RU2701662C9 - Crispr-cas system components, methods and compositions for sequence manipulation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention refers to biotechnology. Disclosed are compositions and systems containing vectors comprising polynucleotide sequences coding components of CRISPR-Cas type II complex, as well as a kit containing one of such compositions or systems.

EFFECT: present invention enables the change of genomic loci of the eukaryotic cell by using Cas9 nuclease fused with one or more nuclear localization sequences (NLS).

25 cl, 74 dwg, 16 ex

Description

Related applications and inclusion by reference

This application claims the priority of provisional patent applications US 61/736527, 61/748427, 61/768959, 61/791409 and 61/835931 C. general reference BI-2011/008 / WSGR, registry number 44063-701.101, BI-2011/008 / WSGR, registry number 44063-701.102, general reference BI-2011/008 / VP, registry number 44790.01.2003, BI -2011 / 008 / VP, registry number 44790.02.2003, and BI-2011/008 / VP, registry number 44790.03.2003, respectively, all of which are entitled "SYSTEMS, METHODS AND COMPOSITIONS FOR MANIPULATION WITH SEQUENCES" (SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION) filed December 12, 2012, January 2, 2013, February 25, 2013, March 15, 2013, and June 17, 2013, respectively.

Reference is made to provisional patent applications US 61/758468; 61/769046; 61/802174; 61/806375; 61/814263; 61/819803 and 61/828130, each of which is entitled "DESIGN AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR MANIPULATING WITH SEQUENCES" (ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE, January 30, 2013; February 25, 2013; March 15, 2013; March 28, 2013; April 20, 2013; May 6, 2013 and May 28, 2013, respectively. Reference is also made to provisional patent applications US 61/835936, 61/836127, 61/836101, 61/836080, 61/836123 and 61/835973, each of which was filed June 17, 2013. Reference is also made to the provisional patent application. US 61/842322 and US patent application 14/054414, each with a common reference BI-2011 / 008A, entitled "CRISPR-CAS SYSTEMS AND METHODS FOR CHANGING GENE PRODUCTS EXPRESSION" (CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS ), filed July 2, 2013 and October 15, 2013, respectively.

The aforementioned applications and all documents mentioned in them or during their paperwork (“documents referred to in an application”), and all documents mentioned or referred to in documents mentioned in an application, and all documents mentioned or referred to in this document ("documents referred to in this document"), and all documents referred to or referenced in the documents referred to in this document, along with any manufacturer's instructions, descriptions, product specifications and product descriptions for any application ucts mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference and can be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if it were specifically and separately indicated that each individual document is incorporated by reference.

Technical field

The present invention generally relates to systems, methods and compositions used to control gene expression, including targeted effects on the sequence, such as making changes to the genome or editing the gene, in which you can use vector systems that are close to short palindromic repeats, regularly arranged in groups (CRISPR), and their components.

Approval of Federally Funded Study

The present invention was developed with government support according to the NIH Pioneer Award DP1MH100706 issued by national health institutes. The government has certain rights to the present invention.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing technologies and analysis methods have greatly accelerated the ability to catalog and map genetic factors associated with a wide range of biological functions and diseases. Accurate targeted genome targeting technologies are needed to ensure systematic reverse engineering of casual genetic changes by enabling selective changes to individual genetic elements, as well as promoting applications in synthetic biology, biotechnology and medicine. Although genome editing technologies such as zinc finger domain constructor, transcriptional activator-like effectors (TALEs), or homing meganucleases are available for making changes to the target genome, there is still a need for new genome constructing technologies that are affordable, easy to implement, scalable and targeted for targeting several positions in the eukaryotic genome.

SUMMARY OF THE INVENTION

There is an urgent need for alternative and functional systems and technologies for targeted sequencing with a wide range of applications. The present invention satisfies this need and provides related benefits. CRISPR / Cas or the CRISPR-Cas system (both expressions are used interchangeably throughout this application) does not provide for the production of individualized proteins to target specific sequences, but rather one Cas enzyme can be programmed with a short RNA molecule to recognize a specific target DNA, in other words The Cas enzyme can bind to a specific target DNA using the indicated short RNA molecule. Adding a CRISPR-Cas system to the spectrum of genome sequencing technologies and analysis methods can greatly simplify the technique and speed up the ability to catalog and map genetic factors associated with a wide range of biological functions and diseases. In order to effectively use the CRISPR-Cas system for editing the genome without harmful effects, it is important to understand the design and optimization aspects of these tools for constructing the genome, which are aspects of the claimed invention.

In one aspect, the present invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises (a) a first regulatory element operably linked to a paired tracr sequence and one or more embedding sites to embed one or more guide sequences above the paired tracr sequence, where upon expression the guide sequence controls the sequence-specific binding of the complex CRISPR with the target sequence in a eukaryotic cell, where the CRISPR complex contains the CRISPR enzyme, forming a complex with (1) on ravlyaetsya sequence which hybridizes to the target sequence, and (2) tracr-pair sequence that hybridizes to tracr-sequence; and (b) a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; where components (a) and (b) are in the same or in different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence below the paired tracr sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, where upon expression each of two or more guide sequences controls sequence specific binding of the CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the system comprises a tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the implementation of the tracr sequence is characterized by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. Determining the optimal alignment is the responsibility of a person skilled in the art. For example, there are publicly and commercially available alignment algorithms and programs, such as, without limitation, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan. In some embodiments, the CRISPR complex contains one or more nuclear localization sequences that are effective enough to control the accumulation of the CRISPR complex in a detectable amount in the nucleus of the eukaryotic cell. Not wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for the activity of the CRISPR complex in eukaryotes, but that the inclusion of such sequences increases the activity of the system, especially with respect to targeting nucleic acid molecules in the nucleus. In some embodiments, the CRISPR enzyme is an enzyme of the type II CRISPR system. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 S. pneumoniae, S. pyogenes, or S. thermophilus, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the implementation of the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or from 10 to 30, or from 15 to 25, or from 15 to 20 nucleotides in length. In general and throughout this specification, the expression "vector" refers to a nucleic acid molecule capable of transferring another nucleic acid to which it has been linked. Vectors include, without limitation, nucleic acid molecules that are single-stranded, double-stranded or partially double-stranded; nucleic acid molecules that contain one or more free ends do not contain free ends (for example, ring); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid", which means a double stranded DNA loop in which additional DNA segments can be inserted, such as using standard molecular cloning techniques. Another type of vector is a viral vector, where DNA or RNA sequences obtained from a virus are present in a vector for packaging into a virus (for example, retroviruses, retroviruses with a defective replication system, adenoviruses, adenoviruses with a defective replication system and adeno-associated viruses). Viral vectors also include polynucleotides carried by viruses to transfect the host cell. Certain vectors are capable of self-regulating replication in the host cell into which they are introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and are thus replicated along with the host genome. Moreover, certain vectors are able to control the expression of genes with which they are functionally linked. Such vectors are referred to herein as “expression vectors”. Conventional expression vector vectors suitable for recombinant DNA technologies are often in the form of plasmids.

Recombinant expression vectors may contain the nucleic acid according to the present invention in a form suitable for expression of the nucleic acid in the host cell, which means that the recombinant expression vectors include one or more regulatory elements that can be selected taking into account the host cells to be used for expression that are operably linked to the nucleic acid sequence that is expected to be expressed. In the context of a recombinant expression vector, the expression “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element (s) in such a way as to allow expression of the nucleotide sequence (for example, in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

The expression "regulatory element" is intended to include promoters, enhancers, internal ribosome binding sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that control the constitutive expression of the nucleotide sequence in many types of host cells, and those that control the expression of the nucleotide sequence only in certain host cells (for example, tissue-specific regulatory sequences). A tissue-specific promoter can drive expression primarily in the target tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or certain types of cells (e.g., lymphocytes). Regulatory elements can also control expression in a time-dependent manner, such as, for example, a cell cycle-dependent or stage-dependent manner, which may or may not also be tissue specific or cell type specific. In some embodiments, the implementation of the vector contains one or more pol III promoters (for example, 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (for example, 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (for example, 1, 2, 3, 4, 5 or more pol I promoters), or combinations 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, the Rouse sarcoma virus (RSV) retroviral LTR promoter (optional with an RSV enhancer), the cytomegalovirus promoter (CMV) (optional with a CMV enhancer) [see, for example, Boshart et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, glycerophosphate kinase (PGK) promoter, and EF1α promoter. Also, the expression "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancers; R-U5 'segment in LTR HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466-472, 1988); enhancer SV40; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981). Those skilled in the art will understand that the structure of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like. The vector can be introduced into host cells, thereby obtaining transcripts, proteins or peptides, including fusion proteins or peptides encoded by nucleic acids, as described herein (for example, transcripts of short palindromic repeats arranged regularly in groups (CRISPR ), proteins, enzymes, their mutant forms, their fusion proteins, etc.).

Preferred vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected to target specific types of cells.

In one aspect, the present invention provides a vector comprising a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme containing one or more nuclear localization sequences. In some embodiments, said regulatory element controls the transcription of the CRISPR enzyme in a eukaryotic cell, such that the CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the CRISPR enzyme is an enzyme of the type II CRISPR system. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 S. pneumoniae, S. pyogenes, or S. thermophilus, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

In one aspect, the present invention provides a CRISPR enzyme comprising one or more nuclear localization sequences effective enough to control the accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is an enzyme of the type II CRISPR system. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 S. pneumoniae, S. pyogenes, or S. thermophilus, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme lacks the ability to cleave one or more strands of the target sequence to which it binds.

In one aspect, the present invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a paired tracr sequence and one or more insertion sites for embedding one or more guide sequences above the paired tracr sequence, where, upon expression, the guide sequence controls the sequence-specific binding of the CRISPR complex to the target sequence in a eukaryotic cell, where the CRISPR complex is holds the CRISPR enzyme, complexing with (1) a guide sequence that hybridizes to the target sequence, and (2) a paired tracr sequence that hybridizes to the tracr sequence; and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the host cell contains components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into the genome of a eukaryotic host cell. In some embodiments, component (a) further comprises a tracr sequence below the paired tracr sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, where upon expression each of two or more guide sequences controls sequence specific binding of the CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the eukaryotic host cell further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the implementation of the tracr sequence is characterized by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. In some embodiments, the CRISPR enzyme contains one or more nuclear localization sequences that are effective enough to control the accumulation of said CRISPR enzyme in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the CRISPR enzyme is an enzyme of the type II CRISPR system. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 S. pneumoniae, S. pyogenes, or S. thermophilus, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the implementation of the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or from 10 to 30, or from 15 to 25, or from 15 to 20 nucleotides in length. In one aspect, the present invention provides a non-human eukaryotic organism, preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. In other aspects, the present invention provides a eukaryotic organism, preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. The organism in some embodiments of these aspects may be an animal, for example, a mammal. Also, the body can be an arthropod, such as an insect. The organism may also be a plant. In addition, the body may be a mushroom.

In one aspect, the present invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a paired tracr sequence and one or more embedding sites to embed one or more guide sequences above the paired tracr sequence, where when expressed, the guide sequence controls the sequence-specific binding a CRISPR complex with a target sequence in a eukaryotic cell, where the CRISPR complex contains a CRISPR enzyme that forms a comp CEN (1) a guide sequence which hybridizes to the target sequence, and (2) tracr-pair sequence that hybridizes to tracr-sequence; and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the implementation of the kit contains components (a) and (b) located in the same or in different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence below the paired tracr sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, where upon expression each of two or more guide sequences controls sequence specific binding of the CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the implementation of the tracr sequence is characterized by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. In some embodiments, the CRISPR enzyme contains one or more nuclear localization sequences that are effective enough to control the accumulation of said CRISPR enzyme in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the CRISPR enzyme is an enzyme of the type II CRISPR system. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Cas9 S. pneumoniae, S. pyogenes, or S. thermophilus, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the implementation of the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or from 10 to 30, or from 15 to 25, or from 15 to 20 nucleotides in length.

In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method includes providing binding of the CRISPR complex to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex contains a CRISPR enzyme that is complexed with a guide sequence that hybridizes to the target sequence in the specified target polynucleotide, where the specified guide sequence is associated with a paired tracr sequence, which, in turn, iziruetsya with tracr-sequence. In some embodiments, said cleavage comprises cleaving one or two strands at a specific point in the target sequence with said CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair leads to a mutation comprising the insertion, deletion or replacement of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions in a protein expressed from a gene containing the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein one or more vectors control the expression of one or more of: a CRISPR enzyme, a guide sequence linked to a paired tracr sequence, and a tracr sequence. In some embodiments, the vectors are delivered to a eukaryotic cell in a subject. In some embodiments, said modification takes place in said eukaryotic cell in cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modification. In some embodiments, the method further comprises returning said eukaryotic cell and / or cells derived from a subject to said subject.

In one aspect, the present invention provides a method for modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises providing binding of the CRISPR complex to a polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; where the CRISPR complex contains the CRISPR enzyme, which forms a complex with a guide sequence that hybridizes with the target sequence in the specified target polynucleotide, where the specified guide sequence is associated with a paired tracr sequence, which, in turn, hybridizes with the tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein one or more vectors control the expression of one or more of: a CRISPR enzyme, a guide sequence linked to a paired tracr sequence, and a tracr sequence.

In one aspect, the present invention provides a method for producing a model eukaryotic cell containing a mutated gene responsible for the development of a disease. In some embodiments, the gene responsible for the development of the disease is any gene associated with an increased risk of the presence or development of the disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein one or more vectors control the expression of one or more of: a CRISPR enzyme, a guide sequence linked to a paired tracr sequence, and a tracr sequence; and (b) providing binding of the CRISPR complex to the target polynucleotide to effect cleavage of the target polynucleotide in the specified gene responsible for the development of the disease, where the CRISPR complex contains the CRISPR enzyme, complexing with (1) a guide sequence that hybridizes with the target sequence in the target polynucleotide, and (2) a paired tracr sequence that hybridizes to the tracr sequence, thereby obtaining a model eukaryotic cell containing the mutated gene, the response for the development of venous disease. In some embodiments, the implementation of the cleavage includes the cleavage of one or two strands at a certain point in the target sequence indicated by the CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair leads to a mutation comprising the insertion, deletion or replacement of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions when expressing a protein from a gene containing a target sequence.

In one aspect, the present invention provides a method for producing a biologically active agent that modulates a cell signaling process associated with a gene responsible for the development of a disease. In some embodiments, the gene responsible for the development of the disease is any gene associated with an increased risk of the presence or development of the disease. In some embodiments, the method comprises (a) bringing the test compound into contact with a model cell according to any one of the described embodiments, and (b) detecting a reading change that indicates a decrease or increase in the signaling process in the cell associated with the mutation in the specified gene responsible for the development of the disease, thereby obtaining the specified biologically active agent that modulates the specified process of signal transmission in the cell, asso iirovanny with this gene, responsible for the development of the disease.

In one aspect, the present invention provides a recombinant polynucleotide containing a guide sequence above a paired tracr sequence, where the guide sequence upon expression controls sequence specific binding of the CRISPR complex to the corresponding target sequence present in the eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. In some embodiments, the target sequence is a proto-oncogen or oncogene.

In one aspect, the present invention provides a method for selecting one or more prokaryotic cells by introducing one or more mutations into a gene in one or more prokaryotic cells, the method comprising introducing one or more vectors into prokaryotic cell (s), (s) where one or several vectors control the expression of one or more of: the CRISPR enzyme, a guide sequence associated with a paired tracr sequence, a tracr sequence, and an editing matrix; where the editing matrix contains one or more mutations that stop the cleavage of the CRISPR enzyme; ensuring homologous recombination of the editing matrix with the target polynucleotide in the selected cell (s); providing binding of the CRISPR complex to the target polynucleotide to carry out the cleavage of the target polynucleotide in the specified gene, where the CRISPR complex contains the CRISPR enzyme, which forms a complex with (1) a guide sequence that hybridizes with the target sequence in the target polynucleotide, and (2) a paired tracr sequence, which hybridizes with the tracr sequence, where the binding of the CRISPR complex to the target polynucleotide induces cell death, thereby ensuring the selection of one or more okaryotic cells into which one or more mutations have been introduced. In a preferred embodiment, the CRISPR enzyme is Cas9. In another aspect of the present invention, the selected cell may be a eukaryotic cell. Aspects of the present invention provide the ability to select specific cells without the need for a selection marker or a two-step method, which may include a negative selection system.

Accordingly, it is not an object of the present invention to encompass within the scope of the present invention any previously known product, a method for producing a product, or a method for using the product, so that applicants reserve the right and hereby disclose a waiver of any previously known product, process or method. It should be further noted that the present invention is not intended to cover, within the scope of the present invention, any product, method of producing a product, or method of using a product that does not meet the written description and the requirements of sufficient disclosure of the USPTO invention (first paragraph of § 112 of Article 35 of the USC) or EPO ( Article 83 of the EPC), so that the applicants reserve the right and hereby disclose the waiver of rights to any previously described product, method of obtaining the product or method of using the product.

It should be noted that in this disclosure and especially in the claims and / or paragraphs, expressions such as “contains”, “contained”, “comprising”, etc., may have the meaning ascribed to them in US patent law, for example, they can mean “includes,” “included,” “including,” and the like, and that expressions such as “consisting essentially of” and “consists essentially of” have the meanings ascribed to them in the patent US law, for example, they allow elements not explicitly stated, but exclude elements that are known smoother art or that affect the basic and novel characteristics of the present invention. These and other embodiments are disclosed or are apparent from and encompassed by the following detailed description.

Brief description of graphic materials

New features of the present invention set forth with characteristic features in the attached claims. A better understanding of the features and advantages of the present invention will be available through reference to the following detailed description, which sets forth representative embodiments in which the principles of the present invention are used, and to the accompanying drawings.

Figure 1 shows a schematic model of a CRISPR system. Cas9 nuclease from Streptococcus pyogenes (yellow) targets genomic DNA using a synthetic RNA guide (sgRNA) consisting of a 20-nucleotide guide sequence (blue) and scaffold (red). The guide sequence forms base pairs with the target DNA (cyan) immediately above the desired motif adjacent to the proto-spacer (PAM; magenta) 5'-NGG, and Cas9 mediates double-stranded cleavage (DSB) by ~ 3 bp. above RAM (red triangle).

Figures 2A-F depict a representative CRISPR system, a possible mechanism of action, illustrative adaptation for expression in eukaryotic cells, and test results evaluating nuclear localization and CRISPR activity.

3 shows an exemplary expression cassette for the expression of CRISPR system elements in eukaryotic cells, predicted structures of illustrative guide sequences and CRISPR system activity as measured in eukaryotic and prokaryotic cells.

In figures 4A-D shows the results of the assessment of the specificity of SpCas9 in relation to an illustrative target.

Figures 5A-G depict an exemplary vector system and the results of its use in controlling homologous recombination in eukaryotic cells.

Figure 6 presents a table of protospacer sequences and summarizes the results regarding the effectiveness of modifications for target protospacer developed based on illustrative CRISPR systems of S. pyogenes and S. thermophilics with corresponding PAM to loci in the human and mouse genomes. Cells were transfected with Cas9 and either pre-crRNA / tracrRNA or chimeric RNA and analyzed 72 hours after transfection. The percentage of insertions / deletions was calculated based on the results of the analysis using Surveyor with the indicated cell lines (N = 3 for all target protospacer, the errors are standard errors of the mean, ND means "not detected by analysis using the Surveyor", and NT means " not tested in this study ").

Figures 7A-C show a comparison of various tracrRNA transcripts for Cas9-mediated targeted gene targeting.

Figure 8 shows a schematic representation of a Surveyor nuclease assay for detecting double-stranded break microinserts and microdeletions.

Figures 9A-B show representative bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells.

The figure 10 shows the analysis of interference during transformation with a bacterial plasmid, expression cassettes and plasmids used in it, and indicators of the efficiency of transformation of cells used in it.

Figures 11A-C show histograms of the distances between adjacent PAM (NGG) of locus 1 of S. pyogenes SF370 (Figure 10A) and PAM (NNAGAAW) of locus 2 of LMD9 S. thermophilus (Figure 10B) in the human genome and the distance for each RAM in the chromosomes (Chr) (Figure 10C).

Figures 12A-C depict a representative CRISPR system, illustrative adaptation for expression in eukaryotic cells, and test results evaluating CRISPR activity.

Figures 13A-C illustrate illustrative manipulations with the CRISPR system to target genome loci in mammalian cells.

Figures 14A-B show the results of a Northern blot analysis of crRNA processing in mammalian cells.

The figure 15 shows an illustrative selection of protospacer in the PVALB loci of the human and Th mouse.

Figure 16 shows an illustrative protospacer and corresponding target PAM sequences of the S. thermophilus CRISPR system at the human EMX1 locus.

