of about 26 bp to about 36 bp.

[0459] Embodiment 9. The composition of embodiment 8, wherein each of the second nucleic acid target sequence and the first nucleic acid target sequence has a spacer spacing of about 29 bp to about 35 bp.

[0460] Embodiment 10. The composition of embodiment 9, wherein each of the second nucleic acid target sequence and the first nucleic acid target sequence has a spacer distance of about 30 bp to about 34 base bp.

[0461] Embodiment 11. The composition of any preceding embodiment, wherein the first FokI and the second FokI are monomeric subunits capable of associating to form a homodimer.

[0462] Embodiment 12. The composition of any one of embodiments 1-10, wherein the first FokI and the second FokI are different monomeric subunits capable of associating to form a heterodimer.

[0463] Embodiment 13. The composition of any preceding embodiment, wherein the N-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus of the first FokI via a first linker polypeptide.

[0464] Embodiment 14. The composition of any one of embodiments 1-12, wherein the C-terminus of the first Cas8 subunit protein is covalently linked to the N-terminus of the first FokI via a first linker polypeptide.

[0465] Embodiment 15. The composition of any preceding embodiment, wherein the N-terminus of the second Cas8 subunit protein is covalently linked to the C-terminus of the second FokI via a second linker polypeptide.

[0466] Embodiment 16. The composition of any one of embodiments 1-14, wherein the C-terminus of the second Cas8 subunit protein is covalently linked to the N-terminus of the second FokI via a second linker polypeptide.

[0467] Embodiment 17. The composition of any preceding embodiment, wherein each of the first Cas8 subunit protein and the second Cas8 subunit protein comprises the same amino acid sequence.

[0468] Embodiment 18. The composition of any preceding embodiment, wherein each of the first Cse2 subunit protein and the second Cse2 subunit protein comprises the same amino acid sequence, each of the first Cas5 subunit protein and the second Cas5 subunit protein comprises the same amino acid sequence, each of the first Cas6 subunit protein and the second Cas6 subunit protein comprises the same amino acid sequence, and each of the first Cas7 subunit protein and the second Cas7 subunit protein comprises the same amino acid sequence.

[0469] Embodiment 19. The composition of any preceding embodiment, wherein the first guide polynucleotide comprises RNA.

[0470] Embodiment 20. The composition of any preceding embodiment, wherein the second guide polynucleotide comprises RNA.

[0471] Embodiment 21. The composition of any preceding embodiment, wherein the genomic DNA comprises the PAM of the second nucleic acid target sequence and the PAM of the first nucleic acid target sequence.

[0472] Embodiment 22. A cell comprising: the composition of any preceding embodiment.

[0473] Embodiment 23. The cell of embodiment 22, wherein the genomic DNA of the cell comprises the PAM of the second nucleic acid target sequence and the PAM of the first nucleic acid target sequence.

[0474] Embodiment 24. The cell of embodiment 22 or 23, wherein the cell is a prokaryotic cell.

[0475] Embodiment 25. The cell of embodiment 22 or 23, wherein the cell is a eukaryotic cell.

[0476] Embodiment 26. One or more nucleic acid sequences encoding the first Cse2 subunit protein, the first Cas5 subunit protein, the first Cas6 subunit protein, the first Cas7 subunit protein, the first fusion protein and the first guide polynucleotide of any one of embodiments 1-21.

[0477] Embodiment 27. One or more nucleic acid sequences encoding the second Cse2 subunit protein, the second Cas5 subunit protein, the second Cas6 subunit protein, the second Cas7 subunit protein, the second fusion protein and the second guide polynucleotide of any one of embodiments 1-21.

[0478] Embodiment 28. One or more expression cassettes comprising one or more nucleic acid sequences of embodiment 26, embodiment 27, or both.

[0479] Embodiment 29. One or more vectors comprising one or more expression cassettes of embodiment 28.

[0480] Embodiment 30. A method of binding a polynucleotide comprising a first nucleic acid target sequence and a second nucleic acid target sequence, the method comprising:

[0481] Providing the composition of any one of embodiments 1-21 for introduction into a cell or biochemical reaction; and

[0482] The composition is introduced into a cell or biochemical reaction to promote contact of the first engineered first type I CRISPR-Cas effector complex with the first nucleic acid target sequence, and contact of the second engineered first type I CRISPR-Cas effector complex with the second nucleic acid target sequence, resulting in binding of the first engineered first type I CRISPR-Cas effector complex to the first nucleic acid target sequence, and binding of the second engineered first type I CRISPR-Cas effector complex to the second nucleic acid target sequence in the polynucleotide.

[0483] Embodiment 31. The method of embodiment 30, wherein the genomic DNA comprises polynucleotides.

[0484] Embodiment 32. A method of cleaving a polynucleotide comprising a first nucleic acid target sequence and a second nucleic acid target sequence, the method comprising:

[0485] Providing the composition of any one of embodiments 1-21 for introduction into a cell or biochemical reaction; and

[0486] The composition is introduced into a cell or biochemical reaction to promote contact of a first engineered first type I CRISPR-Cas effector complex with a first nucleic acid target sequence, and contact of a second engineered first type I CRISPR-Cas effector complex with a second nucleic acid target sequence, resulting in cleavage of the first nucleic acid target sequence by the first engineered first type I CRISPR-Cas effector complex, and cleavage of the second nucleic acid target sequence by the second engineered first type I CRISPR-Cas effector complex.

[0487] Embodiment 33. The method of embodiment 32, wherein the genomic DNA comprises polynucleotides.

[0488] Embodiment 34. A kit comprising: the composition of any one of embodiments 1-21; and a buffer.

[0489] Embodiment 35. A kit comprising: one or more nucleic acid sequences of embodiment 26, embodiment 27, or embodiment 26 and embodiment 27; and a buffer.

[0490] Embodiment 36. A composition comprising:

[0491] The first engineered type I CRISPR-Cas effector complex comprises:

[0492] Cse2 subunit protein, Cas5 subunit protein, Cas6 subunit protein and Cas7 subunit protein,

[0493] A first fusion protein comprising a Cas8 subunit protein and a first FokI, wherein the N-terminus of the first Cas8 subunit protein or the C-terminus of the first Cas8 subunit protein is covalently linked to the C-terminus or N-terminus of the first FokI through a first linker polypeptide, and

[0494] a guide polynucleotide comprising a spacer capable of binding to a nucleic acid target sequence; and

[0495] A second fusion protein comprising a modified first type I CRISPR-Cas3 fusion protein comprising a dCas3* protein and a second FokI, wherein the N-terminus of the dCas3* protein or the C-terminus of the dCas3* protein is covalently linked to the C-terminus or N-terminus of the second FokI, respectively, via a second linker polypeptide, and wherein the first linker polypeptide has a length of about 10 amino acids to about 40 amino acids, and the effector complex comprises,

[0496] Embodiment 37. The composition of embodiment 36, wherein the first linker polypeptide has a length of about 5 amino acids to about 40 amino acids.

[0497] Embodiment 38. The composition of embodiment 36, wherein the second linker polypeptide has a length of about 5 amino acids to about 40 amino acids.

[0498] Embodiment 39. A cell comprising: the composition of any one of embodiments 36-38.

[0499] Embodiment 40. The cell of embodiment 39, wherein the cell is a prokaryotic cell.

[0500] Embodiment 41. The cell of embodiment 39, wherein the cell is a eukaryotic cell.

[0501] Embodiment 42. One or more nucleic acid sequences encoding the Cse2 subunit protein, Cas5 subunit protein, Cas6 subunit protein, Cas7 subunit protein, first fusion protein and guide polynucleotide of any one of embodiments 36-38.

[0502] Embodiment 43. One or more nucleic acid sequences encoding the second fusion protein of any one of embodiments 36-38.

[0503] Embodiment 44. One or more expression cassettes comprising one or more nucleic acid sequences of embodiment 42, embodiment 43, or embodiment 42 and embodiment 43.

[0504] Embodiment 45. One or more vectors comprising one or more expression cassettes of embodiment 44.

[0505] Embodiment 46. A method of binding a polynucleotide comprising a nucleic acid target sequence, the method comprising:

[0506] Providing the composition of any one of embodiments 36-38 for introduction into a cell or biochemical reaction; and

[0507] The composition is introduced into a cell or biochemical reaction to promote contact of the engineered first type I CRISPR-Cas effector complex with the nucleic acid target sequence, and contact of the second fusion protein with the engineered first type I CRISPR-Cas effector complex, resulting in binding of the engineered first type I CRISPR-Cas effector complex and the second fusion protein to the nucleic acid target sequence in the polynucleotide.

[0508] Embodiment 47. The method of embodiment 46, wherein the genomic DNA comprises polynucleotides.