17 shows a sequence table for primers and probes used for Surveyor, RFLP, genomic sequencing, and Northern blot analyzes.

Figures 18A-C show illustrative manipulation of the CRISPR system with chimeric RNAs and the results of analyzes using SURVEYOR regarding the activity of the system in eukaryotic cells.

Figures 19A-B are graphical representations of SURVEYOR assay results for CRISPR system activity in eukaryotic cells.

The figure 20 presents a representative display of some Cas9 target sites of S. pyogenes in the human genome, obtained using the genomic browser UCSC.

Figure 21 shows the predicted secondary structures for representative chimeric RNAs containing a guide sequence, a paired tracr sequence and a tracr sequence.

22 illustrates exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells.

Figure 23 shows that the activity of Cas9 nuclease with respect to endogenous targets can be used to edit the genome, (a) The concept of editing the genome using the CRISPR system. The targeted CRISPR construct controlled the cleavage of the chromosomal locus and was co-transformed with an editing matrix that recombined with the target to prevent cleavage. Kanamycin-resistant transformants that withstood the effects of CRISPR contained modifications introduced using an editing matrix, tracr, transactivating CRISPR RNA; aphA-3, cantamycin resistance gene. (b) Transformation of crR6M DNA in R6 ^8232.5 cells without an editing matrix, srtA R6 wild-type, or R6370.1 editing matrices. Recombination of srtA with either R6 or R6 ^370.1 prevented cleavage with Cas9. Transformation efficiency was calculated as colony forming units (cfu) per μg of crR6M DNA; mean values are shown with standard deviations from at least three independent experiments. PCR analysis was performed on 8 clones at each transformation. "Un." mean unedited srtA locus of strain R6 ^8232.5 ; "Ed." shows the editing matrix. Targets R6 ^8232.5 and R6 ^370.1 differ in EaeI restriction.

24 shows an analysis of PAM and seed sequences that preclude Cas9 cleavage. (a) PCR products with randomized RAM sequences or randomized seed sequences transformed crR6 cells. These cells expressed Cas9 loaded with crRNA, which was targeted to the chromosome region of R6 ^8232.5 cells (highlighted in pink), missing from the R6 genome. More than 2 × 105 chloramphenicol-resistant transformants carrying inactive PAM or seed sequences were combined for amplification and deep sequencing of the target site. (b) Relative fraction of the number of read fragments after transformation with random PAM constructs of crR6 cells (compared to the number of read fragments in R6 transformants). The relative prevalence of each 3-nucleotide sequence of PAM is shown. Extremely underrepresented sequences (NGGs) are shown in red; partially underrepresented in orange (NAG), (c) Relative fraction of the number of read fragments after transformation of random seed sequences of crR6 cells with constructs (compared to the number of read fragments in R6 transformants). The relative prevalence of each nucleotide at each position of the first 20 nucleotides of the protospacer sequence is shown. High prevalence indicates the absence of Cas9 cleavage, i.e. CRISPR inactivating mutation. The gray line shows the level of the WT sequence. The dashed line represents the level above which the mutation significantly disrupts cleavage (see "deep sequencing data analysis" section in Example 5)

Figure 25 shows the introduction of single and multiple mutations using the CRISPR system in S. pneumoniae, (a) Nucleotide and amino acid sequences of wild-type bgaA and edited bgaA (green nucleotides; underlined amino acid residues). The protospacer, PAM, and restriction sites are shown. (b) Transformation efficiency of cells transformed with constructs for targeted exposure in the presence of an editing or control matrix, (c) PCR analysis of 8 transformants of each editing experiment, followed by cleavage of BtgZI (R → A) and TseI (NE → AA). The bgaA deletion was identified as a smaller PCR product, (d) Miller analysis to measure β-galactosidase activity of WT strains and edited, (f) For a one-step, double deletion, the design for targeted exposure contained two spacers (in this case, matching srtA and bgaA ), and it was co-transformed with two different editing matrices, (f) PCR analysis of 8 transformants to detect deletions at the srtA and bgaA loci. 6/8 transformants contained deletions of both genes.

The figure 26 presents the mechanisms underlying the editing using the CRISPR system. (a) A stop codon was introduced into the ermAM erythromycin resistance gene to create the JEN53 strain. The wild-type sequence can be restored by targeting the stop codon with the CRISPR :: ermAM (stop) construct and using the wild-type ermAM sequence as the editing matrix. (b) The ermAM sequences are mutant and wild-type, (c) The erythromycin-resistant (erm ^R ) CFU fraction calculated from total or kanamycin-resistant (kan ^R ) CFU. (d) A fraction of the total number of cells that received both the CRISPR construct and the editing matrix. Cotransformation with the CRISPR construct for targeted exposure provided more transformants (t-test, p = 0.011). In all cases, the values show the mean ± standard deviation for three independent experiments.

Figure 27 illustrates the editing of the genome by the CRISPR system in E. coli. (a) A kanamycin resistance plasmid carrying the CRISPR array (pCRISPR), targeting the gene for editing, can transform the engineered strain HME63 containing the chloramphenicol resistance plasmid carrying cas9 and tracr (pCas9), together with the mutation-determining oligonucleotide. (b) A K42T mutation providing streptomycin resistance was introduced into the rpsL gene. (c) Streptomycin-resistant (strep ^R ) CFU fraction was calculated by total or kanamycin-resistant (kan ^R ) CFU. (d) The fraction of all cells that received both the plasmid pCRISPR and the edited oligonucleotide. Cotransformation with pCRISPR plasmid for targeted exposure provided more transformants (t-test, p = 0.004). In all cases, the values show the mean ± standard deviation for three independent experiments.

Figure 28 shows that the transformation of the genomic DNA of crR6 leads to the editing of the locus of targeted exposure, (a) Element IS1167 R6 S. pneumoniae was replaced by the locus CRISPR01 SF370 S. pyogenes to create a strain of crR6. This locus encodes a six-spacer CRISPR array for Cas9 nuclease, tracrRNA, necessary for crRNA biogenesis, and Cas1, Cas2, and Csn2 proteins, which are not necessary for targeted exposure. Strain crR6M contains a minimal functional CRISPR system without cas1, cas2 and csn2. The aphA-3 gene encodes resistance to cantamycin. The protospacers of streptococcal bacteriophages φ8232.5 and φ370.1 hybridized with the chloramphenicol resistance gene (cat) and integrated into the srtA gene of strain R6 to create strains R68232.5 and R6370.1. (b) Left panel: transformation of the genomic DNA of crR6 and crR6M into R6 ^8232.5 and R ^6370.1 . As a control, cell competencies were also transformed with the streptomycin resistance gene. Right panel: PCR analysis of 8 transformants of R6 ^8232.5 with genomic DNA crR6. For PCR, primers were used to amplify the srtA locus. In 7/8 genotyped colonies, the srtA locus R68232.5 was replaced by a WT locus from crR6 genomic DNA.

The figure 29 presents the chromatograms of the DNA sequences of the edited cells obtained in this study. In all cases, wild-type sequences and mutant protospacer and PAM sequences (or their reverse complementary strand) were detected. If necessary, an amino acid sequence encoded by the protospacer is provided. For each editing experiment, all strains were sequenced for which PCR and restriction analysis confirmed the introduction of the desired modification. A representative chromatogram is shown. (a) Chromatogram of the introduction of the PAM mutation in the target R6 ^8232.5 (figure 23d). (b) Chromatograms of the introduction of mutations R> A and NE> AA into the β-galactosidase gene (bgaA) (Figure 25c). (c) Chromatogram of the introduction of a 6664 bp deletion in ORF bgaA (figures 25c and 25f). The dashed line indicates the boundaries of the deletion. (d) Chromatogram of the introduction of a 729 bp deletion in ORF srtA (Figure 25f). The dashed line indicates the boundaries of the deletion. (e) Chromatograms of the formation of a premature stop codon in ermAM (Figure 33). (f) Editing rpsL in E. coli (Figure 27).

Figure 30 illustrates CRISPR resistance to random S. pneumoniae targets containing various PAMs. (a) Position of 10 random targets in the R6 genome of S. pneumoniae. Selected targets contain different PAMs and are located on both strands. (b) Spacers corresponding to the targets were cloned into the minimal CRISPR array of plasmid pLZ12 and transformed with them the crR6Rc strain, which provides processing and targeted mechanism in transformants, (c) Transformation efficiency of various plasmids of the R6 and crR6Rc strains. No colonies were identified for the transformation of pDB99-108 (T1-T10) into crR6Rc. The dashed line represents the detection limit of the analysis.

The figure 31 presents the General scheme of targeted editing of the genome. To ensure targeted editing of the genome, crR6M was additionally designed to contain tracrRNA, Cas9 and only one repeat of the CRISPR array, followed by a cantamycin resistance marker (aphA-3), creating a crR6Rk strain. The DNA of this strain was used as a template for PCR with primers designed to introduce a new spacer (green block, designated N). PCRs with left and right primers were combined using the Gibson method to create a construct for targeted exposure. The constructs for both targeted exposure and editing then transformed the crR6Rc strain, which is the crR6Rk equivalent strain but contains a cantamycin resistance marker replaced by a chloramphenicol resistance marker (cat). About 90% of kanamycin-resistant transformants contained the desired mutation.

Figure 32 illustrates the distribution of distances between PAMs. NGG and CCN, which are considered valid PAM. Data are presented for the S. pneumoniae genome R6, as well as for random sequences of the same length and with the same GC content (39.7%). The dashed line represents the average distance (12) between the PAM in the R6 genome.

Figure 33 illustrates CRISPR-mediated editing of the ermAM locus using genomic DNA as a construct for targeted exposure. To use genomic DNA as a construct for targeted exposure, autoimmune activity on CRISPR must be avoided, and thus a spacer corresponding to a sequence not present on the chromosome (in this case, ermAM erythromycin resistance gene) should be used. (a) Nucleotide and amino acid sequences of the wild-type and mutated ermAM gene (red letters). The protospacer and PAM sequences are shown. (b) Schematic representation of CRISPR-mediated editing of the ermAM locus using genomic DNA. A construct carrying an ermAM-targeted spacer (blue block) was obtained by PCR and assembly using the Gibson method, and the crR6Rc strain was transformed by it, creating the JEN37 strain. The JEN37 genomic DNA was used as a construct for targeted exposure, and together with the editing matrix, it co-transformed JEN38, a strain in which the srtA gene is replaced by a wild-type ermAM copy. Kanamycin-resistant transformants contain the edited genotype (JEN43). (c) The number of kanamycin-resistant cells was obtained after cotransformation with targeted exposure and editing matrices or control matrices. In the presence of a control matrix, 5.4 × 10 ³ CFU / ml and 4.3 × 10 ⁵ CFU / ml were obtained using the editing matrix. This difference indicates an approximately 99% editing efficiency [(4.3 × 10 ⁵ −5.4 × 10 ³ ) / 4.3 × 10 ⁵ ]. (d) To check for edited cells, seven kanamycin-resistant clones and JEN38 were streaked onto plates with agar medium with erythromycin (erm +) or without (erm-) erythromycin. Only a positive control displayed erythromycin resistance. The mutated ermAM genotype of one of these transformants was also confirmed by DNA sequencing (Figure 29e).

Figure 34 illustrates sequential introduction of mutations by CRISPR-mediated genome editing, (a) Schematic illustration of sequential introduction of mutations by CRISPR-mediated genome editing. First of all, R6 was designed to create crR6Rk. crR6Rk was co-transformed with a construct for targeted exposure to srtA hybridized with cat to select edited cells for chloramphenicol, together with an editing construct with ΔsrtA deletion within the reading frame. The strain ΔsrtA crR6 was obtained by selection by chloramphenicol. The ΔsrtA strain was then co-transformed with a construct for targeted exposure to bgaA hybridized with aphA-3 for selection of edited cells by kanamycin, and an editing construct containing a ΔbgaA deletion within the reading frame. Finally, the constructed CRISPR locus can be removed from the chromosome by first cotransforming R6 DNA containing the wild-type IS1167 locus and a plasmid carrying the bgaA protospacer (pDB97), as well as by spectinomycin selection. (b) PCR analysis of 8 chloramphenicol resistant (Cam) transformants to detect deletion at the srtA locus. (c) β-galactosidase activity, as measured by Miller’s assay. In S. pneumoniae, this enzyme is attached to the cell wall by sortase A. Deletion of the srtA gene is manifested in the release of β-galactosidase into the supernatant. The ΔbgaA mutants are not active, (d) PCR analysis of 8 spectinomycin-resistant (Spec) transformants to detect wild-type CRISPR IS1167 locus substitution.

Figure 35 shows the background of the CRISPR mutation frequency in S. pneumoniae. (a) Transformation of JEN53 constructs for targeted impact of CRISPR :: ∅ or CRISPR :: erm (stop) with or without an ermAM editing matrix. The difference in kan ^R CFU between CRISPR :: ∅ and CRISPR :: erm (stop) indicates that Cas9 cleavage destroys unedited cells. Mutants avoiding CRISPR interference in the absence of an editing matrix were observed at a frequency of 3 × 10 ⁻³ . (b) PCR analysis of the CRISPR locus of escaping cells indicates that 7/8 contain a spacer deletion. (c) The cell that escaped exposure No. 2 carries a point mutation in cas9.

Figure 36 shows that important elements of locus 1 of CRISPR S. pyogenes were reproduced in E. coli using pCas9. The plasmid contained tracrRNA, Cas9, as well as a leader sequence driving the crRNA array. The pCRISPR plasmids contained only the leader sequence and array. Spacers can be inserted into the crRNA array between BsaI sites using annealed oligonucleotides. The structure of the oligonucleotide is shown below. pCas9 is resistant to chloramphenicol (CmR) and is based on the backbone of the low copy plasmid pACYC184. pCRISPR is based on a large number of copies of the plasmid pZE21. Two plasmids were required because the pCRISPR plasmid containing the spacer targeted on the E. coli chromosome cannot be constructed using this organism as a host for cloning if Cas9 is also present (this will result in death of the host).

The figure 37 shows the editing in MG1655 E. coli running CRISPR. An oligonucleotide (W542) carrying a point mutation, which provides resistance to streptomycin, and eliminates the immune activity of CRISPR, together with a plasmid that specifically targets rpsL (pCRISPR :: rpsL), or a control plasmid (pCRISPR :: ∅) wild-type strain was co-transformed MG1655 E. coli containing pCas9. Transformants were selected on a medium containing either streptomycin or kanamycin. The dashed line indicates the detection limit of the transformation analysis.

Figure 38 shows the background frequency of the CRISPR mutation in E. coli HME63. (a) Transformation of competent HME63 cells with plasmids pCRISPR :: ∅ or pCRISPR :: rpsL. Mutants that escaped CRISPR interference were observed with a frequency of 2.6 × 10 ^-4 . (b) Amplification of the CRISPR array of cells that escaped exposure showed that the spacer was removed in 8/8.

Figure 39A-D shows a circular image of a phylogenetic analysis revealing five Cas9 families, including three groups of large Cas9 (~ 1400 amino acids) and two small Cas9 (~ 1100 amino acids).

40A-F shows a linear display of a phylogenetic analysis revealing five Cas9 families, including three large Cas9 groups (~ 1400 amino acids) and two small Cas9 (~ 1100 amino acids) groups.

Figure 41A-M shows sequences where mutation points are located in the SpCas9 gene.

Figure 42 shows a schematic representation of a construct in which a transcription activation domain (VP64) is fused to Cas9 with two mutations in the catalytic domains (D10 and H840).

Figure 43A-D shows genome editing through homologous recombination, (a) Schematic representation of SpCas9 nickase with D10A mutation in the catalytic domain of RuvC I. (b) Schematic representation of homologous recombination (HR) at the human EMX1 locus using sense or antisense single-stranded oligonucleotides as matrices for repair. The red arrow at the top indicates the cleavage site for sgRNA; PCR primers for genotyping (tables J and K) are indicated by arrows in the right panel, (c) The sequence of the site modified by HR. d, Analysis of insertions / deletions at target locus 1 of EMX1 (n = 3) mediated by wild-type SpCas9 (wt) and SpCas9 nickase (D10A) using SURVEYOR. The arrows indicate the position of the fragments of the expected size.

44A-B show single vector structures for SpCas9.

Figure 45 shows a quantitative analysis of the cleavage of the constructs NLS-Csn1, Csn1, Csn1-NLS, NLS-Csn1-NLS, NLS-Csn1-GFP-NLS and UnTFN.

The figure 46 shows the frequency index NLS-Cas9, Cas9, Cas9-NLS and NLS-Cas9-NLS.

Figure 47 presents a gel analysis demonstrating that SpCas9 with nickase mutations (alone) does not induce double-stranded breaks.

Figure 48 shows the structure of the oligonucleotide DNA used as the homologous recombination (HR) matrix in this experiment, and a comparison of HR efficiency induced by various combinations of Cas9 protein and HR matrix.

49A shows a vector map for targeted exposure with condition-dependent expression of Cas9, Rosa26.

49B shows a vector map for targeted exposure with constitutive expression of Cas9, Rosa26.

Figure 50A-H shows the sequences for each element present on the vector maps of figures 49A-B.

Figure 51 shows a schematic representation of important elements in the constructs for constitutive and condition-dependent expression of Cas9.

The figure 52 shows the confirmation of the function of the constructs for constitutive and dependent on the conditions of expression of Cas9.

Figure 53 shows confirmation of the nuclease activity of Cas9 using a Surveyor.

The figure 54 shows a quantitative analysis of the nuclease activity of Cas9. Figure 55 shows the structure of the structure and the strategy of homologous recombination (HR).

Figure 56 shows the results of PCR genotyping of the genome of constructs for constant (right) and condition-dependent (left) expression at two different exposure times (upper row for 3 minutes and lower row for 1 minute).

Figure 57 shows Cas9 activation in mESC.

Figure 58 shows a schematic representation of the strategy used to mediate gene knockout via NHEJ using the nickel variant of Cas9 in conjunction with two RNA guides.

Figure 59 shows how DNA double-strand breakage (DSB) repair facilitates gene editing. In a path of error-prone non-homologous end-junction (NHEJ), the ends of the DSB are processed by endogenous DNA repair mechanisms and joined together, which can lead to random mutations like insertion or deletion (insertion / deletion) at the junction. Insertion / deletion mutations that occur in the coding region of a gene can cause a reading frame shift and the appearance of a premature stop codon, which leads to gene knockout. Alternatively, a repair matrix in the form of a plasmid or single-stranded oligodeoxynucleotides (ssODN) can be provided to efficiently use a homologous recombination (HDR) repair pathway, which provides high quality and accurate editing.

Figure 60 shows a timeline and a general description of the experiments. The stages of development, creation, confirmation of the reagent and the propagation of the cell line. Individual sgRNAs (light blue labels) for each target, as well as genotyping primers, were constructed in silico using an online construction tool (available at genome-engineering.org/tools). SgRNA expression vectors were then cloned into a plasmid containing Cas9 (PX330) and verified by DNA sequencing. Prepared plasmids (pCRISPR) and optional repair matrices to facilitate homologous targeted repair were then transfected with cells and analyzed for the ability to mediate targeted cleavage. And finally, it is possible to carry out clonal propagation of transfected cells to obtain isogenic cell lines with certain mutations.

Figure 61A-C shows the selection of the target and preparation of the reagents, (a) For Cas9 S. pyogenes, for targets of 20 bp (highlighted in blue) should be 5'-NGG, which can be found in any strand of genomic DNA. It is recommended that you use the online tool described in this protocol to help you select a target (www.genome-engineering.org/tools). (b) Schematic representation of the co-transfection of the expression plasmid Cas9 (PX165) and PCR-amplified U6 expression cassette sgRNA. Using an U6 promoter containing a PCR matrix and a fixed forward primer (U6 Fwd), sgRNA-coding DNAs can be attached to the reverse primer U6 (U6 Rev) and synthesized as an extended DNA oligonucleotide (Ultramer oligos from IDT). It should be noted that the guide sequence (blue N) in U6 Rev is the back-complementary flanking target sequence of 5'-NGG. (c) Schematic representation of the scarless cloning of the oligonucleotide guide sequence into a plasmid containing Cas9 and sgRNA framework (PX330). Guide oligonucleotides (blue N) contain sticky ends for ligation into a pair of BbsI sites in PS330 with orientation to the upper and lower strands matching the given genome targets (i.e., the upper oligonucleotide is 20 bp in front of the 5'-NGG sequence in genomic DNA). The cleavage of PX330 BbsI allows for the substitution of type IIs restriction sites (blue outline) with direct insertion of annealed oligonucleotides. It is worth noting that an additional G was placed in front of the first base of the guide sequence. Applicants have found that the additional G in front of the guide sequence does not adversely affect the effectiveness of targeted exposure. In cases where the selected 20-nt guide sequence does not start with guanine, additional guanine will efficiently transcribe sgRNA using the U6 promoter, for which guanine is preferred in the first base of the transcript.