[0509] Embodiment 48. A method of cleaving a polynucleotide comprising a nucleic acid target sequence, the method comprising:

[0510] Providing the composition of any one of embodiments 36-38 for introduction into a cell or biochemical reaction; and

[0511] introducing the composition into a cell or biochemical reaction to promote contact of a first engineered first class I type CRISPR-Cas effector complex with a first nucleic acid target sequence and contact of a second engineered first class I type CRISPR-Cas effector complex with a second nucleic acid target sequence,

[0512] The composition is introduced into a cell or biochemical reaction to promote contact of the engineered first type I CRISPR-Cas effector complex with the nucleic acid target sequence, and contact of the second fusion protein with the engineered first type I CRISPR-Cas effector complex, resulting in cleavage of the nucleic acid target sequence by the engineered first type I CRISPR-Cas effector complex and the second fusion protein.

[0513] Embodiment 49. The method of embodiment 48, wherein the genomic DNA comprises polynucleotides.

[0514] Embodiment 50. A kit comprising: the composition of any one of embodiments 36-38; and a buffer.

[0515] Embodiment 51. A kit comprising one or more nucleic acid sequences of embodiment 42, embodiment 43, or embodiment 42 and embodiment 43; and a buffer.

[0516] Embodiment 52. An engineered type I CRISPR Cas3 mutant protein ("mCas3 protein") capable of reduced movement along DNA relative to a wild-type type I CRISPR Cas3 protein ("wtCas3 protein"), the mCas3 protein comprising:

[0517] about 95% or greater sequence identity to the corresponding wtCas3 protein,

[0518] A nuclear localization signal is covalently linked at the amino terminus, the carboxyl terminus, or both, and

[0519] One or more mutations capable of downregulating helicase activity, wherein the engineered type I CRISPR Cas3 mutant protein retains nuclease activity;

[0520] wherein the DNA is double-stranded DNA (dsDNA) comprising a target region comprising a nucleic acid target sequence;

[0521] wherein when the wtCas3 protein is associated with a corresponding Cascade nucleoprotein complex ("Cascade NP complex / wtCas3 protein"), and the Cascade NP complex comprises a guide comprising a spacer region complementary to a nucleic acid target sequence, binding of the Cascade NP complex / wtCas3 protein to the nucleic acid target sequence facilitates cleavage in the DNA target region, resulting in a deletion ("wtCas3-deletion"); and

[0522] The mCas3 protein, when associated with the Cascade NP complex ("Cascade NP complex / mCas3 protein") and bound to a nucleic acid target sequence, facilitates cleavage in the DNA target region, resulting in shorter deletions relative to wtCas3-deletions.

[0523] Embodiment 53. The mCas3 protein of embodiment 53, wherein the one or more mutations are amino acid substitutions.

[0524] Embodiment 54. The mCas3 protein of any preceding embodiment, wherein the one or more mutations are in the RecA1 region or the RecA2 region of the helicase domain.

[0525] Embodiment 55. The mCas3 protein of any preceding embodiment, wherein the one or more mutations downregulate the binding of the mCas3 protein to single-stranded DNA (ssDNA) relative to the wtCas3 protein.

[0526] Embodiment 56. The mCas3 protein of any preceding embodiment, wherein the one or more mutations downregulate the hydrolysis of adenosine triphosphate (ATP) by the mCas3 protein, or downregulate the binding of ATP to the mCas3 protein.

[0527] Embodiment 57. The mCas3 protein of any preceding embodiment, wherein the coding sequence of the mCas3 protein is covalently linked to the amino terminus or carboxyl terminus of the coding sequence of the Cas protein of the Cascade NP complex.

[0528] Embodiment 58. The mCas3 protein of any preceding embodiment, wherein the one or more mutations downregulate the binding of the mCas3 protein to single-stranded DNA (ssDNA) relative to the wtCas3 protein.

[0529] Embodiment 59. The mCas3 protein of any preceding embodiment, wherein the coding sequence of the mCas3 protein is covalently linked to the amino terminus or carboxyl terminus of the coding sequence of the Cas protein of the Cascade RNP complex.

[0530] Embodiment 60. The mCas3 protein of any preceding embodiment, wherein the Cas protein is selected from the group consisting of: Cse2, Cas8 protein, Cas7 protein, Cas6 protein, and Cas5 protein.

[0531] Embodiment 61. The mCas3 protein of any preceding embodiment, wherein the wtCas3 protein is an E. coli class 1 CRISPR Cas3 protein.

[0532] Embodiment 62. The mCas3 protein of embodiment 61, wherein the one or more mutations are selected from D452H, A602V, and D452H and A602V.

[0533] Embodiment 63. The mCas3 protein of any preceding embodiment, wherein the DNA is in a cell.

[0534] Embodiment 64. The mCas3 protein of embodiment 63, wherein the cell is a eukaryotic cell.