62A-D show the expected multiplex NHEJ results. (a) Schematic representation of the analysis using SURVEYOR used to determine the percentage of insertions / deletions. First of all, genomic DNA from a heterogeneous population of cells with targeted Cas9 was amplified by PCR. The amplicons were then slowly annealed to create heteroduplexes. Re-annealed heteroduplexes were digested with SURVEYOR nuclease, while homoduplexes remained intact. The efficiency of Cas9-mediated cleavage (% insert / deletion) was calculated based on the fraction of cleaved DNA, which was determined using the integrated intensity of the bands in the gel. (b) Two sgRNAs (orange and blue labels) were designed to target human GRIN2B and DYRK1A loci. SURVEYOR gel analysis showed modifications at both loci in transfected cells. Colored arrows indicated the expected fragment sizes for each locus. (c) A pair of sgRNA (light blue and green tags) was designed to excon exon (dark blue) at the human EMX1 locus. Target sequences and PAM (red) are shown in corresponding colors, and cleavage sites are marked with a red triangle. The predicted junction is shown below. Individual clones were isolated from cell populations transfected with sgRNA, 3, 4, or both were also analyzed by PCR (OUT Fwd, OUT Rev), displaying a deletion of ~ 270 bp Typical clones without modification (12/23), with mono-allelic (10/23) and biallelic (1/23) modifications are shown. IN Fwd and IN Rev primers were used to display inversion events (Fig. 6d). (d) Quantification of clonal lines with deletion in exon EMX1. Two sgRNA pairs (sgRNA 3.1, 3.2 flanking on the left; sgRNA 4.1, 4.2 flanking on the right) were used to mediate deletions of various sizes near exon EMX1. Transfected cells were cloned and propagated for genotyping analysis by deletion and inversion events. Of the 105 clones that were screened, 51 (49%) and 11 (10%) carried heterozygous and homozygous deletions, respectively. The approximate sizes of the deletions are given, since the junction may vary.

63A-C illustrate the use of ssODN and targeted exposure vector to mediate HR with both wild-type Cas9 and Cas9 mutant nickase in HEK293FT and HUES9 cells with efficiencies ranging from 1.0 to 27%.

The figure 64 shows a schematic representation of a PCR-based method for fast and effective targeted exposure to CRISPR in mammalian cells. A plasmid containing the human U6 RNA polymerase III promoter was amplified using PCR using a U6 specific forward primer and a reverse primer carrying the reverse complementary strand of the U6 promoter, sgRNA scaffold (+85) with a guide sequence and 7 T nucleotides for termination transcription. The resulting PCR product was purified and delivered together with a plasmid carrying Cas9 driven by the CBh promoter.

Figure 65 shows the results of using the Transgenomics SURVEYOR mutation detection kit for each RNA guide and the corresponding controls. A positive analysis result using SURVEYOR represents one large band corresponding to genomic PCR and two smaller bands, which are products of SURVEYOR nuclease, which produces a double-stranded break at the mutation site. Each RNA guide was evaluated in a mouse cell line, Neuro-N2a, using liposomal unstable cotransfection of hSpCas9. 72 hours after transfection, genomic DNA was purified using Epicentre QuickExtract DNA. PCR was performed to amplify the locus of interest.

Figure 66 shows the results of a Surveyor analysis for 38 live calves (lanes 1-38), 1 dead calf (lane 39), and 1 wild-type calf for comparison (lane 40). Cubs 1-19 were injected with gdNA Chd8.2, and pups 20-38 were injected with gRNA Chd8.3. Of the 38 live pups, 13 were positive for mutation. One dead calf also had a mutation. No mutation was detected in the wild-type sample. PCR genome sequencing was consistent with analysis using SURVEYOR.

Figure 67 shows the structure of various Cas9-NLS designs. All Cas9s were a human codon-optimized version of Sp Cas9. NLS sequences are linked to the cas9 gene at either the N-terminus or the C-terminus. All Cas9 variants with different NLS structures were cloned into a vector containing the backbone, so that it was driven by the EF1a promoter. In the same vector, there was a chimeric RNA that deliberately acts on the human EMX1 locus, driven by the U6 promoter, which forms a two-component system with it.

Figure 68 shows the genome cleavage efficiency induced by Cas9 variants carrying various NLS structures. The percentage indicates the portion of the human genomic EMX1 DNA that was digested by each construct. All experiments were obtained from 3 biological replicates. n = 3, error means standard error of the mean.

69A shows the structure of CRISPR-TF (transcription factor) having a transcription activation function. Chimeric RNA is expressed using the U6 promoter, and the codon-optimized variant of the Cas9 double mutant protein (hSpCas9m), functionally linked to the three-component NLS and VP64 functional domain, is expressed using the EF1a promoter. The double mutations, D10A and H840A, make the Cas9 protein incapable of introducing any cleavage, but support its ability to bind to the target DNA in the direction of chimeric RNA.

69B shows the activation of transcription of the human SOX2 gene by the CRISPR-TF system (chimeric RNA and Cas9-NLS-VP64 fusion protein). 293FT cells were transfected with plasmids carrying two components: (1) U6-driven different chimeric RNAs that specifically target 20 bp sequences. at or near the locus of the human genome SOX2, and (2) driven by EF1 hSpCas9m (double mutant), an NLS-VP64 fusion protein. 96 hours after transfection, 293FT cells were harvested and the level of activation was measured by induction of mRNA expression using qRT-PCR analysis. All expression levels are normalized compared to the control group (earring column), which represents the results of cells transfected with a CRISPR-TF plasmid without a chimeric RNA. QRT-PCR probes were used to detect SOX2 mRNA in the analysis of Taqman Human gene expression (Life Technologies). The data of all experiments of 3 biological replicates, n = 3, are presented; error bars show the standard error of the mean.

Figure 70 shows the optimization of the NLS structure for SpCas9.

71 shows a quantile-quantile diagram for NGGNN sequences.

Figure 72 shows a histogram of data density with a matched normal distribution (black line) and a quantile of 0.99 (dashed line).

Figure 73A-C shows RNA-directed repression of bgaA expression using dgRNA :: cas9 **. a. The Cas9 protein binds to tracrRNA and the CRISPR RNA precursor. which is processed by RNase III to form crRNA. crRNA controls the binding of Cas9 to the bgaA promoter and represses transcription. b. The targets used to direct Cas9 ** to the bgaA promoter are presented. Estimated codons -35, -10, as well as the starting bgaA don donor, are shown in bold, c. Beta-galactosidase activity as a measure in Miller’s analysis in the absence of targeted exposure and for four different targets.

Figure 74A-E shows a characteristic of Cas9 ** mediated repression, a. The gfpmut2 gene and its promoter, including the -35 and -10 signals, are presented together with the position of the various target sites used in this study. b. Relative fluorescence with a targeted effect on the coding thread, p. Relative fluorescence with a targeted effect on a non-coding thread. d. Northern blot with probes B477 and B478 of RNA extracted from T5, T10, B10 or control strain without target, e. The effect of increasing the number of mutations at the 5'-end of crRNA B1, T5 and B10.

The figures in this document are for illustrative purposes only and do not need to be drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

The expressions "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They denote the polymeric form of nucleotides of any length, both deoxyribonucleotides and ribonucleotides or their analogues. Polynucleotides can have any spatial structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are: coding or non-coding regions of a gene or gene fragment, loci (locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transport RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), miRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic probes islot and primers. A polynucleotide may contain one or more modified nucleotides, such as, for example, methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after polymer assembly. The nucleotide sequence may be interrupted by components other than nucleotides. The polynucleotide can be further modified after polymerization, such as, for example, by conjugation with a labeling component.

In aspects of the present invention, the expressions “chimeric RNA”, “chimeric RNA guide”, “RNA guide”, “single RNA guide” and “synthetic RNA guide” are used interchangeably and denote a polynucleotide sequence containing a guide sequence, a tracr sequence and a pair tracr sequence. The expression "guide sequence" means a sequence of approximately 20 bp within the RNA guide that defines the target site, and it can be used interchangeably with the terms “guide” or “spacer”. The expression “paired tracr sequence” can also be used interchangeably with the expression “direct repeat (s)”.

Used in this document, the expression "wild type" is an expression from this field, understandable to a person skilled in the art, and means a typical form of an organism, strain, gene or characteristics that are found in nature in contrast to mutant or variant forms.

Used in this document, the expression "option" should be understood as meaning a manifestation of qualities that are characterized by a pattern that differs from that found in nature.

The expression "not found in nature" or "constructed" is used interchangeably, and it indicates human intervention. Expressions, when they relate to nucleic acid molecules or polypeptides, mean that the nucleic acid molecule or polypeptide at least practically does not contain at least one distinct component with which they are naturally associated in nature and found in nature.

“Complementarity” means the ability of a nucleic acid to form hydrogen (s) linkage (s) with another nucleic acid sequence using either the traditional Watson-Crick base pairing or other non-traditional types. The percent complementarity shows the percentage of residues in the nucleic acid molecule that can form hydrogen bonds (for example, the formation of Watson-Crick pairs) with the second nucleic acid sequence (for example, 5, 6, 7, 8, 9, 10 of 10 will be 50%, 60%, 70%, 80%, 90% and 100% complementary). “Exact complementarity” means that all adjacent residues of the nucleic acid sequence will be hydrogen bonded to the same number of adjacent residues in the second nucleic acid sequence. The expression “substantially complementary” as used herein means a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% within the area of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 , 45, 50 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” with respect to hybridization mean conditions under which a nucleic acid that is complementary to the target sequence preferentially hybridizes to the target sequence and practically does not hybridize to non-target sequences. Severe conditions, as a rule, are sequence dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to the target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.

"Hybridization" means a reaction in which one or more polynucleotides react to form a complex that is stabilized by the formation of hydrogen bonds between the bases of the nucleotide residues. The formation of hydrogen bonds can occur according to the principle of pairing according to Watson-Crick, the Hugstein binding, or in any other sequence-specific manner. The complex may contain two strands forming a duplex structure, three or more strands forming a multi-strand complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction may be a step in a broader method, such as an initial PCR step or cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is called a "complementary sequence" of this sequence.

As used herein, the expression “expression” means a process in which a polynucleotide is transcribed from a DNA template (such as an mRNA or other RNA transcript), and / or a method by which a transcribed mRNA is further translated into peptides, polypeptides or squirrels. Transcripts and encoded polypeptides can collectively be called a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing mRNA in a eukaryotic cell.

The expressions "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and its structure may be interrupted by components other than amino acids. Expressions also encompass a polymer of amino acids that has been modified; for example, the formation of disulfide bonds, glycosylation, lipidization, acetylation, phosphorylation or any other manipulation, such as the connection with the component for labeling. As used herein, the expression “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both D- and L-optical isomers, amino acid analogs, and peptidomimetics.

The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, without limitation, mice, monkeys, humans, farm animals, animals for sport, and domestic animals. Also covered are tissues, cells and their offspring of a biological organism obtained in vivo or cultured in vitro.

The terms “therapeutic agent”, “therapeutic agent” or “treatment agent” are used interchangeably, and they mean a molecule or compound that has some beneficial effect when administered to a subject. Beneficial effects include the implementation of diagnostic determinations; relief of a disease, symptom, disorder or pathological condition; weakening or preventing the onset of a disease, symptom, disorder or condition; as well as general resistance to disease, symptom, disorder or pathological condition.

As used herein, the terms “treatment,” or “treatment” or “temporary relaxation” or “relief” are used interchangeably. These expressions mean an approach for obtaining favorable or desirable results, including, without limitation, therapeutic effect and / or prophylactic effect. The therapeutic effect is understood to mean any therapeutically significant improvement or effect in relation to one or more diseases, conditions or symptoms in the treatment. For the prophylactic effect, the compositions can be administered to a subject at risk of developing a particular disease, condition or symptom, or to a subject who reports one or more physiological symptoms of the disease, even if the disease, condition or symptom may not yet have manifested.

The expression “effective amount” or “therapeutically effective amount” means an amount of an agent that is sufficient to provide favorable or desired results. A therapeutically effective amount may vary depending on one or more of: the subject and the disease state to be treated, the weight and age of the subject, the severity of the disease state, route of administration and the like, which one skilled in the art can easily determine. The expression also applies to the dose by which an image can be obtained for determination by any one of the visualization methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosage regimen to be followed, whether it is administered in combination with other agents, the timing of administration, the visualized tissue, and the physical delivery system it is enclosed in.

The practical application of the present invention provides, unless otherwise indicated, traditional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA technology, which are within the skill of a person skilled in this field. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. Eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Some aspects of the present invention relate to vector systems containing one or more vectors, or vectors as such. Vectors can be designed to express CRISPR transcripts (e.g., transcripts of nucleic acids, proteins or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells, such as, for example, Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example, using the regulatory sequences of the T7 promoter and T7 polymerase.

Vectors can be introduced and propagated in prokaryote. In some embodiments, a prokaryote is used to amplify copies of a vector that is intended to be introduced into a eukaryotic cell, or as an intermediate vector for the preparation of a vector that is intended to be introduced into a eukaryotic cell (for example, by amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as, for example, to provide a source of one or more proteins for delivery to a host cell or host organism. The expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters that control the expression of either fusion proteins or other than fusion proteins. Fusion vectors add a certain amount of amino acids to the protein encoded therein, such as, for example, to the amino terminus of a recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to enhance expression of a recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to facilitate the purification of the recombinant protein by functioning as a ligand in affinity purification. Often, the proteolytic cleavage site is introduced into the fusion expression vectors at the junction of the fusion fragment and the recombinant protein to facilitate separation of the recombinant protein from the fusion fragment after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. Illustrative fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ) in which glutathione -8-transferase (GST), maltose-binding protein E or protein A, respectively, are fused to the target recombinant protein.

Examples of suitable inducible non-fusion expression vectors of E. coli include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al. ., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, California) and picZ (InVitrogen Corp, San Diego, California).

In some embodiments, the vector controls the expression of the protein in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc group (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL group (Lucklow and Summers 1989. Virology 170: 31-39).

In some embodiments, a vector is capable of controlling the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When using mammalian cells, expression vector control functions are typically provided by one or more regulatory elements. For example, widely used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, monkey virus 40, and others, are disclosed herein and are known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, chapters 16 and 17 in Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In some embodiments, recombinant mammalian expression vectors are capable of driving expression of nucleic acid predominantly in a particular type of cell (for example, tissue-specific regulatory elements are used for expression of nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin gene promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific tissue promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular, promoters of T-cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore , 1983. Cell 33: 741-748), neuron-specific promoters (e.g., neurofilament gene promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), specific for pancreatic cells promoters (Edlund, et al., 1985. Science 230: 912-916) and cn breast cell specific promoters (for example, a whey promoter; US patent No. 4873316 and publication of European application No. 264166). Stage-regulated promoters are also encompassed, for example, mouse hox gene promoters (Kessel and Gruss, 1990. Science 249: 374-379) and α-fetoprotein gene promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546) .

In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system so as to control the expression of one or more elements of the CRISPR system. In general, CRISPR (short palindromic repeats regularly arranged in groups), also known as SPIDRs (direct repeats interrupted by spacers), make up a family of DNA loci that are typically specific for a particular bacterial species. The CRISPR locus includes a certain class of alternating short sequence repeats (SSRs) that were found in E. coli (Ishino et al., J. Bacteriol., 169: 5429-5433 [1987]; and Nakata et al., J. Bacteriol. 171: 3553-3556 [1989]), and associated genes. Similar alternating SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (see Groenen et al., Mol. Microbiol 10: 1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., Dis., 5: 254-263 [1999]; Masepohl et al., Biochim. Biophys. Acta 1307: 26-30 [1996]; and Mojica et al., Mol. Microbiol., 17: 85-93 [1995]). CRISPR loci typically differ from other SSRs in repeat structure, which were called short intervals at regular intervals (SRSR) (Janssen et al., OMICS J. Integ. Biol., 6: 23-33 [2002]; and Mojica et al., Mol. Microbiol., 36: 244-246 [2000]). In general, repeats are short elements that occur in groups that are regularly separated by unique insertion sequences with an almost constant length (Mojica et al., [2000], supra). Although repeat sequences are highly conserved between strains, a number of alternating repeats and spacer sequences are generally different from strain to strain (van Embden et al., J. Bacteriol., 182: 2393-2401 [2000]) . CRISPR loci have been identified in more than 40 species of prokaryotes (see, for example, Jansen et al., Mol. Microbiol., 43: 1565-1575 [2002]; and Mojica et al., [2005]), including , but are not limited to, Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Bloom, Mycobacterium, Streptomyces Chromium, Thermifeus, Aquifexomius, Aquifex Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methureomomocomella Chollocella

In general, “CRISPR system” means collectively transcripts and other elements involved in the expression of CRISPR-associated (“Cas”) genes or the control of their activity, including sequences encoding the Cas gene, tracr (trans-activated CRISPR) sequence ( for example, tracrRNA or active partial tracrRNA), a paired tracr sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of the endogenous CRISPR system), a directing sequence (also called a “spacer” in the context of the endogenous system s CRISPR) or other sequence and transcripts with locus CRISPR. In some embodiments, one or more elements of a CRISPR system are derived from a Type I, Type II, or Type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism containing an endogenous CRISPR system, such as, for example, Streptococcus pyogenes. In general, the CRISPR system is characterized by elements that contribute to the formation of the CRISPR complex at the site of the target sequence (also called the protospacer in the context of the endogenous CRISPR system). In the context of the formation of a CRISPR complex, “target sequence” means a sequence with respect to which the guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Full complementarity is not necessary provided that there is sufficient complementarity for hybridization and the formation of the CRISPR complex. The target sequence may contain any polynucleotide, such as, for example, DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the implementation of the target sequence may be in the organelle of a eukaryotic cell, for example, mitochondria or chloroplast. A sequence or matrix that can be used for recombination at a target locus containing the target sequences is called an “editing matrix” or “editing polynucleotide” or “editing sequence”. In an aspect of the present invention, an exogenous matrix polynucleotide may be referred to as an editing matrix. In one aspect of the present invention, recombination is homologous recombination.

Typically, in the context of the endogenous CRISPR system, the formation of a CRISPR complex (containing a guide sequence that hybridizes with the target sequence and forms a complex with one or more Cas proteins) results in cleavage of one or both strands in or around (for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from) the target sequence. Not wishing to be bound by theory, it is believed that the tracr sequence, which may contain or consist of all or part of the wild-type tracr sequence (for example, approximately or more than approximately 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence) can also form part of a CRISPR complex, such as by hybridizing along at least part of a tracr sequence with all or part of a paired tracr sequence that is operably linked to a guide sequence. In some embodiments, the tracr sequence is sufficiently complementary to the paired tracr sequence for hybridization and participation in the formation of the CRISPR complex. As with the target sequence, it is believed that full complementarity is not necessary provided that it is sufficient to perform the function. In some embodiments, the implementation of the tracr sequence is characterized by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. In some embodiments, one or more vectors that control the expression of one or more elements of the CRISPR system are introduced into the host cell, so that the expression of elements of the CRISPR system controls the formation of the CRISPR complex at one or more target sites. For example, each of the Cas enzyme, the guide sequence associated with the paired tracr sequence, and the tracr sequence can be functionally linked to separate regulatory elements in separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in one vector with one or more additional vectors, providing any components of the CRISPR system that are not included in the first vector. Elements of the CRISPR system, which are combined into one vector, can be located in any convenient orientation, such as, for example, one element located 5 '("above") relative to or 3' ("below") relative to the second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of the second element and directed in the same or opposite direction. In some embodiments, a single promoter controls expression of a transcript encoding a CRISPR enzyme and one or more of a guide sequence, a paired tracr sequence (optionally operably linked to a guide sequence), and a tracr sequence inserted in one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in one intron). In some embodiments, the CRISPR enzyme, the directing sequence, the paired tracr sequence, and the tracr sequence are operably linked to and expressed from the same promoter.

In some embodiments, the vector contains one or more embedding sites, such as, for example, a restriction endonuclease recognition sequence (also called a "cloning site"). In some embodiments, one or more embedding sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more embedding sites) are located above and / or below one or several elements of the sequence of one or more vectors. In some embodiments, the vector comprises an embedment site above the paired tracr sequence and optionally below a regulatory element operably linked to the paired tracr sequence, so that after embedding the guide sequence into the embedment site and upon expression, the guide sequence controls the sequence-specific binding of the CRISPR complex to the target sequence in a eukaryotic cell. In some embodiments, the vector contains two or more embedding sites, with each embedding site located between two paired tracr sequences so as to enable embedding of the guide sequence into each site. In such an arrangement, two or more guide sequences may comprise two or more copies of one guide sequence, two or more different guide sequences, or combinations thereof. In cases where several different guide sequences are used, a single expression construct can be used to specifically target CRISPR activity on several different corresponding target sequences in the cell. For example, one vector may contain about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of these guiding sequence vectors can be provided and optionally delivered to the cell.

In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence for a CRISPR enzyme, such as, for example, a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5 , Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csf1, Csf1 , Csf4, their homologues or their modified variants. These enzymes are known; for example, the amino acid sequence of Cas9 protein S. pyogenes can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as, for example, Cas9. In some embodiments, the CRISPR enzyme is Cas9, and it can be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme controls cleavage of one or both strands at a specific point in the target sequence, such as, for example, within the target sequence and / or within the complementary sequence of the target sequence. In some embodiments, the CRISPR enzyme controls cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that is mutated with respect to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, replacing aspartate with alanine (D10A) in the catalytic domain of RuvC I Cas9 from S. pyogenes transforms Cas9 from a nuclease that cleaves both strands to nikase (cleaves one strand). Other examples of mutations that convert Cas9 to nickase include, but are not limited to, H840A, N854A, and N863A. In some embodiments, Cas9 nicase can be used in combination with the guide sequence (s), for example, two guide sequences that deliberately act on the sense and antisense strands of the target DNA, respectively. This combination allows both threads to be incised and used to induce NHEJ. The authors of this application showed (data not shown) the effectiveness of two targets for nickases (i.e., sgRNAs that specifically target the same point, but on different DNA strands) when inducing mutagenic NHEJ. A single nicase (Cas9-D10A with one sgRNA) is not able to induce NHEJ and create insertions / deletions, but the authors of this application showed that double nikase (Cas9-D10A and two sgRNAs, purposefully acting on different strands at the same point) is capable of do this in human embryonic stem cells (hESC). Efficiency is approximately 50% of that nuclease (i.e., normal Cas9 without D10 mutation) in hESC.

As an additional example, two or more Cas9 catalytic domains (RuvC I, RuvC II, and RuvC III) can be mutated to produce mutated Cas9, which has virtually no DNA splitting activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce the Cas9 enzyme, which has virtually no DNA splitting activity. In some embodiments, the implementation of the CRISPR enzyme is considered as one in which there is practically no all activity for DNA cleavage, in cases where the activity for DNA cleavage of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less with respect to its non-mutated form. Other mutations may be appropriate; in cases where Cas9 or another CRISPR enzyme is derived from a species other than S. pyogenes, mutations in the corresponding amino acids can be made to achieve similar effects.

In some embodiments, an enzyme coding sequence for a CRISPR enzyme is codon-optimized for expression in certain cells, such as eukaryotic cells. Eukaryotic cells can be cells of a particular organism or obtained from it, such as, for example, a mammal, including, without limitation, a human, mouse, rat, rabbit, dog or non-human primate. In general, codon optimization means a method of modifying a nucleic acid sequence to increase expression in host cells of interest by replacing at least one codon (e.g., approximately or more than approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with codons that are used most often or most often in the genes of this host cell, while preserving the native amino acid sequence. Different species exhibit a certain "preference" for specific codons of a particular amino acid. The “preference” of codons (differences in the frequency of use of codons between organisms) is often correlated with the translation efficiency of messenger RNA (mRNA), which, in turn, is believed to depend, inter alia, on the properties of the codons that are translated and the availability of specific transport molecules RNA (tRNA). The predominance of selected tRNA in the cell, as a rule, is a reflection of the codons used most often in the synthesis of peptides. Accordingly, genes can be adapted for optimal gene expression in a given organism using codon optimization. Codon usage tables are publicly available, for example, in the Codon Usage Database, and these tables can be adapted in various ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28: 292 (2000). Computer algorithms are also available for optimizing codons of a specific sequence for expression in a specific host cell, such as, for example, Gene Forge (Aptagen; Jacobus, PA) also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in the sequence encoding the CRISPR enzyme correspond to the most commonly used codon for a certain amino acid.

In some embodiments, the vector encodes a CRISPR enzyme containing one or more nuclear localization sequences (NLS), such as, for example, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLS In some embodiments, the CRISPR enzyme contains about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the amino end, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the carboxy end, or a combination of this (for example, one or more NLS at the amino end and one or more NLS at the carboxy -end). In cases where multiple NLSs are present, each can be selected independently of the others, so that one NLS can be present in several copies and / or in combination with one or more other NLS present in one or more copies. In a preferred embodiment of the present invention, the CRISPR enzyme contains at most 6 NLS. In some embodiments, NLS is believed to be adjacent to the N- or C-terminus when the closest amino acid NLS is in the range of about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypetid chain from the N- or C-terminus. Typically, NLS consists of one or more short sequences of positively charged lysine or arginine molecules located on the surface of a protein, but other types of NLS are known. Non-limiting examples of NLS include NLS sequences derived from: NLS from the SV40 virus large T antigen with the PKKKRKV amino acid sequence; NLS from nucleoplasmin (for example, double NLS from nucleoplasmin with the sequence KRPAATKKAGQAKKKK); NLS from c-myc with the amino acid sequence PAAKRVKLD or RQRRNELKRSP; NLS from hRNPA1 M9 with the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMMZFKNKGKDTAELRRRRRVEVSVELRKAKXDEQILKRRNV of the IBB domain from importin-alpha; sequences of VSRKRPRP and PPKKARED from fibroid T-protein; POPKKKPL sequence from human p53; the SALIKKKKKMAP sequence from mouse c-abl IV; DRLRR and PKQKKRK sequences from NS1 influenza virus; the sequence of RKLKKKIKKL from the hepatitis virus delta antigen; the REKKKFLKRR sequence from mouse Mx1 protein; the KRKGDEVDGVDEVAKKKSKK sequence from human poly (ADP-ribose) polymerase; and the RKCLQAGMNLEARKTKK sequence from steroid hormone receptors for glucocorticoids (human).

In general, one or more NLSs are effective enough to control the accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, the degree of manifestation of nuclear localization activity may be the result of the following: the amount of NLS in the CRISPR enzyme, the specific (used) used NLS, or a combination of these factors. Detection of accumulation in the core can be performed using any suitable technique. For example, a detectable marker can be fused to the CRISPR enzyme, so that the location in the cell can be visualized, such as in combination with means for detecting the location of the nucleus (for example, a stain-specific agent, such as DAPI). Examples of detectable markers include fluorescent proteins (such as green fluorescent proteins or GFP; RFP; CFP) and epitope tags (HA tag, flag tag, SNAP tag). Cell nuclei can also be isolated from cells, the contents of which can then be analyzed using any suitable method for detecting a protein, such as immunohistochemistry, western blotting, or analysis of enzyme activity. Accumulation in the nucleus can also be determined indirectly, such as, for example, by analyzing the effect of the formation of the CRISPR complex (for example, by analyzing the DNA cleavage or mutation in the target sequence or by analyzing the gene expression activity modified by the formation of the CRISPR complex and / or CRISPR enzyme activity) ) compared to a control that was not exposed to an enzyme or CRISPR complex or was exposed to a CRISPR enzyme that lacks one or more NLS.

In general, the guide sequence is any polynucleotide sequence having sufficient

complementarity with the target polynucleotide sequence for hybridization with the target sequence and control sequence specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm is approximately or more than approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97 5%, 99% or more. Optimal alignment can be determined using any suitable algorithm for aligned sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burroughs-Wheeler transform (e.g. Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. The ability of the guide sequence to control sequence specific binding of the CRISPR complex to the target sequence can be assessed using any suitable assay. For example, components of the CRISPR system sufficient for the formation of the CRISPR complex, including the guiding sequence to be investigated, can be delivered to the host cell with the corresponding target sequence, such as, for example, by transfection with vectors encoding the components of the CRISPR sequence, followed by evaluating a preferred cleavage within the target sequence, such as, for example, using the Surveyor analysis described herein. Similarly, cleavage of the target polynucleotide sequence can be established in vitro by providing the target sequence, components of the CRISPR complex, including the guide sequence to be examined, and a control guide sequence other than the test guide sequence, and comparing the effects of the test and control guide sequence binding or cleavage rate of the target sequence. Other analyzes are possible and will be apparent to a person skilled in the art.

A guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the cell genome. Illustrative target sequences include those that are unique in the target genome. For example, for Cas9 S. pyogenes, a unique target sequence in the genome may include a target site for Cas9 in the form MMMMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N represents A, G, T or C; and X can be any) is characterized by a single occurrence in the genome. A unique target sequence in the genome can include the target site for S. pyogenes Cas9 in the form of MMMMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (N is A, G, T or C; and X can be any) is characterized by a single occurrence in the genome. For Cas9 CRISPR1 S. thermophilus, the unique target sequence in the genome may include the target site for Cas9 in the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N represents A, G, T or C; X can be any A; X can be any A; X can be any A or X) appearance in the genome. The unique target sequence in the genome may include the target site for Cas9 CRISPR1 S. thermophilus as MMMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N represents A, G, T or C; X can be any; and W represents a single occurrence of A or T) genome. For Cas9 S. pyogenes, a unique target sequence in the genome can include a target site for Cas9 in the form MMMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNKNNNNXGGXG (N is A, G, T or C; and X can be any) is characterized by a single occurrence in the genome. The unique target sequence in the genome may include the target site for S. pyogenes Cas9 in the form MMMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T or C; and X can be any) is characterized by a single occurrence in the genome. In each of these sequences, “M” may be A, G, T or C and should not be taken into account when identifying a sequence as unique.

In some embodiments, a guide sequence is selected to reduce the proportion of the secondary structure in the guide sequence. The secondary structure can be determined using any suitable polynucleotide folding algorithm. Some programs are based on calculating Gibbs minimum free energy. An example of one such algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of the folding algorithm is the online RNAfold web server developed at the Institute of Theoretical Chemistry at the University of Vienna using the centroid method for predicting the structure (see, for example, AR Graber et al., 2008, Cell 106 (1 ): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62). Additional algorithms can be found in the application for a US patent with a serial number of TBA (case number of the patent attorney 44790.11.2022; general reference BI-2013 / 004A); incorporated herein by reference.

In general, a paired tracr sequence includes any sequence that is sufficiently complementary to a tracr sequence to facilitate one or more of: (1) excision of a guide sequence flanked by paired tracr sequences in a cell containing the corresponding tracr sequence; and (2) the formation of the CRISPR complex on the target sequence, where the CRISPR complex contains a paired tracr sequence that hybridizes with the tracr sequence. In general, the degree of complementarity is indicated based on the optimal alignment of the paired tracr sequence and the tracr sequence along the length of the shorter of the two sequences. Optimal alignment can be determined using any suitable alignment algorithm and can be additionally calculated for secondary structures, such as, for example, self-complementarity within either a tracr sequence or a paired tracr sequence. In some embodiments, the degree of complementarity between the tracr sequence and the paired tracr sequence along the shorter of the two with optimal alignment is approximately or more than approximately 25%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95%, 97.5%, 99% or more. Exemplary illustrations of optimal alignment between the tracr sequence and the paired tracr sequence are shown in FIGS. 12B and 13B. In some embodiments, the implementation of the tracr sequence is approximately or more than approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 50 or more nucleotides in length. In some embodiments, the tracr sequence and the paired tracr sequence are contained in a single transcript, so that hybridization between the two gives a transcript with a secondary structure, such as a hairpin. Preferred looping sequences for use in hairpin structures are four nucleotides in length and most preferably have a GAAA sequence. However, shorter or longer loop sequences can be used, as well as alternative sequences. The sequences preferably include a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop forming sequences include CAAA and AAAG. In one embodiment of the present invention, the transcript or transcribed polynucleotide sequence is characterized by at least two or more hairpins. In preferred embodiments, the transcript is characterized by two, three, four, or five hairpins. In a further embodiment of the present invention, the transcript is characterized by at most five hairpins. In some embodiments, a single transcript further includes a transcription termination sequence; preferably, it is a polyT sequence, for example, of six nucleotides T. An exemplary illustration of such a “hairpin” structure is presented in the lower part of Figure 13B, where part of the sequence in the 5 ′ direction with respect to the terminal “N” and above the loop corresponds to the paired tracr sequence , and part of the sequence in the 3 'direction with respect to the loop corresponds to the tracr sequence. Additional non-limiting examples of individual polynucleotides containing a guide sequence, a paired tracr sequence and a tracr sequence are the following (listed from 5 ′ to 3 ′), where “N” is the base of the guide sequence, the first block of lowercase letters is a paired tracr -sequence, and the second block of lowercase letters is a tracr sequence, and the final poly-T sequence is a transcription terminator: (1)

(2)

(2)

(3)

(3)

(4)

(four)

(5)

(5)

и (6)

. В некоторых вариантах осуществления последовательности (1)-(3) используют в комбинации с Cas9 из CRISPR1 S. thermophilus. В некоторых вариантах осуществления последовательности (4)-(6) используют в комбинации с Cas9 из S. pyogenes. В некоторых вариантах осуществления tracr-последовательность является транскриптом, отдельным от транскрипта, содержащего парную tracr-последовательность (как, например, показанная в верхней части фигуры 13B).

and (6)

. In some embodiments, sequences (1) to (3) are used in combination with Cas9 from CRISPR1 S. thermophilus. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a transcript separate from the transcript containing the paired tracr sequence (such as shown at the top of FIG. 13B).

In some embodiments, a recombination matrix is also provided. The matrix for recombination may be a component of another vector, which is described herein, may be contained in a separate vector or provided as a separate polynucleotide. In some embodiments, the recombination matrix is designed to serve as a matrix for homologous recombination, such as within or adjacent to a target sequence cut or cleaved with a CRISPR enzyme as part of a CRISPR complex. The matrix polynucleotide may be of any suitable length, such as, for example, approximately or more than approximately 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. With optimal alignment, the template polynucleotide can overlap with one or more nucleotides of the target sequences (for example, with approximately or more than approximately 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, with optimal alignment of the matrix sequence and the polynucleotide containing the target sequence, the closest nucleotide of the template polynucleotide is in the range of about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500 , 1000, 5000, 10000 or more nucleotides from the target sequence.

In some embodiments, the CRISPR enzyme is part of a fusion protein containing one or more domains of a heterologous protein (for example, approximately or more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A fusion protein containing a CRISPR enzyme may contain any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope labels, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, repression activity transcription, transcription release factor activity, histone modification activity, DNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (YA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, blue fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence that encodes a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including, without limitation, maltose binding protein (MBP), S-tag, Lex A fusion products, and DNA binding domain (DBD), fusion products of GAL4 and DNA binding domain and fusion products of herpes simplex virus BP16 protein (HSV). Additional domains that can form part of a fusion protein containing the CRISPR enzyme are described in US 20110059502, incorporated herein by reference. In some embodiments, the labeled CRISPR enzyme is used to identify the location of the target sequence.

(eds) (1995); и Yu et al., Gene Therapy 1: 13-26 (1994).In some aspects, the present invention provides methods comprising delivering one or more polynucleotides, such as, for example, one or more vectors described herein, one or more of their transcripts and / or one protein or proteins transcribed from them, into host cell. In some aspects, the present invention further provides cells obtained by such methods and organisms (such as animals, plants or fungi) containing or derived from such cells. In some embodiments, the CRISPR enzyme, in combination with (and optionally complexing with) the guide sequence, is delivered to the cell. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to introduce nucleic acids encoding components of the CRISPR system into cells in a culture and in a host organism. Delivery systems based on non-viral vectors include DNA plasmids, RNA (for example, a transcript of the vector described herein), a “bare” nucleic acid and a nucleic acid that is complexed with a delivery vehicle, such as a liposome. Viral vector-based delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11: 211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8: 35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al., In Current Topics in Microbiology and Immunology, Doerfler and

(eds) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994).

Methods other than viral nucleic acid delivery include lipofection, nucleofection, microinjection, ballistic transfection, virosomes, liposomes, immunoliposomes, polycation or conjugates lipid: nucleic acid, naked DNA, artificial virions and enhanced DNA uptake. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787 and 4,897,355), and lipofection reagents are sold commercially (for example, Transfectam ™ and Lipofectin ™). Cationic and neutral lipids that are suitable for effective lipofection with recognition of the polynucleotide receptor include those from Feigner, WO 91/17424; WO 91/16024. Delivery can take place in cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

The preparation of lipid: nucleic acid complexes, including targeted liposomes, such as immunolipid complexes, is well known to specialists in this field (see, for example, Crystal, Science 270: 404-410 (1995); Blaese et al. Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al. Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995); Ahmad et al., Cancer Res. 52: 4817-4820 (1992); U.S. Pat. and 4946787).

When applying systems based on RNA and DNA viruses for the delivery of nucleic acids, carefully developed methods are used to ensure the targeted action of the virus on specific cells in the body and the transfer of useful virus sequences to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and modified cells can optionally be administered to patients (ex vivo). Conventional virus-based systems may include retroviral, lentiviral, adenoviral vectors, vectors based on adeno-associated virus and herpes simplex virus for gene transfer. Integration into the host genome is possible with gene transfer methods based on retrovirus, lentivirus and adeno-associated virus, which often leads to long-term expression of the inserted transgene. In addition, high transduction efficiencies were observed in many different types of cells and target tissues.

Tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding a possible target population of target cells. Lentiviral vectors are retroviral vectors that are able to transfect or infect non-dividing cells and, as a rule, give high viral titers. The choice of a retrovirus-based gene transfer system will thus depend on the target tissue. Retroviral vectors consist of long terminal repeats active in the cis position with a packing capacity of up to 6-10 bp alien sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of vectors, which are then used to integrate the therapeutic gene into the target cell to obtain constant transgene expression. Widely used retroviral vectors include those based on leukemia virus (MuLV), gibbon leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., J. Virol. 66: 2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176: 58-59 (1990); Wilson et al ., J. Virol. 63: 2374-2378 (1989); Miller et al., J. Virol. 65: 2220-2224 (1991); PCT / US94 / 05700). In applications in which transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of exhibiting very high transduction efficiencies in many types of cells and do not require cell division. High titers and expression levels were obtained with such vectors. Such a vector can be obtained in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce target nucleic acids into cells, for example, in the preparation of in vitro nucleic acids and peptides and for gene therapy procedures in vivo and ex vivo (see, for example, West et al., Virology 160: 38-47 (1987); U.S. Patent No. 4,793,768; WO 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 ( 1994) The creation of recombinant AAV vectors is described in a number of publications, including US Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4: 2072-2081 (1984); Hermonat & Muzyczka, PNAS 81: 6466-6470 (1984); and Samulski et al., J. Virol. 63: 038 22-3828 (1989).

Packaging cells are typically used to produce viral particles that are capable of infecting a host cell. Such cells include 293 cells that pack the adenovirus, and ψ2 cells or PA317 cells that pack the retrovirus. Viral vectors used in gene therapy are typically created by producing a cell line that packs a nucleic acid vector into a viral particle. The vectors usually contain the minimum viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences replaced on the expression cassette for expression of polynucleotide (s). Missing viral functions, as a rule, are provided in an auxiliary object by means of a line of packaging cells. For example, AAV vectors used in gene therapy, as a rule, have only ITR sequences from the AAV genome, which are necessary for packaging and integration into the host genome. Viral DNA is packaged in a cell line that contains an auxiliary plasmid encoding other AAV genes, namely rep and cap, but without ITR sequences. The cell line can also be infected with adenovirus as a helper virus. The helper virus facilitates the replication of the AAV vector and the expression of AAV genes from an auxiliary plasmid. Auxiliary plasmid is not packaged in significant quantities due to the lack of ITR sequences. Adenovirus infection can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Additional methods for delivering nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

In some embodiments, the host cell is transiently or non-transiently transfected with one or more of the vectors described herein. In some embodiments, the cell is transfected as in vivo in a subject. In some embodiments, the cell to be transfected is taken from a subject. In some embodiments, a cell is derived from cells taken from a subject, such as a cell line. A wide range of cell lines for tissue culture are known in the art. Examples of cell lines include, without limitation, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1 , COS-6, COS-M6A, monkey kidney epithelial cells BS-C-1, mouse embryonic fibroblasts BALB / 3T3, 3T3 Swiss, 3T3-L1, human fetal fibroblasts 132-d5; mouse fibroblasts 10.1, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3 cells , BHK-21, BR 293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr - / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2 , EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells , Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II , MDCK II, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM- 1, NW-145, cell lines OPCN / OPCT, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2, Sf-9, SkBr3, T2, T-47D, T84 cells, line cells THP1, U373, U87, U937, VCaP, cell Ki Vero, WM39, WT-49, X63, YAC-1, YAR and their transgenic variants. Cell lines are available from a number of sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassas, Virginia)). In some embodiments, a cell transfected with one or more of the vectors described herein is used to produce a new cell line containing one or more sequences derived from a vector. In some embodiments, a cell transiently transfected with CRISPR system components as described herein (such as by transient transfection with one or more vectors or RNA transfection) and modified by CRISPR complex activity is used to produce a new cell line containing cells that contain a modification, but which lack any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more of the vectors described herein, or cell lines derived from such cells, were used to evaluate one or more test compounds.