[0535] Embodiment 65. The mCas3 protein of Embodiment 64, wherein the eukaryotic cell is a mammalian cell (eg, a human cell).

[0536] Embodiment 66. One or more polynucleotides encoding the mCas3 protein of any one of embodiments 52-65.

[0537] Embodiment 67. A plasmid comprising a polynucleotide sequence encoding the mCas3 protein of any one of embodiments 52-65 operably linked to a regulatory sequence for expression in a mammalian cell.

[0538] Embodiment 68. One or more plasmids comprising a polynucleotide sequence encoding the mCas3 protein of any one of embodiments 52-65, and one or more polynucleotides encoding the protein components of the corresponding type I CRISPR Cascade operably linked to a regulatory sequence for expression in a mammalian cell.

[0539] Embodiment 69. The one or more plasmids of embodiment 68, further comprising a plasmid encoding one or more guide polynucleotides operably linked to regulatory sequences for expression in mammalian cells.

[0540] Embodiment 70. A type I CRISPR Cascade nucleoprotein complex comprising the mCas3 protein of any one of embodiments 52-65.

[0541] Embodiment 71. The type I CRISPR Cascade nucleoprotein complex of embodiment 70, wherein the nucleoprotein complex is an RNP.

[0542] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. From this specification and the Examples, those skilled in the art can determine the essential characteristics of the present invention and, without departing from the spirit and scope of the present invention, can change, substitute, alter and modify the present invention to adapt it to various uses and conditions. Such changes, substitutions, variations and modifications are also intended to fall within the scope of this disclosure.

[0543] Examples

[0544] Aspects of the present invention are shown in the following examples. Efforts have been made to ensure the accuracy of the numerals used (e.g., quantity, concentration, percentage change, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, temperatures are degrees Celsius and pressures are at or near atmospheric pressure. It should be understood that these examples are provided by way of example only and are not intended to limit the scope of the present invention.

[0545] Example 1

[0546] Computational design of polynucleotides encoding Cascade components

[0547] This example provides a description of the design of polynucleotide components encoding Cascade using genes, proteins, and CRISPR sequences derived from the Type IE CRISPR-Cas system.

[0548] Table 15 shows the polynucleotide DNA sequences encoding five Cascade type IE proteins, specifically from the E. coli strain K-12MG1655, and the amino acid sequences of the resulting protein components. The genomic sequence was obtained from NCBI reference sequence NZ_CP014225.1. In Table 15, the polynucleotide sequences were amplified from E. coli gDNA or were manufacturer-produced polynucleotides encoding Cascade protein components that were codon-optimized specifically for expression in E. coli and for expression in human cells.

[0549]

[0550] In addition, several fusion proteins comprising the Cascade protein were designed. Table 16 shows the polynucleotide DNA sequence of the gene encoding the Cascade protein fusion protein, as well as the amino acid sequence of the resulting protein component. In most cases, the fusion protein described in Table 16 includes a shorter three-amino acid linker connecting the two polypeptide sequences in the fusion construct; the linker typically comprises glycine-glycine-serine (GGS) or glycine-serine-glycine (GSG). The accurate three-amino acid linker sequence used in each specific fusion protein can be found in the full-length amino acid sequence in Table 16.

[0551]

[0552] When co-expressed with other Cascade proteins, His6 (hexahistidine; SEQ ID NO: 418) and Strep-tag on Cse2 protein TM II (GE Healthcare Bio-Sciences, Pittsburgh, PA) (SEQ ID NO: 419) peptide tag enables cleavage via nickel-nitriloacetic acid (Ni-NTA) resin or Strep-Tactin, respectively. TM (IBA GMBH LLC, The complex was purified using ELISA (Germany) resin. The HRV3C (human rhinovirus 3C) protease recognition sequence (SEQ ID NO: 420) is cleaved by HRV3C protease and can be used to remove N-terminal fusions from the target protein. The NLS (nuclear localization signal; SEQ ID NO: 421) peptide tag on the Cas6, Cas7, and / or Cas8 proteins enables nuclear transport in eukaryotic systems. The HA (hemagglutinin; SEQ ID NO: 422) peptide tag on the Cas6 or Cas7 protein enables detection of heterologous protein expression by Western blotting using an anti-HA antibody. The MBP (maltose binding protein; SEQ ID NO: 423) peptide fusion is a solubilization tag that facilitates purification of the Cas8 protein. The TEV (tobacco etch virus) protease ...

Claims

1. An engineered type I CRISPR Cas3 mutant protein ("mCas3 protein") capable of reduced movement along DNA relative to a wild-type type I CRISPR Cas3 protein ("wtCas3 protein"), the mCas3 protein comprising: The nuclear localization signal is covalently linked to the amino terminus, the carboxyl terminus, or both the amino terminus and the carboxyl terminus, and wherein the DNA is a double-stranded DNA (dsDNA) comprising a target region containing a nucleic acid target sequence; wherein the mCas3 protein is an Escherichia coli mCas3 protein selected from G317A mCas3, S318A mCas3, G319A mCas3, K320N mCas3, T321N mCas3, Q297E mCas3, D452E mCas3, E453N mCas3, R662A mCas3, R665Q mCas3, T346AmCas3, Q347N mCas3, G375A mCas3, K412G mCas3, T423A mCas3, D425H mCas3, Q426T mCas3, H601A mCas3, A602V mCas3, R603Q mCas3, R609S mCas3, T635A mCas3, Q636A mCas3 and Q640H mCas3, or is a mCas3 protein selected from D448A Pseudomonas sp. S-6-2 mCas3 protein of mCas3 and D448A D75A mCas3.

2. The mCas3 protein of claim 1, wherein the coding sequence of the mCas3 protein is covalently linked to the amino terminus or carboxyl terminus of the coding sequence of the Cas protein of the Cascade NP complex.

3. The mCas3 protein of claim 1, wherein the DNA is intracellular.

4. The mCas3 protein of claim 3, wherein the cell is a eukaryotic cell.

5. A type I CRISPR Cascade nucleoprotein complex comprising the mCas3 protein of any one of claims 1 to 4.

6. An engineered type I CRISPR-Cas effector composition comprising: Type I CRISPR-Cas subunit proteins; Type I guide polynucleotides; and A modified type I CRISPR mCas3 protein, which is the Pseudomonas S-6-2D448AmCas3 protein.

7. The modified type I CRISPR-Cas effector composition of claim 6, wherein the Pseudomonas S-6-2mCas3 protein comprises SEQ ID NO: 1919, or SEQ ID NO: 1921.

8. The modified type I CRISPR-Cas effector composition of claim 6, further comprising a linker polypeptide, which covalently links the modified type I CRISPR mCas3 protein to the type I CRISPR-Cas subunit protein.

9. The modified type I CRISPR-Cas effector composition of claim 6, wherein the type I CRISPR-Cas subunit protein is selected from Cas8 protein, Cas5 protein and Cas7 protein.

10. The engineered type I CRISPR-Cas effector composition of claim 9, further comprising a type I CRISPR Cas6 protein.

11. The modified type I CRISPR-Cas effector composition of claim 9, further comprising a type I CRISPR Cse2 protein.

12. A cell comprising the engineered type I CRISPR-Cas effector composition of any one of claims 6-11.

13. The cell of claim 12, further comprising: A second engineered type I CRISPR-Cas effector composition comprising: Type II CRISPR-Cas subunit protein; a second type I guide polynucleotide; and The second engineered type I CRISPR mCas3 protein is the Pseudomonas S-6-2D448A mCas3 protein.

14. The cell of claim 12, further comprising a donor polynucleotide.

15. The cell of claim 12, wherein the cell comprises a eukaryotic cell.

16. The cell of claim 15, wherein the eukaryotic cell comprises an induced pluripotent stem cell.

17. An in vitro or ex vivo method for nicking double-stranded DNA (dsDNA), comprising: contacting a first nicking site in a first target region in the dsDNA with a first engineered Type I CRISPR-Cas effector composition comprising: The first type I CRISPR-Cas subunit protein; a first Type I guide polynucleotide; and The first engineered type I CRISPR mCas3 protein is a Pseudomonas S-6-2D448A mCas3 protein; Thereby, the first modified type I CRISPR-Cas effector composition binds to the first target region, and the first modified type I CRISPR mCas3 protein binds to the dsDNA at the first nicking site and causes a nick therein.

18. The in vitro or ex vivo method of claim 17, further comprising: contacting a second nicking site in a second target region in the dsDNA with a second engineered Type I CRISPR-Cas effector composition, the second engineered Type I CRISPR-Cas effector composition comprising: Type II CRISPR-Cas subunit protein; a second type I guide polynucleotide; and a second engineered type I CRISPR mCas3 protein, which is a Pseudomonas S-6-2D448A mCas3 protein; Thereby, the second modified type I CRISPR-Cas effector composition binds to the second target region, and the second modified type I CRISPR mCas3 protein binds to the dsDNA at the second nicking site and causes a nick therein.

19. The in vitro or ex vivo method of claim 18, wherein the distance between the first nicking site and the second nicking site on the dsDNA is 1 to 120 base pairs.

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