In some embodiments, one or more of the vectors described herein is used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as, for example, a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism, or subject, or plant is an alga. Methods for producing transgenic plants and animals are known in the art and typically begin with a method for transfecting a cell, such as described herein.

In one aspect, the present invention provides methods for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method includes providing binding of the CRISPR complex to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex contains a CRISPR enzyme that is complexed with a guide sequence that hybridizes to the target sequence in the specified target polynucleotide, where the specified guide sequence is associated with a paired tracr sequence, which, in turn, iziruetsya with tracr-sequence.

In one aspect, the present invention provides a method for modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises providing binding of the CRISPR complex to a polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; where the CRISPR complex contains the CRISPR enzyme, which forms a complex with a guide sequence that hybridizes with the target sequence in the specified target polynucleotide, where the specified guide sequence is associated with a paired tracr sequence, which, in turn, hybridizes with the tracr sequence.

Given recent advances in crop genomics, the possibility of using the CRISPR-Cas system for efficient and cost-effective gene editing and manipulation will provide the ability to quickly select and compare single and multiplex genetic manipulations to transform such genomes in relation to increased production and improved traits. In this regard, reference is made to US patents and publications of US patents: US patent No. 6603061 - Agrobacterium-mediated Plant Transformation Method (Agrobacterium-Mediated Plant Transformation Method); US patent No. 7868149 - sequences of the plant genome and their use (Plant Genome Sequences and Uses Thereof) and US 2009/0100536 - transgenic plants with improved agrotechnical characteristics (Transgenic Plants with Enhanced Agronomic Traits), all contents and disclosures of each of which are included in this document by reference in full. In practicing the present invention, the contents and disclosure of Morrell et al "Crop genomics: advances and applications" Nat Rev Genet. 2011 Dec 29; 13 (2): 85-96 are also incorporated herein by reference in full. In an advantageous embodiment of the present invention, the CRISPR / Cas9 system is used to construct microalgae (Example 15). Accordingly, in this document, reference to animal cells may also be applicable, subject to necessary changes, in relation to plant cells, unless explicitly stated otherwise.

In one aspect, the present invention provides methods for modifying a target polynucleotide in a eukaryotic cell, which may occur in vivo, ex vivo or in vitro. In some embodiments, the method comprises collecting a cell or population of cells from a person, or an animal or plant other than a person (including microscopic algae), and modifying the cell or cells. Cultivation can be carried out at any ex vivo stage. A cell or cells can even be reintroduced to a non-human animal or plant (including microscopic algae).

In plants, pathogens are often specific for the host. For example, Fusarium oxysporum f. sp. lycopersici causes the Fusarium wilt of the tomato, but only the tomato, a F. oxysporum f. dianthii and Puccinia graminis f. sp. tritici only affects wheat. Plants possess inherent and induced protective responses that provide resistance to most pathogens. Mutations and recombination events in plant generations lead to genetic variability, which causes susceptibility, especially since pathogens multiply with greater frequency than plants. In plants, resistance of non-host species may be observed, for example, the host and pathogen are incompatible. There may also be horizontal resistance, for example, partial resistance to all races of a pathogen, usually controlled by many genes, and vertical resistance, for example, full resistance to some races of a pathogen, but not to other races, usually controlled by several genes. At the level of gene interaction, plants and pathogens evolve together, and genetic changes in one balance the changes in the other. Accordingly, using natural variability, breeders combine the most useful genes for productivity, quality, uniformity, endurance, stability. Sources of resistance genes include native or foreign varieties, vintage varieties, related wild plants, and induced mutations, for example, when processing plant material with mutagenic agents. Using the present invention, plant breeders receive a new tool for the induction of mutations. Accordingly, a person skilled in the art can analyze the genome of sources of resistance genes, and for varieties having the desired characteristics or characteristics, use the present invention to induce the appearance of resistance genes with greater accuracy than in the case of previously used mutagenic agents, and, therefore, to accelerate and improving plant breeding programs.

In one aspect, the present invention provides kits comprising any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a paired tracr sequence and one or more embedding sites for embedding a guide sequence above a paired tracr sequence, where upon expression the guide sequence controls the sequence-specific binding of the CRISPR complex to target sequence in a eukaryotic cell, where the CRISPR complex contains the CRISPR enzyme, forming a complex with (1) the guide The sequence which hybridizes with the target sequence, and (2) tracr-pair sequence that hybridizes to tracr-sequence; and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. Elements may be provided separately or in combination and may be provided in any suitable container, such as, for example, a vial, vial or tube. In some embodiments, a kit includes instructions in one or more languages, for example, in more than one language.

In some embodiments, the kit contains one or more reagents for use in a method that uses one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may include one or more reaction buffers or storage buffers. Reagents may be provided in a form that is applicable in a particular assay, or in a form that involves the addition of one or more other components before use (for example, in the form of a concentrate or lyophilized form). The buffer may be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the implementation of the buffer is alkaline. In some embodiments, the buffer has a pH of from about 7 to about 10. In some embodiments, the kit contains one or more oligonucleotides corresponding to the guide sequence for insertion into the vector so that the guide link and regulatory element are functional. In some embodiments, the kit comprises a template polynucleotide for homologous recombination.

In one aspect, the present invention provides methods for using one or more elements of a CRISPR system. The CRISPR complex of the present invention provides an effective means of modifying the target polynucleotide. The CRISPR complex of the present invention is characterized by a wide variety of useful properties, including the modification (for example, deletion, insertion, translocation, inactivation, activation) of the target polynucleotide in many cell types. The CRISPR complex of the present invention as such has a wide range of applications, for example, in gene therapy, drug screening, diagnosis and prognosis of diseases. The exemplary CRISPR complex contains the CRISPR enzyme, which forms a complex with a guide sequence that hybridizes with the target sequence in the target polynucleotide. The directing sequence is linked to a paired tracr sequence, which in turn hybridizes to the tracr sequence.

The target polynucleotide of the CRISPR complex may be any polynucleotide endogenous or exogenous to a eukaryotic cell. For example, the target polynucleotide may be a polynucleotide located in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein), or a non-coding sequence (e.g., a regulatory polynucleotide or excess DNA). Not wishing to be bound by theory, it is believed that the target sequence should be associated with PAM (a motif adjacent to the protospacer); that is, the short sequence recognized by the CRISPR complex. Certain requirements regarding the sequence and length of PAM vary depending on the CRISPR enzyme used, but PAM is usually a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). Examples of PAM sequences are provided in the Examples section below, and one skilled in the art will be able to identify additional PAM sequences for use with this CRISPR enzyme.

The CRISPR target polynucleotide may include a number of disease-associated genes and polynucleotides, as well as genes and polynucleotides associated with biochemical signaling pathways, which are listed in provisional patent applications US 61/736527 and 61/748427 with general reference BI-2011/008 / WSGR, registry number 44063-701.101, and BI-2011/008 / WSGR, registry number 44063-701.102, respectively, both are entitled "SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCES" filed December 12 January 12 and January 2, 2013, respectively, the contents of all of which are included in this document by reference in full.

Examples of target polynucleotides include a sequence associated with biochemical signaling pathways, for example, associated with biochemical signaling pathways of a gene or polynucleotide. Examples of target polynucleotides include disease-associated genes or polynucleotides. “Disease-associated” gene or polynucleotide means any gene or polynucleotide that provides transcription or translation products at an abnormal or abnormal form in cells derived from diseased tissues compared to non-diseased tissues or control cells. This may be a gene that begins to be expressed at an abnormally high level; it may be a gene that begins to be expressed at an abnormally low level, where altered expression correlates with the onset and / or development of the disease. A disease-associated gene also means a gene that carries a mutation (s) or genetic change that is directly responsible or is in disequilibrium linkage with the gene (s) that (s) are responsible for the etiology of the disease. The products being translated or translated may be known or unknown and may be at a normal level or at a deviating level.

Examples of disease-associated genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information at the National Library of Medicine (Bethesda, Maryland), available on the World Wide Web.

Examples of disease-associated genes and polynucleotides are listed in Tables A and B. Specific disease information is available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Baltimore, Maryland) and the National Center for Biotechnology Information, the National Library of Medicine (Bethesda, MD), available on the Internet. Examples of gene and polynucleotide associated signals with biochemical pathways are listed in Table C.

Mutations in these genes and pathways can lead to the production of abnormal proteins or proteins in inappropriate amounts that affect function. Additional examples of genes, diseases, and proteins are thus incorporated by reference from provisional patent application US 61/736527, filed December 12, 2012, and 61/748427, filed February 2, 2013. Such genes, proteins, and pathways may be the target polynucleotide of the CRISPR complex.

hybrids. Mclvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct; 7(5): 551-8). Система CRISPR-Cas может быть приспособлена для корректировки таких дефектов геномной нестабильности.Embodiments of the present invention also relate to methods and compositions related to knocking out genes, amplifying genes, and repairing specific mutations associated with DNA repeat instability and neurological disorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, Oct 13, 2011 - Medical). It has been found that certain aspects of tandem repeat sequences are responsible for more than twenty human diseases (New insights into repeat instability: role of

hybrids. Mclvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct; 7 (5): 551-8). The CRISPR-Cas system can be adapted to correct for such defects in genomic instability.

An additional aspect of the present invention relates to the use of a CRISPR-Cas system for correcting defects in the EMP2A and EMP2B genes that have been found to be associated with Laforet disease. Lafora disease is an autosomal recessive condition characterized by progressive myoclonus epilepsy, which can begin in the form of epileptic seizures in adolescence. Some cases of the disease can be caused by mutations in genes that have already been identified. The disease causes seizures, muscle cramps, difficulty walking, dementia and, ultimately, death. Currently, there is no therapy that has been shown to be effective against the development of the disease. Other genetic disorders associated with epilepsy can also be targeted with the CRISPR-Cas system, and the underlying genetics are further described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology: twenty; 2009).

In yet another aspect of the present invention, the CRISPR-Cas system can be used to correct ophthalmic defects that result from several genetic mutations that are further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

Some additional aspects of the present invention relate to the correction of defects associated with a wide range of genetic diseases, which are further described on the website of the National Institutes of Health in the thematic subsection "Genetic Disorders" (website at health.nih.gov/topic/GeneticDisorders). Inherited brain diseases may include, without limitation, adrenoleukodystrophy, corpus callosum agenesis, Aicardi syndrome, Alpers syndrome, Alzheimer's disease, Bart syndrome, Butten disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstman-Straussler-Shanker syndrome, and other Huntington's disease triplet repetition-related disorders, Leu disease, Lesch-Nyhan syndrome, Menkes disease, types of mitochondrial myopathy and colpocephaly according to the NINDS criteria. Such diseases are further described on the website of the National Institutes of Health under the subject heading “Genetic Brain Disorders”.

In some embodiments, the condition may be neoplasia. In some embodiments, where the condition is neoplasia, the targeted genes are any of those listed in Table A (in this case, PTEN and so on). In some embodiments, the condition may be age-related macular degeneration. In some embodiments, the condition may be a schizophrenic disorder. In some embodiments, the condition may be a trinucleotide repeat disorder. In some embodiments, the condition may be a fragile X chromosome syndrome. In some embodiments, the condition may be a secretory-related disorder. In some embodiments, the condition may be a prion-related disorder. In some embodiments, the condition may be ALS. In some embodiments, the condition may be addictive to drugs. In some embodiments, the condition may be autism. In some embodiments, the condition may be Alzheimer's disease. In some embodiments, the condition may be inflammation. In some embodiments, the condition may be Parkinson's disease.

Examples of proteins associated with Parkinson's disease include, but are not limited to, α-synuclein, DJ-1, LRRK2, PINK1, parkin, UCHL1, synfilin-1 hNURRI.

Examples of addictive proteins may include, for example, ABAT.

Examples of inflammation-related proteins may include, for example, monocyte chemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, CC chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, IgG receptor IIB (IgG receptor IIB) (FCGR2b, also called CD32), encoded by the Fcgr2b gene, or Fc-epsilon-R1g protein (Fc epsilon R1g) (FCER1g), encoded by the Fcer1g gene.

Examples of proteins associated with diseases of the cardiovascular system may include, for example, IL1B (interleukin 1, beta (interleukin 1, beta)), XDH (xanthine dehydrogenase), TP53 (tumor protein p53 (tumor protein p53)), PTGIS (prostaglandin-12 (prostacyclin) synthase (prostaglandin I2 (prostacyclin) synthase)), MB (myoglobin (myoglobin)), IL4 (interleukin 4 (interleukin 4)), ANGPT1 (angiopoietin 1 (angiopoietin 1)), ABCG8 (A -binding cassette, subfamily G (WHITE), representative 8 (ATP-binding cassette, sub-family G (WHITE), member 8)) or CTSK (cathepsin K (cathepsin K)).

Examples of proteins associated with Alzheimer's disease can include, for example, a protein that is a very low density lipoprotein receptor protein (VLDLR), encoded by the VLDLR gene, ubiquitin-like modifier activating enzyme 1 (ubiquitin-like modifier activating enzyme 1) (UBA1) encoded by the UBA1 gene, or a protein that is a catalytic subunit of the NEDD8-aKTHBHpyioinero enzyme E1 (NEDD8-activatirig enzyme E1 catalytic subunit protein) (UBE1C) encoded by the UBA3 gene.

Examples of proteins associated with autism spectrum disorders may include, for example, protein 1 associated with the benzodiazapine receptor (peripheral) associated protein 1) (BZRAP1), encoded by the BZRAP1 gene, protein, member 2 of the AF4 / FMR2 family (AF4 / FMR2 family member 2 protein) (AFF2), encoded by the AFF2 gene (also called MFR2), an autosomal homolog 1 protein associated with the fragile X mental retardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or protein-autosomal homolog 2, assoc ovanny with mental retardation associated with Fragile X chromosome (fragile X mental retardation autosomal homolog 2 protein) (FXR2), encoded gene FXR2.

Examples of proteins associated with macular degeneration may include, for example, an ATP binding cassette, a representative protein of 4 subfamilies A (ABC1) (ATP-binding cassette, sub-family A (ABC1) member 4 protein) (ABCA4) encoded ABCR genome, apolipoprotein E protein (APOE) encoded by the APOE gene, or chemokine ligand 2 protein (CC motif) (chemokine (CC motif) Ligand 2 protein) (CCL2) encoded by the gene CCL2.

Examples of proteins associated with schizophrenia may include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

Examples of proteins involved in tumor suppression may include, for example, ATM (mutated, ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related (ataxia telangiectasia and Rad3 related)), EGFR (epidermal receptor growth factor (epidermal growth factor receptor)), ERBB2 (homolog 2 of v-erb-b2 erythroblastic leukemia of viral oncogen (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2)), ERBB3 (homolog 3 of v-erb-b2 erythroblastic leukemia of viral oncogen (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3)), ERBB4 (homolog 4 of v-erb-b2 erythroblastic viral oncogen leukemia (v-erb-b2 erythr oblastic leukemia viral oncogene homolog 4)), Notch 1, Notch2, Notch 3 or Notch 4.

Examples of proteins associated with a disorder associated with secretase activity may include, for example, PSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B (cathepsin B)), PSEN1 (presenilin 1 (presenilin 1)), APP (amyloid beta (A4) precursor protein (amyloid beta (A4) precursor protein)), APH1B (anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4) (presenilin 2 ( Alzheimer disease 4)) or BACE1 (APP-cleaving enzyme 1).

Examples of proteins associated with amyotrophic lateral sclerosis may include, for example, SOD1 (superoxide dismutase 1 (superoxide dismutase 1)), ALS2 (protein associated with amyotrophic lateral sclerosis 2 (amyotrophic lateral sclerosis 2)), FUS (FUS RNA binding protein (fused in sarcoma)), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B (vascular endothelial growth factor B)) and VAGFC (vascular endothelial growth factor C (vascular endothelial growth factor C)) and any combination thereof.

Examples of proteins associated with prion diseases can include SOD1 (superoxide dismutase 1), ALS2 (protein associated with amyotrophic lateral sclerosis 2 (amyotrophic lateral sclerosis 2)), FUS (RNA-binding protein FUS (fused in sarcoma)), TARDBP ( TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B) and VAGFC (endothelial growth factor vascular C (vascular endothelial growth factor C)) and any combination thereof.

Examples of proteins associated with neurodegenerative conditions in prion diseases may include, for example, A2M (Alpha-2-Macroglobulin), AATF (Apoptosis antagonizing transcription factor), ACPP (prostate-specific acid phosphatase (Acid phosphatase prostate)), ACTA2 (alpha-actin 2 smooth muscle aorta (Actin alpha 2 smooth muscle aorta)), ADAM22 (ADAM, metallopeptidase domain), ADORA3 (type A3 adenosine receptor (Adenosine A3 receptor)) or ADRA1D (alpha-ID adrenergic receptor for alpha-ID adrenergic receptor (Alpha-ID adrenergic receptor for Alph a-ID adrenoreceptor)).

Examples of proteins associated with immunodeficiency may include, for example, A2M [alpha-2-macroglobulin (alpha-2-macroglobulin)]; AANAT [arylalkylamine-N-acetyltransferase (arylalkylamine N-acetyltransferase)]; ABCA1 [ATP-binding cassette, subfamily A (ABC1), representative 1 (ATP-binding cassette, sub-family A (ABC1), member 1)]; ABCA2 [ATP-binding cassette, subfamily A (ABC1), representative 2 (ATP-binding cassette, sub-family A (ABC1), member 2)] or ABCAZ [ATP-binding cassette, subfamily A (ABC1), representative 3 ( ATP-binding cassette, sub-family A (ABC1), member 3)].

Examples of proteins associated with disorders associated with trinucleotide repeat include, for example, AR (androgen receptor), FMR1 (protein 1, associated with mental retardation associated with the fragility of the X chromosome (fragile X mental retardation 1)) , HTT (huntingtin) or DMPK (protein kinase associated with muscular dystrophy (dystrophia myotonica-protein kinase)), FXN (frataxin (frataxin)), ATXN2 (ataxin 2 (ataxin 2)).

Examples of proteins associated with impaired transmission of nerve impulses include, for example, SST (somatostatin (somatostatin)), NOS1 (nitric oxide synthase 1 (neuronal) (nitric oxide synthase 1 (neuronal)), ADRA2A (adrenergic, alpha-2A-, receptor (adrenergic, alpha-2A-, receptor)), ADRA2C (adrenergic, alpha-2C-, receptor (adrenergic, alpha-2C-, receptor)), TACR1 (tachykinin receptor 1 (tachykinin receptor 1)) or HTR2c (5- hydroxytryptamine (serotonin) receptor 2C (5-hydroxytryptamine (serotonin) receptor 2C)).

Examples of sequences associated with neurological development include, for example, A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine-N-acetyltransferase (arylylamine acetyltransferase)], ABAT [4-aminobutyrate aminotransferase (4-aminobutyrate aminotransferase)], ABCA1 [ATP binding cassette, subfamily A (ABC1), representative 1 (ATP-binding cassette, sub-family A (ABC1), member 1)] or ABCA13 [ATP-binding cassette, subfamily A (ABC1), representative 13 (ATP-binding cassette, sub-family A (ABC1), member 13)].

Additional examples of preferred conditions that are to be treated with this system include those that can be selected from Aicardi-Gutieres syndrome; Alexander's disease; Allan-Herndon-Dudley Syndrome; POLG gene related disorders; alpha-mannosidosis (type II and III); Alstrom's syndrome; Angelman syndrome; ataxia-telangiectasia; high neuronal lipofuscinosis; beta thalassemia; bilateral optic atrophy and type 1 (infantile) optic atrophy; retinoblastoma (bilateral); Canavan disease cerebro-oculo-facies-skeletal syndrome 1 [COFS1]; cerebroendinal xanthomatosis; Cornelia de Lange syndrome; MART-related disorders; hereditary prion diseases; Drave syndrome; Alzheimer's familial disease with early onset; Friedreich's ataxia [FRDA]; frins syndrome; fucosidosis; congenital muscular dystrophy of Fukuyama; galactosialidosis; Gaucher disease organic acididemia; hemophagocytic lymphohistiocytosis; Hatchinson-Guildford Progeria Syndrome; mucolipidosis II; infantile disease accumulation of free sialic acid; neurodegeneration associated with the PLA2G6 gene; Jerwell-Lange-Nielsen syndrome; nodular congenital bullous epidermolysis; Huntington's disease; Crabbe disease (infantile); mitochondrial DNA associated Lee syndrome and NARP; Lesch-Nyhan syndrome; associated with the LIS1 gene of lissencephaly; low syndrome; maple syrup diseases; duplication syndrome MECP2; ATP7A-related copper metabolic disorders; muscle dystrophy associated with the LAMA2 gene; aryl sulfatase A deficiency; mucopolysaccharidosis of types I, II or III; disorders associated with biogenesis of peroxisis, a spectrum of diseases like Zellweger syndrome; disorders of the type of neurodegeneration with the accumulation of iron in the brain; acid sphingomyelinase deficiency; Nimann-Peak disease type C; glycine encephalopathy; ARX gene related disorders; disorders of the ornithine cycle; imperfect osteogenesis associated with the COL1A1 / 2 gene; mitochondrial DNA removal syndromes; PLP1 gene related disorders; perry syndrome; Phelan-McDermid syndrome; type II glycogen accumulation diseases (Pompe disease) (infantile); MART-related disorders; MECP2 gene related disorders; epiphyseal point chondrodysplasia of the 1st type of bones of the upper limbs or femur; Roberts syndrome; Sandhoff disease; Schindler's disease - type 1; adenosine deaminase deficiency; Smith-Lemley-Opitz syndrome; spinal muscular atrophy; spinal cerebellar ataxia with occurrence in infancy; hexosaminidase deficiency; thanatophore dysplasia type 1; type VI collagen-related disorders; Asher syndrome type I; congenital muscular dystrophy; Wolf-Hirschhorn syndrome; insufficiency of lysosomal acid lipase and pigment xeroderma.

As will be appreciated, it is contemplated that the present system can be used to target any polynucleotide sequence of interest. Some conditions or diseases that can be effectively treated using the present system are included in the tables above, and examples of currently known genes associated with such conditions are also provided therein. However, the genes given as examples are not exhaustive.

Examples

The following examples are provided to illustrate various embodiments of the present invention, and are not intended to limit the present invention in any way. These examples, together with the methods described herein, currently reflect preferred embodiments, are illustrative and are not intended to limit the scope of the present invention. Changes in this document and other applications that are covered by the essence of the present invention, as defined by the claims, will be apparent to experts in this field.

Example 1: activity of the CRISPR complex in the nucleus of a eukaryotic cell

An example of a type II CRISPR system is a type II CRISPR locus from Streptococcus pyogenes SF370, which contains a group of 4 genes Cas9, Cas1, Cas2 and Csn1, as well as 2 non-coding RNA elements, tracrRNA and a characteristic array of repeating sequences (direct repeats), alternating with short fragments of non-repeating sequences (spacers, approximately 30 bp each). In this system, double-stranded cleavage (DSB) of the target DNA is formed during four consecutive stages (Figure 2A). First, two non-coding RNAs, an array of pre-crRNA and tracrRNA, are transcribed from the CRISPR locus. Secondly, tracrRNA hybridizes with direct pre-crRNA repeats, which are then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas9 to the target DNA, consisting of a protospacer and the corresponding PAM, through the formation of a heteroduplex between the spacer region of crRNA and protospacer DNA. Finally, Cas9 mediates cleavage of the target DNA above PAM with the formation of DSB inside the protospacer (Figure 2A). This example describes an illustrative method of adapting this RNA-programmable nuclease system to control the activity of the CRISPR complex in the nuclei of eukaryotic cells.

Cell Culture and Transfection

The human embryo kidney cell line (HEK), HEK 293FT (Life Technologies), was maintained in Eagle's Dulbecco modification (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 units / ml penicillin and 100 μg / ml streptomycin, at 37 ° C with incubation at 5% CO ₂ . The neuro2A (N2A) mouse cell line (ATCC) was maintained in DMEM supplemented with 5% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 units / ml penicillin and 100 μg / ml streptomycin, at 37 ° C 5% CO ₂ .

HEK 293FT or N2A cells were seeded in 24-well plates (Corning) one day prior to transfection with a density of 200,000 cells per well. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer's recommended protocol. A total of 800 ng plasmids were used for each well of the 24-well plate.

Surveyor analysis and sequencing analysis for genome modification

HEK 293FT or N2A cells were transfected with plasmid DNA as described above. After transfection, cells were incubated at 37 ° C for 72 hours before extraction of genomic DNA. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer's protocol. Briefly, cells were resuspended in a QuickExtract solution and incubated at 65 ° C for 15 minutes and at 98 ° C for 10 minutes. The extracted genomic DNA was immediately processed or stored at -20 ° C.

The genomic region surrounding the CRISPR target site was PCR amplified for each gene and the products were purified using a QiaQuick Spin Column (Qiagen) column following the manufacturer's protocol. A total of 400 ng of purified PCR products were mixed with 2 μl of 10X Taq polymerase PCR buffer (Enzymatics) and ultra-high purity water to a final volume of 20 μl and subjected to a re-annealing process to ensure heteroduplex formation: 95 ° C for 10 min ., a linear decrease in temperature from 95 ° C to 85 ° C at a speed of 2 ° C / s, from 85 ° C to 25 ° C at a speed of 0.25 ° C / s and with holding at 25 ° C for 1 minute. After repeated annealing, the products were treated with Surveyor nuclease and S Surveyor enhancer (Transgenomics), following the manufacturer's recommended protocol, and analyzed in 4-20% Novex TBE polyacrylamide gels (Life Technologies). The gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and the image was obtained using the Gel Doc gel imaging system (Bio-rad) image processing system. Quantitative analysis was based on the relative intensities of the bands as a unit of measure for the fraction of cleaved DNA. Figure 8 is a schematic illustration of this analysis using Surveyor.

Analysis of restriction fragment length polymorphism to detect homologous recombination.

HEK 293FT and N2A cells were transfected with plasmid DNA and incubated at 37 ° C for 72 hours before extraction of genomic DNA, as described above. The target genomic region was PCR amplified using primers outside the homologous arms of the template for homologous recombination (HR). PCR products were separated on a 1% agarose gel and extracted using the MinElute GelExtraction Kit (Qiagen). The purified products were digested with Hindlll (Fermentas) and analyzed on a 6% Novex TBE polyacrylamide gel (Life Technologies).

Prediction and analysis of the secondary structure of RNA

The secondary structure of RNA was predicted using the online RNAfold web server developed at the Institute of Theoretical Chemistry at the University of Vienna using the centroid method for predicting structure (see, for example, AR Gruber et al., 2008, Cell 106 ( 1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

Analysis of interference during transformation with bacterial plasmids Elements of the S. pyogenes CRISPR locus 1 sufficient for CRISPR activity were reproduced in E. coli using the plasmid pCRISPR (schematically depicted in Figure 10A). pCRISPR contained tracrRNA, SpCas9, and a leader sequence running the crRNA array. As shown, spacers (also referred to as "guide sequences") were inserted into the crRNA array between BsaI sites using hybridized oligonucleotides. The control plasmids used in the interference analysis were created by embedding the protospacer sequence (also referred to as the “target sequence”) together with the adjacent CRISPR motif sequence (PAM) in pUC19 (see FIG. 10B). The control plasmid contained ampicillin resistance genes. 10C is a schematic illustration of an interference analysis. Chemically competent E. coli strains already bearing pCRISPR and the corresponding spacer were transformed with a control plasmid containing the corresponding protospacer-PAM sequence. pUC19 was used to evaluate the transformation efficiency of each competent strain bearing pCRISPR. CRISPR activity led to cleavage of the pPSP plasmid carrying the protospacer, which eliminated ampicillin resistance, otherwise provided by pUC19 lacking a protospacer. Figure 10D presents the competence of each E. coli strain bearing pCRISPR used in the assays shown in Figure 4C.

RNA purification

HEK 293FT cells were maintained and transfected as described above. Cells were harvested by trypsinization followed by washing in phosphate buffered saline (PBS). Total cellular RNA was extracted using TRI reagent (Sigma) following the manufacturer's protocol. Quantitative analysis of total extracted RNA was performed using Naonodrop (Thermo Scientific) and the data was normalized to the same concentration;

Analysis of the expression of crRNA and tracrRNA in mammalian cells using Northern blotting

RNA was mixed with equal volumes of 2X loading buffer (Ambion), heated to 95 ° C for 5 minutes, cooled on ice for 1 minute, and then loaded into 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics) after a preliminary run gel for at least 30 minutes. Samples were subjected to electrophoresis for 1.5 hours at a maximum power of 40 watts. After that, the RNA was transferred to a Hybond N + membrane (GE Healthcare) at a current strength of 300 mA in a semi-dry transfer device (Bio-rad) at room temperature for 1.5 hours. RNA was crosslinked with the membrane using the “auto-crosslink” button on the auto-crosslinker using the Stratagene UV Crosslinker the Stratalinker (Stratagene). The membrane was pre-hybridized in the ULTRAhyb-Oligo Hybridization Buffer (Ambion) for 30 minutes. rotating at 42 ° C, and then probes were added and hybridization was performed overnight. Probes were ordered from IDT and labeled [gamma- ³² P] ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs). The membrane was washed once with preheated (42 ° C) 2xSSC, 0.5% SDS for 1 min. followed by two washes for 30 minutes at 42 ° C. The membrane was exposed on a fluorescent screen for one hour or overnight at room temperature, and then scanned using an image forming apparatus on a phosphor phosphor coating (Typhoon).

Creation and evaluation of the bacterial system CRISPR

Elements of the CRISPR locus, including tracrRNA, Cas9, and the leader sequence, were PCR amplified from Streptococcus pyogenes SF370 genomic DNA with flanking homologous arms for assembly using the Gibson method. Two BsaI sites of type IIS were introduced between two direct repeats to ensure insertion of spacers (Figure 9). PCR products were cloned into EcoRV digested pACYC184 downstream of the tet promoter using the Gibson Assembly Master Mix (NEB) master mix for assembly. Other endogenous elements of the CRISPR system were missed, with the exception of the last 50 bp. Csn2. Oligonucleotides (Integrated DNA Technology) encoding spacers with complementary sticky ends were cloned into a BsaI cleaved pDC000 vector (NEB) and then ligated using T7 ligase (Enzymatics) to obtain pCRISPR plasmids. Control plasmids containing spacers with PAM sequences (also referred to herein as CRISPR motif sequences) were created by ligation of hybridized oligonucleotides carrying compatible sticky ends (Integrated DNA Technology) into BamHI cleaved pUC19. Cloning for all constructs was performed in E. coli strain JM109 (Zymo Research).

Cells carrying pCRISPR were made competent using the Z-Competent E. coli Transformation Kit and Buffer Set (Zymo Research, T3001) in accordance with the manufacturer's instructions. In the transformation analysis, 50 μl aliquots of competent cells bearing pCRISPR were thawed on ice and 1 ng of plasmid with spacer or pUC19 was transformed for 30 minutes followed by 45 seconds of heat shock at 42 ° C and 2 minutes on ice. Then, 250 μl of SOC (Invitrogen) was added, followed by incubation with shaking at 37 ° C for 1 h, and 100 μl of cells obtained after SOC were plated on double selection plates (12.5 μg / ml chloramphenicol, 100 μg / ml ampicillin). To obtain CFU / ng DNA, the total number of colonies was multiplied by 3.

To improve the expression of CRISPR components in mammalian cells, two genes from Locus 1 of Streptococcus pyogenes SF370 (S. pyogenes) were codon-optimized, Cas9 (SpCas9) and RNase III (SpRNase III). To ensure nuclear localization, the cellular nuclear localization signal (NLS) was included in the amino (N) or carboxyl (C) terminal regions of both SpCas9 and SpRNase III (Figure 2B). To ensure visualization of protein expression, the fluorescent protein gene as a marker was also included in the N- or C-terminal regions of both proteins (Figure 2B). A SpCas9 variant was also created with NLS attached to both the N- and C-terminal domains (2xNLS-SpCas9). Constructs containing NLC fused SpCas9 and SpRNase III were transfected into human embryonic kidney cells (HEK) 293FT, and it was found that the relative position of NLS relative to SpCas9 and SpRNase III affects their nuclear localization efficiency. Although C-terminal NLS was sufficient to target SpRNase III into the nucleus, attaching one copy of these specific NLS to either the N- or C-terminal regions of SpCas9 was not able to provide adequate nuclear localization in this system. In this example, the C-terminal NLS was from nucleoplasmin (KRPAATKKAGQAKKKK), and the C-terminal NLS was from the large T40 antigen SV40 (PKKKRKV). Of the tested variants of SpCas9, only 2xNLS-SpCas9 showed nuclear localization (Figure 2B).

tracrRNA from the CRISPR locus of S. pyogenes SF370 contained two transcription initiation sites giving rise to two transcripts of 89 nucleotides (nt) and 171 nt, which were then processed into identical mature 75 nt tracrRNAs. Shorter 89 nt tracrRNAs were selected for expression in mammalian cells (the expression construct is depicted in Figure 7A, with functionality as determined by a Surveyor analysis shown in Figure 7B). Transcription initiation sites are designated +1, and the transcription terminator and sequence analyzed by Northern blotting are also indicated. The expression of the processed tracrRNA was also confirmed by Northern blotting. Figure 7C shows the results of Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1 (1) -DR. The left and right sections were obtained with 293FT cells transfected without SpRNase III or with one, respectively. U6 is an indicator for monitoring loading during blotting with a probe targeting small nuclear RNA (snRNA) of human U6. Transfection of the expressing construct with short tracrRNA led to high levels of the processed form of tracrRNA (~ 75 bp). Very low amounts of long tracrRNAs were detected by Northern blotting.

To stimulate the precise initiation of transcription, the U6 promoter based on RNA polymerase III was chosen to control the expression of tracrRNA (Figure 2C). Similarly, a U6 promoter-based construct was designed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DR, also included in the expression “paired tracr sequences”; FIG. 2C). The initial spacer was designed to target a target site of 33 base pairs (bp) (a protospacer of 30 bp, as well as a 3-bp CRISPR sequence (PAM) corresponding to the NGG recognition motif of Cas9) at the human EMX1 locus (Figure 2C), a key gene in the development of the cerebral cortex.

HEK 293FT cells were transfected with a combination of CRISPR components to determine if heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA and pre-crRNA) in mammalian cells allows targeted mammalian chromosome cleavage to be achieved. Since DSBs in mammalian nuclei are partially repaired using the non-homologous terminal junction (NHEJ) pathway, which leads to the formation of inserts / deletions, SURVEYOR analysis was used to identify potential activity for cleavage at the target EMX1 locus (Figure 8) (see, for example, Guschin et al., 2010, Methods Mol Biol 649: 247). Joint transfection of all four CRISPR components was able to induce cleavage of up to 5.0% in the protospacer (see Figure 2D). Co-transfection of all CRISPR components, with the exception of SpRNase III, also induced up to 4.7% insertion / deletion in the protospacer, indicating that mammalian endogenous RNases may exist that could aid crRNA maturation, such as, for example, related enzymes Dicer and Drosha. Removal of any of the three remaining components eliminated the activity of the CRISPR system for genome cleavage (Figure 2D). Sanger sequencing of amplicons containing the target locus confirmed activity for cleavage: 5 mutated alleles were detected in 43 cloned sequencers (11.6%). In such experiments using a series of guide sequences, percentages of insertion / deletion were up to 29% (see figures 4-7, 12 and 13). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells. To optimize cleavage efficiency, applicants also determined whether various tracrRNA isoforms influenced cleavage efficiency and found that in this illustrative system only a short (89 bp) transcript was able to mediate cleavage of the human EMX1 genome locus (Figure 6B).

The figure 14 presents an additional analysis of the processing of crRNA in mammalian cells using Northern blotting. Figure 14A shows a schematic representation of the expression vector for a single spacer flanked by two direct repeats (DR-EMX1 (1) -DR). A 30 bp spacer aimed at the protospacer 1 of the human EMX1 locus (see FIG. 6) and direct repeat sequences are shown in the sequence at the bottom of FIG. 14A. The line indicates a region whose inverse complementary sequence was used to create Northern blot probes to detect crRNA EMX1 (1). Figure 14B shows the results of Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1 (1) -DR. The left and right sections were obtained with 293FT cells transfected without SpRNase III or with one, respectively. DR-EMX1 (1) -DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA and was not dependent on the presence of SpRNase III. Mature crRNA found in total RNA transfected with 293FT was ~ 33 bp in length. and was shorter than the mature crRNA from S. pyogenes 39-42 bp in length These results demonstrate that the CRISPR system can be transferred to eukaryotic cells and reprogrammed to facilitate cleavage of the endogenous target mammalian polynucleotides.

Figure 2 shows the CRISPR bacterial system described in this example. Figure 2A shows a schematic representation of CRISPR locus 1 from Streptococcus pyogenes SF370 and a putative mechanism of CRISPR-mediated DNA cleavage using this system. The mature crRNA, which has undergone processing from an array of direct repeat spacers, directs Cas9 to targets in the genome consisting of complementary protospacer and protospacer adjacent motif (PAM). Upon base pairing, the Cas9 spacer target mediates double stranded cleavage in the target DNA. Figure 2B shows the construction of Cas9 S. pyogenes (SpCas9) and RNase III (SpRNase III) with nuclear localization cell signals (NLS) for import into the mammalian nucleus. Figure 2C shows the expression of SpCas9 and SpRNase III in mammals, driven by the constitutive promoter EF1a, and an array of tracrRNA and pre-crRNA (DR-cneftcep-DR), driven by the U6 promoter of RNA polymerase 3 to stimulate precise initiation and termination of transcription. A protospacer from a human EMX1 locus with a satisfactory PAM sequence was used as a spacer in a pre-crRNA array. Figure 2D shows a Surveyor nuclease assay for SpCas9-mediated minor insertions and deletions. SpCas9 was expressed with SpRNase III, tracrRNA, and a pre-crRNA array carrying the target spacer for EMX1, and without them. Figure 2E shows a schematic representation of base pairing between the target locus and EMR1-targeted crRNA, as well as an illustrative chromatogram showing microdeletion adjacent to the SpCas9 cleavage site. Figure 2F shows mutated alleles identified by sequencing analysis of 43 clonal amplicons, showing a variety of microinserts and microdeletions. Dashes indicate deleted bases, and unaligned or mismatched bases indicate insertions or mutations. Scale mark = 10 μm.

To further simplify the three-component system, a chimeric crRNA-tracrRNA hybrid structure was adapted in which mature crRNA (containing the guiding sequence) was fused to partial tracrRNA via a stem-loop structure to simulate the natural crRNA: tracrRNA duplex (Figure 3A). To increase the efficiency of co-delivery, a bicistronic expression vector was created to control coexpression of chimeric RNA and SpCas9 in transfected cells (Figures 3A and 8). In parallel, bicistronic vectors were used to express pre-crRNA (DR-directing sequence-DR) with SpCas9 to induce processing in crRNA involving separately expressed tracrRNA (comparison in Figure 13B: upper and lower). Figure 9 is a schematic illustration of bicistronic expression vectors for a pre-crRNA array (Figure 9A) or chimeric crRNA (represented by a short line below the insertion site of the guide sequence and above the EF1α promoter in Figure 9B) with hSpCas9 showing the position of the various elements and the points of insertion of the guide sequence . The extended sequence around the position of the insertion site of the guide sequence in Figure 9B also shows the partial DR sequence (GTTTAGAGCTA) and the partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT). Guide sequences can be inserted between BbsI sites using hybridized oligonucleotides. The sequence structures for the oligonucleotides are shown below in the schematic illustrations in figure 9 with the indicated suitable ligation adapters. WPRE is a post-transcriptional regulatory element of the marmot hepatitis virus. The effectiveness of the mediated chimeric RNA cleavage was investigated by targeted exposure to the same EMX1 locus described above. Using both Surveyor analysis and Sanger amplicon sequencing, the authors of the present invention confirmed that the chimeric structure of RNA facilitates cleavage of the human EMX1 locus with a degree of modification of approximately 4.7% (Figure 4).

The generalization of CRISPR-mediated cleavage in eukaryotic cells was studied by targeting additional genome loci in both human and mouse cells by constructing chimeric RNAs that target multiple sites in human EMX1 and PVALB as well as mouse Th loci. Figure 15 shows the selection of some additional protospeakers serving as targets at the human PVALB loci (Figure 15A) and Th mouse (Figure 15B). Schematic images of gene loci and the positions of three protospacer in the last exon of each of them are presented. Underlined sequences include a 30 bp protospeiser sequence. and 3 bp at the 3'-end corresponding to the PAM sequences. The protospacers on the sense and antisense strands are indicated above and below the DNA sequences, respectively. The degree of modification for the human PVALB and Th loci of the mouse reached 6.3% and 0.75%, respectively, demonstrating the wide applicability of the CRISPR system for the modification of various loci in many organisms (figures 3B and 6). Although using chimeric constructs, cleavage was detected with only one of the three spacers for each locus, all target sequences were cleaved with insertion / deletion efficiencies as high as 27% using the co-expressed pre-crRNA scheme (Figure 6)

Figure 13 provides a further illustration of the fact that SpCas9 can be reprogrammed to target multiple loci of the genome in mammalian cells. Figure 13A is a schematic representation of the human EMX1 locus, which shows the position of the five protospacer indicated by the underlined sequences. 13B is a schematic representation of a pre-crRNA / trcrRNA complex, showing hybridization between the direct repeat region of pre-crRNA and tracrRNA (above), and a schematic representation of a chimeric RNA structure containing a 20 bp guide sequence. and a paired tracr sequence and a tracr sequence consisting of an incomplete direct repeat and tracrRNA sequences hybridized to a hairpin structure (bottom). The results of a Surveyor analysis comparing the efficacy of Cas9-mediated cleavage in five protospacer at the human EMX1 locus are shown in Figure 13C. A targeted effect on each protospacer was carried out either using the processed pre-crRNA / tracrRNA complex (crRNA) or using chimeric RNA (chiRNA).

Since the secondary structure of RNA can be important for intermolecular interactions, the structure prediction algorithm based on the minimum free energy and the ensemble of weighted structures according to Boltzmann was used to compare the estimated secondary structure of all directing sequences used in the experiment with targeted action on the gene (Figure 3B) (see. e.g. Gruber et al., 2008, Nucleic Acids Research, 36: W70). The analysis revealed that in most cases, the effective guide sequences in the context of the chimeric crRNA, in fact, did not contain motifs of the secondary structure, while ineffective guide sequences were more likely to form internal secondary structures, which could interfere with the base pairing with the DNA of the target protospacer. Therefore, it is possible that variability in the secondary structure of the spacer may affect the effectiveness of CRISPR-mediated interference using chimeric crRNA.

Figure 3 shows an example of expression vectors. 3A is a schematic illustration of a bicistronic vector for controlling expression of the chimeric synthetic construct crRNA-tracrRNA (chimeric RNA) as well as SpCas9. The chimeric RNA guide contains a 20 bp guide sequence corresponding to the protospacer at the genomic target site. 3B is a schematic view showing guide sequences aimed at the mouse EMX1, PVALB loci of a human, and Th, as well as their predicted secondary structures. The modification efficiency at each target site is shown below the figure of the secondary structure of RNA (EMX1, n = 216 read fragments during amplicon sequencing; PVALB, n = 224 read fragments; Th, n = 265 read fragments). The results are presented for the laying algorithm of each base, colored according to its ability to accept the predicted secondary structure, as indicated by the color scale reproduced in figure 3B as a gray scale.

The structures of the additional vectors for SpCas9 are shown in FIG. 44, which shows individual expression vectors including the U6 promoter linked to the insertion site for the guide oligonucleotide and the Cbh promoter linked to the SpCas9 coding sequence. The vector shown in Figure 44b includes the coding sequence of tracrRNA linked to the H1 promoter.

In order to determine whether spacers with secondary structures are capable of functioning in prokaryotic cells, where CRISPR function in vivo, interference upon transformation with plasmids carrying protospacer was studied in E. coli strain heterologously expressing CRISPR locus 1 of S. pyogenes SF370 (figure 10). The CRISPR locus was cloned into a low copy E. coli expression vector and the crRNA array was replaced with a single spacer flanked by a DR pair (pCRISPR). E. coli strains carrying different pCRISPR plasmids were transformed with control plasmids containing the corresponding protospacer and PAM sequences (Figure 10C). When analyzing bacteria, all spacers contributed to effective CRISPR interference (Figure 4C). These results indicate that there may be additional factors affecting the effectiveness of CRISPR activity in mammalian cells.

To investigate the specificity of CRISPR-mediated cleavage, the effect of single nucleotide mutations in the guide sequence with respect to protospeiser cleavage in the mammalian genome was analyzed using a number of single point mutations targeted to EMX1 chimeric crRNAs (Figure 4A). 4B shows the results of a Surveyor nuclease assay comparing the efficiency of Cas9 cleavage when pairing with various mutant chimeric RNAs. Mismatch of one base in the area up to 12 bp from 5 'in PAM, essentially stopped the SpCas9 genome cleavage, while spacers with mutations at positions located at more distant positions above the transcription progression remained active with respect to the original protospeiser target (Figure 4B). In addition to PAM, SpCas9 is characterized by single base specificity in the last 12 bp. spacer. In addition, CRISPR is able to mediate genome cleavage as efficiently as a pair of TALE nucleases (TALEN), which specifically target the same protospacer EMX1. Figure 4C is a schematic view showing the structure of TALENs targeting EMX1, and Figure 4D shows a comparison of the efficiencies of TALEN and Cas9 (n = 3) in gel gel distillation of products obtained from a Surveyor analysis.

Having established a set of components to achieve CRISPR-mediated gene editing in mammalian cells through the error-prone NHEJ mechanism, CRISPR's ability to stimulate homologous recombination (HR), a high-precision gene repair pathway to create accurate editorial changes in the genome, was investigated. Wild-type SpCas9 is able to mediate site-specific DSBs that can be repaired using both NHEJ and HR. In addition, the replacement of aspartate-on-alanine (D10A) in the RuvC I catalytic domain in SpCas9 was designed to convert nuclease to nickase (SpCas9n; illustrated in Figure 5A) (see, for example, Sapranausaks et al., 2011, Nucleic Acids Resch, 39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109: E2579) so that the incised genomic DNA undergoes highly accurate repair using homologous recombination (HDR). Surveyor analysis confirmed that SpCas9n does not create insertions / deletions in the EMX1 protospacer target. As shown in FIG. 5B, coexpression of the chimeric crRNA with SpCas9 targeting EMX1 specifically resulted in insertion / deletion at the target site, while coexpression with SpCas9n did not (n = 3). Moreover, sequencing 327 amplicons did not reveal any SpCas9n-induced insertions / deletions. To study CRISPR HR-mediated co-transfection of HEK 293FT cells with chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, the same locus was chosen as well as the HR template for introducing a pair of restriction sites (HindIII and NheI) near the protospacer. 5C is a schematic illustration of an HR strategy with relative positions of recombination points and sequences for primer hybridization (arrows). SpCas9 and SpCas9n did catalyze the integration of the HR matrix into the EMX1 locus. PCR amplification of the target site followed by HindIII restriction digestion revealed cleavage products corresponding to the expected fragment sizes (arrows on the results of restriction fragment length polymorphism analysis using gel electrophoresis shown in Figure 5D), with SpCas9 and SpCas9n mediating similar levels of HR efficiency. Applicants further confirmed HR using Sanger genomic amplicon sequencing (Figure 5E). These results demonstrated the usefulness of CRISPR to facilitate targeted insertion of genes into the mammalian genome. Given the specificity of the targeted impact of 14 bp (12 bp from spacer and 2 bp from PAM) SpCas9 wild-type, the availability of nickase can significantly reduce the likelihood of inappropriate modifications, since single-stranded breaks are not substrates for the error-prone NHEJ path.

Expression constructs imitating the natural architecture of CRISPR loci with spacers assembled into an array (Figure 2A) created the possibility of a multiplex targeted effect on sequences. Using a single CRISPR array encoding a pair of spacers targeting EMX1 and PVALB, effective cleavage was detected at both loci (Figure 4F, which shows both the schematic structure of the crRNA array and the blot obtained after analysis by Surveyor, showing efficient cleavage mediation ) A targeted deletion of larger genomic regions was also investigated using simultaneous DSBs using spacers against two targets in EMX1 separated by 119 bp, and the detected deletion efficiency was 1.6% (3 of 182 amplicons; Figure 4G). This demonstrates that the CRISPR system can mediate multiplex editing within a single genome. Example 2: modifications and alternatives to the CRISPR system The possibility of using RNA for programming sequence-specific DNA cleavage defines a new class of tools for constructing the genome for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the effectiveness and versatility of targeted exposure using CRISPR. The optimal activity of Cas9 may depend on the availability of unbound Mg ²⁺ at levels that exceed those available in the mammalian nucleus (see, for example, Jinek et al., 2012, Science, 337: 816), and preference for the NGG motif immediately below the protospacer limits ability to target action on average for every 12 bp in the human genome (figure 11, the evaluation of both plus and minus threads in the chromosome sequences of a person). Some of these difficulties can be overcome by studying the diversity of CRISPR loci in the microbial metagenome (see, for example, Makarova et al., 2011, Nat Rev Microbiol, 9: 467). Other CRISPR loci can be transferred to the microenvironment of a mammalian cell using a method similar to that described in Example 1. For example, Figure 12 shows an adaptation of a CRISPR type II system from CRISPR 1 LMD-9 Streptococcus thermophilus for heterologous expression in mammalian cells to achieve CRISPR-mediated editing genome. Figure 12A is a schematic illustration of CRISPR 1 from S. thermophilus LMD-9. Figure 12B shows the structure of the expression system for the S. thermophilus CRISPR system. The codon-optimized human hStCas9 is expressed using the constitutive promoter EF1α. Mature variants of tracrRNA and crRNA are expressed using the U6 promoter to stimulate the precise initiation of transcription. Sequences from mature crRNA and tracrRNA are shown. One base, indicated by a lowercase letter “a” in the crRNA sequence, was used to remove the polyU sequence, which serves as the polIII RNA transcription terminator. Figure 12C is a schematic representation showing guide sequences aimed at the human EMX1 locus, as well as their predicted secondary structures. The modification efficiency at each target site is indicated below the secondary RNA structures. The algorithm that generates the colors of the structure of each base according to its ability to accept the predicted secondary structure, which is indicated by the color scale, is reproduced in figure 12C as a gray scale. Figure 12D shows the results of hStCas9-mediated cleavage at the target locus using a Surveyor assay. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively. A statistical analysis of the activity for cleavage by biological copies in these two protospacer sites is also shown in Figure 6. Figure 16 shows a schematic representation of an additional protospacer and corresponding PAM sequences that are targets for S. thermophilics CRISPR system at the human EMX1 locus. The sequences of the two protospacers are highlighted and their corresponding PAM sequences satisfying the NNAGAAW motif are indicated by underlining in the 3 'direction relative to the corresponding highlighted sequence. Both protospacer are aimed at antisense thread.

Example 3: an algorithm for selecting samples of the target sequence

A computer program was created to identify candidate CRISPR target sequences on both strands of the inserted DNA sequence based on the length of the desired guide sequence and the CRISPR motif sequence (PAM) for a particular CRISPR enzyme. For example, target sites for Cas9 from S. pyogenes with PAM NGG sequences can be identified by searching for 5'-N _x -NGG-3 'in both the input sequence and the reverse complement sequence. Similarly, target sites for Cas9 CRISPR1 S. thermophilus with the PAM NNAGAAW sequence can be identified by searching for the 5'-N _x -NNAGAAW-3 'on both the input sequence and the reverse complement sequence. Similarly, target sites for Cas9 CRISPR3 S. thermophilus with the PAM NGGNG sequence can be identified by searching for 5'-N _x -NGGNG-3 'in both the input sequence and the back-complementary sequence. The value of "x" in N _x may be fixed by the program or may be determined by the user, such as 20.

Since several cases of the appearance of the target DNA site in the genome can lead to non-specific editing of the genome, after identifying all possible sites, the program filters the sequences based on the number of times they occur in the corresponding reference genome. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence, such as 11-12 bp in the direction 5 'from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Therefore, in order to avoid editing at additional loci of the genome, the results are filtered based on the number of cases when a seed sequence has been detected: PAM in a suitable genome. The user may be able to select the length of the seed sequence. The user may also be able to determine the number of times a seed sequence has been detected: PAM in the genome as applied to the passage of the filter. The default screening is for unique sequences. The filtering level is changed by changing both the length of the seed sequence and the number of cases of sequence detection in the genome. As an addition or alternative, the program can provide a sequence of the guide sequence complementary to the reported target sequence (s) by providing a sequence that is backward complementary to the identified target sequence (s).

Further details of the methods and algorithms for optimizing sequence selection can be found in the US patent application with serial number 61/836080 (case number of the patent attorney 44790.11.2022); incorporated herein by reference.

Example 4: Evaluation of the hybrids of several chimeric crRNA-tracrRNA

This example describes the results obtained for chimeric RNAs (chiRNAs; containing the guide sequence, paired tracr sequence and tracr sequence in one transcript) having tracr sequences that include fragments of the wild-type tracrRNA sequence with different lengths. Figure 18a shows a schematic representation of a bicistronic expression vector for chimeric RNA and Cas9. Cas9 is driven by the CBh promoter, and chimeric RNA is driven by the U6 promoter. The chimeric RNA guide consists of a 20 bp guide sequence (Ns) connected to a tracr sequence (extending from the first “U” in the lower strand to the end of the transcript), which is truncated at different indicated positions. Guides and tracr sequences are separated by the GUUUUAGAGCUA paired tracr sequence, followed by the GAAA loop sequence. SURVEYOR assays for Cas9-mediated insertions / deletions at the human EMX1 and PVALB loci are shown in Figures 18b and 18c, respectively. Arrows indicate the expected fragments resulting from cleavage using SURVEYOR. ChiRNAs are shown by naming them “+ n,” and crRNA refers to hybrid RNA in which the guiding and tracr sequences are expressed as separate transcripts. A quantitative analysis of these results, performed in triplicate, is illustrated using the histograms in figures 19a and 19b, corresponding to figures 18b and 18c, respectively ("N.D." means the absence of detected inserts / deletions). The ID (identification data) of the protospacer and their corresponding target in the genome, the protospacer sequence, the PAM sequence and the position of the strand are shown in Table D. The guide sequences are designed so that they are complementary to the complete protospacer sequence in the case of individual transcripts in the hybrid system or only the underlined part in case of chimeric RNA.

Cell Culture and Transfection

The human embryo kidney cell line (HEK) 293FT (Life Technologies) was maintained in Eagle's Dulbecco modification (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / ml penicillin and 100 μg / ml of streptomycin, at 37 ° C with incubation at 5% CO ₂ . 293FT cells were seeded in 24-well plates (Corning) 24 hours prior to transfection at a density of 150,000 cells per well. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer's recommended protocol. A total of 500 ng plasmids were used for each well of the 24-well plate.

SURVEYOR analysis for genome modification

293FT cells were transfected with plasmid DNA as described above. Cells were incubated at 37 ° C for 72 hours after transfection before genomic DNA extraction. Genomic DNA was extracted with QuickExtract DNA Extraction Solution (Epicentre) following the manufacturer's protocol. Briefly, centrifuged cells were resuspended in a QuickExtract solution and incubated at 65 ° C for 15 minutes and 98 ° C for 10 minutes. The genomic region surrounding the CRISPR target site was PCR amplified for each gene (primers are listed in Table E) and the products were purified using a QiaQuick Spin Column (Qiagen) column following the manufacturer's protocol. A total of 400 ng of purified PCR products were mixed with 2 μl of 10X Taq DNA Enzymatics PCR buffer and ultra-high purity water to a final volume of 20 μl and subjected to a re-annealing process to ensure heteroduplex formation: 95 ° C for 10 min., linear decrease in temperature from 95 ° C to 85 ° C at a rate of 2 ° C / s, from 85 ° C to 25 ° C at a speed of 0.25 ° C / s and withstand at 25 ° C for 1 minute . After repeated annealing, the products were treated with SURVEYOR nuclease and S SURVEYOR enhancer (Transgenomics), following the protocol recommended by the manufacturer, and analyzed in 4-20% Novex TBE polyacrylamide gels (Life Technologies). The gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and the image was obtained using the Gel Doc gel imaging system (Bio-rad) image processing system. Quantitative analysis was based on the relative intensities of the bands.

Computational Identification of Unique CRISPR Target Sites

To identify unique target sites for the Cas9 (SpCas9) S. pyogenes SF370 enzyme in the human, mouse, rat, zebrafish, fruit fly, and C. elegans genome, a software package was developed to scan both strands of the DNA sequence and identify all possible SpCas9 target sites. For this example, each SpCas9 target site was promptly identified as a 20 bp sequence, followed by a sequence of motif adjacent to the NGG proto-spacer (PAM), with all sequences meeting the definition of 5'-N ₂₀ -NGG- 3 'on all chromosomes. To prevent non-specific editing of the genome after identification of all potential sites, all target sites were filtered based on the number of times they are found in the corresponding reference genome. In order to benefit from the specificity for the Cas9 activity sequence provided by the “seed” sequence, which may be, for example, a sequence of about 11-12 bp 5 'from the PAM sequence, wherein the 5'-NNNNNNNNNN-NGG-3' sequences were selected as unique in the corresponding genome. All genomic sequences were downloaded from the UCSC genomic browser (hg19 human genome, mm9 mouse genome, rn5 rat genome, danRer7 zebrafish genome, D. melanogaster dm4 genome, and C. elegans ce10 genome). All search results are available for viewing using information from the genomic browser UCSC. Illustrative visualization of some target sites in the human genome is presented in figure 21.

Three sites within the EMX1 locus in human HEK 293FT cells were initially targeted. The effectiveness of the modification of the genome of each chiRNA was assessed using SURVEYOR nuclease analysis, which allows the detection of mutations resulting from double-stranded DNA breaks (DSB) and their subsequent repair using the DNA damage repair pathway due to non-homologous termination (NHEJ). The constructs denoted by chiRNA (+ n) indicate that nucleotides of up to + n wild type tracrRNA nucleotides are included in the chimeric RNA construct, with values 48, 54, 67 and 85 used for n. Chimeric RNAs containing longer wild-type tracrRNA fragments (chiRNA (+67) and chiRNA (+85)) mediated DNA cleavage at all three EMX1 target sites, with chiRNA (+85), in particular, showing significantly higher levels of DNA cleavage than the corresponding crRNA hybrids / tracrRNA, in which the guiding and tracr sequences are expressed in separate transcripts (figures 18b and 19a). Two sites at the PVALB locus that did not show detectable cleavage using the hybrid system (the guide sequence and the tracr sequence expressed as separate transcripts) were also targeted using chiRNA. ChiRNA (+67) and chiRNA (+85) were able to mediate significant cleavage in the two protospacer in PVALB (figures 18c and 19b).

For all five targets at the EMX1 and PVALB loci, a corresponding increase in the efficiency of genome modification was observed with an increase in the length of the tracr sequence. Without going into any theory, the secondary structure formed by the 3'-end of tracrRNA can play a role in increasing the rate of formation of the CRISPR complex. An illustration of the predicted secondary structures for each chimeric RNA used in this example is shown in Figure 21. The secondary structure was predicted using RNAfold (http://RNA.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) using minimum free energy and distribution function algorithm. The pseudocolor for each base (reproduced in the gray scale) indicates the possibility of mating. Due to the fact that chiRNAs with longer tracr sequences were able to cleave targets that were not cleaved by native crRNA / tracrRNA CRISPR hybrids, it is possible that chimeric RNA can be loaded onto Cas9 more efficiently than its native hybrid counterpart. To ensure the use of Cas9 for site-specific editing of the genome in eukaryotic cells and organisms, all predicted unique target sites for Cas9 S. pyogenes were determined by calculations in the genomes of human, mouse, rat, zebrafish, C. elegans and D. melanogaster. Chimeric RNAs can be developed for Cas9 enzymes from other microorganisms to expand the target space of CRISPR RNA-programmed nucleases.

22 shows an exemplary bicistronic expression vector for chimeric RNA expression, including the wild-type tracr sequence of RNA up to nucleotide +85 and SpCas9 with nuclear localization sequences. SpCas9 is expressed from the CBh promoter and terminated with the bGH polyA signal (bGH pA). The extended sequence shown immediately below the schematic diagram corresponds to the region surrounding the guide sequence of the embedment site, and includes from 5 'to 3', the 3 'part of the U6 promoter (first hatched region), BbsI cleavage sites (arrows), incomplete direct repetition ( paired GTTTTAGAGCTA tracr sequence, underlined), GAAA loop sequence and +85 tracr sequence (underlined sequence following the loop sequence). Illustrative insertion of the guide sequence is shown below the site of insertion of the guide sequence, with the nucleotides of the guide sequence for the selected target are represented as "N".

The sequences described in the above examples are as follows (polynucleotide sequences are from 5 ′ to 3 ′):

U6-short tracrRNA (Streptococcus pyogenes SF370):

(жирный шрифт = последовательность tracrRNA; подчеркивание = терминаторная последовательность)

(bold = tracrRNA sequence; underscore = terminator sequence)

U6-long tracrRNA (Streptococcus pyogenes SF370):

U6-DR-BbsI-ocTOB-DR (Streptococcus pyogenes SF370):

U6-chimeric PHK-BbsI-skeleton (Streptococcus pyogenes SF370)

NLS-SpCas9-EGFP:

SpCas9-EGFP-NLS:

NLS-SpCas9-EGFP-NLS:

NLS-SpCas9-NLS:

NLS-mCherry-SpRNase3:

SpRNase3-mCherry-NLS:

NLS-SpCas9n-NLS (D10A nickase mutation is lowercase):

hEMX1-HR-matrix-HindII-NheI:

NLS-StCsnl-NLS:

U6-St_tracrRNA (7-97):

U6-DR-cneficep-DR (S. pyogenes SF370)

(нижний регистр, подчеркивание = прямой повтор; N = направляющая последовательность; жирный шрифт = терминатор)

(lower case, underline = direct repeat; N = guide sequence; bold = terminator)

Chimeric RNA containing +48 tracrRNA (S. pyogenes SF370)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Chimeric RNA containing +54 tracrRNA (S. pyogenes SF370)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Chimeric RNA containing +67 tracrRNA (S. pyogenes SF370)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Chimeric RNA containing +85 tracrRNA (S. pyogenes SF370)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

CBh-NLS-SpCas9-NLS

(underline = NLS-hSpCas9-NLS)

Illustrative chimeric RNA for Cas9 from CPJSPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilic (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CPJSPR1 LMD-9 S. thermophilus (with PAM NNAGAAW)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Illustrative chimeric RNA for Cas9 from CRISPR3 LMD-9 S. thermophilus (with PAM NGGNG)

(N = направляющая последовательность; первое подчеркивание = парная tracr-последовательность; второе подчеркивание = tracr-последовательность; жирный шрифт = терминатор)

(N = guide sequence; first underscore = paired tracr sequence; second underscore = tracr sequence; bold = terminator)

Codon-optimized version of Cas9 from the CRISPR3 LMD-9 S. thermophilus locus (with NLS at both 5'- and 3'-ends)

Example 5: RNA-directed editing of bacterial genomes using CRISPR-Cas systems

Applicants have used Cas9 CPJSPR-associated endonuclease to introduce precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relied on Cas9-directed cleavage at the target site to kill unmuted cells and eliminated the need for selectable markers or negative selection systems. The specificity of Cas9 was reprogrammed by changing the sequence of short CRISPR RNA (crRNA) to introduce single or multi-nucleotide changes carried on editing matrices. The simultaneous use of two crRNAs provided multiplex mutagenesis. In S. pneumoniae, about 100% of the cells surviving after Cas9 cleavage contained the desired mutation, and 65% when used in combination with recombination engineering in E. coli. Applicants carefully analyzed the planned requirements for Cas9 to determine the range of sequences representing the targets and showed strategies for editing sites that do not meet these requirements, which indicates the universality of this technique for constructing the bacterial genome.

Understanding gene function depends on the ability to change DNA sequences in a cell in a controlled manner. Site-specific mutagenesis in eukaryotes is achieved by using sequence-specific nucleases that activate homologous recombination of a DNA template containing a mutation of interest. Zinc-finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and homing meganucleases can be programmed to cleave genomes at specific locations, but these approaches require the construction of new enzymes for each target sequence. In prokaryotic organisms, mutagenesis methods either involve the introduction of a selection marker at an editable locus, or require a two-step process that includes a negative selection system. More recently, recombinant phage proteins, a technique that provides homologous recombination of linear DNA or oligonucleotides, have been used for recombinant engineering. However, since there is no choice of mutations, the efficiency of recombination engineering can be relatively low (from 0.1-10% for point mutations up to 10 ^-5 -10 ^-6 for more extensive modifications), in many cases requiring screening of a larger number of colonies. Thus, new technologies are still required that are affordable, easy to use and effective for the genetic construction of both eukaryotic and prokaryotic organisms.

Recent work on CRISPR ("short palindromic repeats, regularly arranged in groups) of the prokaryotic adaptive immune system has led to the identification of nucleases in which sequence specificity is programmed by small RNAs. CRISPR loci consist of a series of repeats separated by spacer sequences that correspond to bacteriophage genomes and other mobile genetic elements.The array of repeat spacers is transcribed as a long predecessor and is processed in sequences of second, to create a small crRNA that defines the target sequences (also known as protospacer) cleaved using CRISPR systems. Important for cleavage is the presence of a sequence motif immediately below the target region, known as the adjacent protospeiser motif (PAM). CRISPR-associated (cas) genes typically flank an array of repeat spacers and encode the enzymatic mechanism responsible for crRNA biogenesis and targeted exposure. Cas9 is a dsDNA endonuclease that uses a crRNA guide to determine the cleavage site. The loading of the crRNA guide onto Cas9 occurs during processing of the crRNA precursor and requires the presence of small antisense RNA on the precursor, tracrRNA and RNAse III. Unlike genome editing using ZFN or TALEN, changing the specificity of targeted Cas9 exposure does not require the construction of proteins, but only the development of a short crRNA guide.

Applicants have recently shown to S. pneumoniae that the introduction of a CRISPR system aimed at the chromosomal locus leads to the destruction of transformed cells. It was noted that accidentally surviving cells contained mutations in the target region, which indicated that endonuclease activity directed to dsDNA, Cas9 in relation to endogenous targets could be used to edit the genome. Applicants have shown that markerless mutations can be introduced by transforming a fragment of a DNA template that will recombine in the genome and exclude recognition of Cas9 targets. Cas9 specificity management with several different crRNAs allows multiple mutations to be introduced simultaneously. Applicants have also described in detail the sequence requirements for targeted Cas9 exposure and have shown that the approach can be combined with recombination engineering to edit the genome in E. coli.

RESULTS: Genome editing using Cas9 cleavage of a chromosome target

Strain crR6 S. pneumoniae contains a Cas9-based CRISPR system that cleaves the target sequence present in the bacteriophage φ8232.5. This target was integrated into the srtA chromosomal locus of the second strain R6 ^8232.5 . The altered target sequence containing the mutation in the PAM region was integrated into the srtA locus of the third strain R6 ^370.1 , making this strain "resistant" to cleavage using CRISPR (Figure 28a). Applicants transformed genomic DNA cells from crR6 cells, expecting that successful transformation of R6 ^8232.5 cells should result in cleavage of the target locus and cell death. Contrary to this expectation, the applicants isolated R6 ^8232.5 transformants, although with about 10 times less potency than R6 ^370.1 transformants (Figure 28b). Genetic analysis of eight transformants of R6 ^8232.5 (Figure 28) revealed that the vast majority are the product of a double recombination phenomenon that excludes the toxicity of targeted Cas9 exposure by replacing the φ8232.5 target with the wild-type srtA locus of the crR6 genome that does not contain the protospacer required for Cas9 recognition. These results were evidence that the simultaneous introduction of a CRISPR system aimed at the genome locus (construct for targeted exposure), together with a matrix for recombination at the target locus (editing matrix), led to targeted editing of the genome (Figure 23a).

To create a simplified system for editing the genome, the applicants modified the CRISPR locus in the crR6 strain by deletion of the cas1, cas2 and csn2 genes, which were shown to be optional for targeted CRISPR exposure, to obtain the crR6M strain (Figure 28a). This strain retained the same properties as crR6 (Figure 28b). In order to increase Cas9-based editing efficiency and demonstrate that the selected DNA template can be used to control the introduced mutations, the applicants co-transformed R6 ^8232.5 cells with PCR products of the wild-type srtA gene or mutant target R6 ^370.1 , each of which must be splitting resistant Cas9. This led to a 5 to 10-fold increase in the frequency of transformation compared to the genomic crR6 DNA alone (Figure 23b). Editing efficiency also increased significantly, with 8/8 of the tested transformants containing a wild-type srtA copy, and 7/8 containing the PAM mutation present in target R6 ^370.1 (Figure 23b and Figure 29a). Taken together, these results indicate the ability to edit the genome using Cas9.

Analysis of planned requirements for Cas9. To introduce specific changes in the genome, one needs to use an editing matrix that carries mutations that stop the cleavage mediated by Cas9, thereby preventing cell death. This is easy to achieve when a deletion of the target or its substitution by another sequence (insertion of a gene) is necessary. If the goal is to create gene fusions or to create single-nucleotide mutations, the elimination of the nuclease activity of Cas9 will be possible only by introducing mutations into the editing matrix, which alter the sequences of either PAM or protospacer. To determine the difficulties of CRISPR-mediated editing, applicants performed an exhaustive analysis of PAM and protospacer mutations that suppress the targeted effect of CRISPR.

Previous studies have suggested that Cas9 S. pyogenes requires the presence of PAM NGG directly below the protospacer. However, due to the fact that only a very limited number of mutations inactivating PAM have been described so far, applicants have carried out a systematic analysis to detect all 5-nucleotide sequences located after the protospacer that exclude CRISPR cleavage. Applicants used randomized oligonucleotides to create all possible 1024 PAM sequences in a heterogeneous PCR product that was transformed into crR6 or R6 cells. It was expected that constructs bearing functional PAMs would be recognized and destroyed by Cas9 in crR6 cells, but not in R6 cells (Figure 24a). More than 2 × 10 ⁵ colonies were combined together to extract DNA for use as a template for co-amplification of all targets. The PCR products were deeply sequenced, and it was found that they contain all 1024 sequences, overlapping in the range from 5 to 42472 read fragments (see the section "Analysis of deep sequencing data"). The functionality of each PAM was evaluated by the relative fraction of its read fragments in the sample crR6 relative to sample R6. Analysis of the first three bases of PAM averaged over the last two bases clearly showed that the NGG pattern was not sufficiently represented in crR6 transformants (Figure 24b). In addition, the following two bases did not significantly affect NGM PAM (see "Deep Sequencing Analysis" section), demonstrating that the NGGNN sequence was sufficient to resolve Cas9 activity. Partial targeted exposure was observed for PAM NAG sequences (Figure 24b). Also, the NNGGN pattern partially inactivated the targeted effect of CRISPR (Table G), which indicated that with a shift of 1 bp the NGG motif could still be recognized by Cas9 with reduced efficiency. These data shed light on the molecular mechanism of Cas9 target recognition, and it was found that the NGG sequences (or CCN in the complementary strand) are sufficient for targeted Cas9 exposure, and that NGG mutations on the NAG or NNGGN in the editing matrix should be avoided. Due to the high frequency of these trinucleotide sequences (every 8 bp), this means that almost any position in the genome can be edited. Moreover, the applicants tested ten randomly selected targets carrying different PAMs, and it was found that they were all functional (Figure 30).

Another way of disrupting Cas9-mediated cleavage is to introduce mutations into the protospacer region of the editing matrix. It is known that point mutations in the "seed sequence" (from 8 to 10 nucleotides of the protospacer directly adjacent to PAM) can stop cleavage with CRISPR nucleases. However, the exact length of this region is unknown, and it is not clear whether mutations in any nucleotide in the seed can interfere with Cas9 target recognition. Applicants followed the same deep sequencing approach as described above to randomize the whole protospacer sequence involved in base pair contacts with crRNA and to identify all sequences that interfere with targeted exposure. Each position of 20 matching nucleotides (14) in the spc1 target present in R6 ^8232.5 cells (Figure 23a) was randomized and transformed into crR6 and R6 cells (Figure 24a). In accordance with the presence of the seed sequence, only mutations in 12 nucleotides immediately above PAM suppressed cleavage with Cas9 (Figure 24c). However, different mutations displayed very different effects. Distal (from PAM) seed positions (12 to 7) withstood most mutations, and only one definite substitution of the base suppressed the targeted effect. At the same time, mutations in any nucleotide at proximal positions (from 6 to 1, with the exception of 3) excluded Cas9 activity, but at different levels for each specific substitution. At position 3, only two substitutions affected CRISPR activity and with different strengths. Applicants have concluded that although mutations in the seed sequence can prevent targeted effects of CRISPR, there are limitations to changes in nucleotides that can be made at any position of the seed. Moreover, these limitations are most likely to vary for different spacer sequences. Thus, applicants believe that mutations in the PAM sequence, if possible, should be the preferred editing strategy. Alternatively, multiple mutations in the seed sequence can be introduced to prevent the nuclease activity of Cas9.

Cas9-mediated genome editing in S. pneumonia. To develop a quick and effective method for targeted genome editing, applicants constructed a crR6Rk strain, a strain in which spacers can be easily introduced using PCR (Figure 33). Applicants decided to edit the S. pneumoniae β-galactosidase (bgaA) gene, the activity of which can be easily measured. Applicants introduced alanine amino acid substitutions into the active site of this enzyme: Mutations R481A (R → A) and N563A, E564A (NE → AA). To illustrate various editing strategies, applicants have developed mutations of both the PAM sequence and the protospacer primer. In both cases, the same construct was used for targeted exposure to crRNA, a complementary region of the β-galactosidase gene that is adjacent to the PAM TGG sequence (CCA in a complementary strand, Figure 26). The editing matrix R → A produced a trinucleotide mismatch in the seed sequence of the protospacer (CGT on GCA, also with the introduction of the BtgZI restriction site). Applicants simultaneously introduced a synonymous mutation into the NE → AA editing matrix, which produced an inactive PAM (TGG on TTG), together with mutations that were 218 nucleotides lower from the protospacer site (AAT GAA on GCT GCA, also with the creation of the TseI restriction site). This latest editing strategy has demonstrated the ability to use remote PAM to create mutations in places where a suitable target can be difficult to choose. For example, although the S. pneumoniae R6 gene, which is characterized by a GC content of 39.7%, contains on average one PAM motif per 12 bp, some PAM motifs are divided up to 194 bp. (figure 33). In addition, applicants developed a deletion within the 6,664 bp ΔbgaA reading frame. In all three cases, cotransformation of targeted action and editing matrices produced 10 times more kanamycin-resistant cells than cotransformation with a control editing matrix containing wild-type bgaA sequences (Figure 25b). Applicants genotyped 24 transformants (8 for each editing experiment) and found that all but one included the desired change (Figure 25c). DNA sequencing also confirmed not only the presence of introduced mutations, but also the absence of secondary mutations in the target region (Figure 29b, c). Finally, applicants measured β-galactosidase activity to confirm that all edited cells showed the expected phenotype (Figure 25d).

Cas9-mediated editing can also be used to create multiple mutations to study biological pathways. Applicants have decided to illustrate this for the sortase-dependent pathway by which surface proteins attach to the membrane of gram-positive bacteria. Applicants introduced a deletion of the sortase gene by cotransformation of a chloramphenicol-resistant construct for targeted exposure and an ΔsrtA editing matrix (Figure 33a, b) followed by a deletion of ΔbgaA using a kanamycin-resistant construct for targeted exposure that replaced the previous one. In S. pneumoniae, β-galactosidase covalently binds to the cell wall using sortase. Thus, a srtA deletion leads to the release of a surface protein into the supernatant, while a double deletion does not lead to a noticeable activity of β-galactosidase (Figure 34c). This sequential selection can be repeated as many times as required to create multiple mutations.

These two mutations can also be introduced simultaneously. The applicants developed a design for targeted exposure, containing two spacers, one matching srtA and one matching bgaA, and co-transformed it with two editing matrices (Figure 25e). Genetic analysis of transformants showed that editing occurred in 6/8 cases (figure 251). In particular, each of the two remaining clones contained a deletion ΔsrtA or ΔbgaA, which suggested the possibility of performing combinatorial mutagenesis using Cas9. Finally, to eliminate the CRISPR sequences, applicants introduced a plasmid containing the bgaA target and the spectinomycin resistance gene together with the genomic DNA from the wild-type R6 strain. Spectinomycin-resistant transformants that retained the plasmid eliminated CRISPR sequences (Figure 34a, d).

The mechanism and effectiveness of editing. To understand the mechanisms underlying genome editing using Cas9, applicants modeled an experiment in which editing efficiency was measured independently of Cas9 cleavage. Applicants have integrated the ermAM erythromycin resistance gene into the srtA locus and introduced a premature stop codon using Cas9-mediated editing (Figure 33). The resulting strain (JEN53) contains the ermAM allele (stop) and is sensitive to erythromycin. This strain can be used to assess the effectiveness with which the ermAM gene is repaired by quantifying the fraction of cells in which antibiotic resistance is restored with or without Cas9 cleavage. JEN53 was transformed with an editing matrix that reconstructs the wild-type allele, together with either the kanamycin-resistant CRISPR construct targeting the ermAM (stop) allele (CRISPR :: ermAM (stop)), or with the control construct without a spacer (CRISPR :: ∅) ( figure 26a, b). In the absence of kanamycin selection, the fraction of the edited colonies was of the order of 10 ^-2 (erythromycin-resistant CFU / all CFU) (Figure 26c), which represented the base recombination frequency without Cas9-mediated selection against unedited cells. However, if kanamycin selection was used and the CRISPR control construct was co-transformed, the fraction of the edited colonies increased to about 10 ^-1 (resistant to kanamycin and erythromycin CFU / resistant to kanamycin CFU) (Figure 26c). This result shows that the CRISPR locus was selected for recombination together with the recombination selection at the ermAM locus, regardless of the Cas9 genome cleavage, which indicated that the cell subpopulation was more susceptible to transformation and / or recombination. Transformation of the CRISPR :: ermAM construct (stop) followed by cantamycin selection led to an increase in the fraction of erythromycin-resistant edited cells to 99% (Figure 26c). To determine whether this increase was due to unedited cells, applicants compared kanamycin-resistant colony forming units (CFU) obtained after cotransformation of JEN53 cells with the CRISPR :: ermAM (stop) or CRISPR :: ∅ constructs.

Applicants counted 5.3 times less kanamycin-resistant colonies after transformation with the ermAM (stop) construct (2.5 × 10 ⁴ / 4.7 × 10 ³ , Figure 35a), a result that confirms that a certain targeted effect of Cas9 on the chromosomal the locus leads to the death of unedited cells. Finally, since the introduction of dsDNA breaks into the bacterial chromosome is known to trigger repair mechanisms that increase the level of recombination of damaged DNA, the applicants investigated whether Cas9 cleavage induces recombination of the editing matrix. Applicants counted 2.2 times more colonies after cotransformation with the CRISPR :: erm (stop) construct than with the CRISPR :: ∅ construct (Figure 26d), which indicated a slight induction of recombination. Taken together, these results indicate that both co-selection of transformed cells, and induction of recombination using Cas9-mediated cleavage, and selection against unedited cells contributed to the high efficiency of genome editing in S. pneumoniae.

Since genome cleavage with Cas9 should destroy unedited cells, it was not expected to receive any cells that received a kanamycin resistance cassette containing Cas9, but not an editing matrix. However, in the absence of an editing matrix, applicants received many kanamycin-resistant colonies after transformation with the CRISPR :: ermAM (stop) construct (Figure 35a). These cells, which "escaped" CRISPR-induced death, created a background that determined the limit of the method. This background frequency can be calculated in this experiment as the ratio CRISPR :: ermAM (stop) / CRISPR :: ∅ CFU, 2.6 × 10 ^-3 (7.1 × 10 ¹ / 72.7 × 10 ⁴ ), implying that if the recombination frequency of the editing matrix is less than this value, selection using CRISPR cannot effectively provide the desired mutants in quantities higher than the background. To understand the origin of these cells, applicants genotyped 8 background colonies and found that 7 contained spacer deletions for targeted exposure (Figure 35b), and one carried a supposedly inactivating mutation in Cas9 (Figure 35c).

Editing the Cas9 genome in E. coli. Activation of the targeted action of Cas9 through the chromosomal integration of the CRISPR-Cas system is the only possible in organisms in which a high frequency of recombination is observed. To develop a more general method that is applicable to other microorganisms, the applicants decided to edit the genome of E. coli using a CRISPR-Cas plasmid-based system. Two plasmids were created: the pCas9 plasmid carrying tracrRNA, Cas9 and the chloramphenicol resistance cassette (Figure 36), and the kanamycin resistance plasmid pCRISPR carrying the array of CRISPR spacers. To evaluate the effectiveness of editing, regardless of the selection using CRISPR, the applicants sought to introduce a transversion of A to C in the rpsL gene, causing resistance to streptomycin. Applicants created the plasmid pCRISPR :: rpsL carrying a spacer that would direct the Cas9 cleavage of the wild-type, but not mutant, rpsL allele (Figure 27b). The E. coli MG1655 plasmid pCas9 was first introduced and the resulting strain was co-transformed with the plasmid pCRISPR :: rpsL and W542, an editing oligonucleotide containing a mutation of substitution A to C. Streptomycin-resistant colonies after transformation with plasmid pCRISPR :: rpsL were the only ones obtained, which indicates that Cas9 cleavage induces the recombination of this oligonucleotide (Figure 37). However, the number of streptomycin-resistant colonies was two orders of magnitude less than the number of kanamycin-resistant colonies, which are presumably cells that avoid cleavage with Cas9. Thus, under these conditions, Cas9 cleavage facilitated the introduction of a mutation, but with an efficiency that was insufficient to select mutant cells in an amount higher than the background number of "cells that escaped exposure."

To increase the efficiency of genome editing in E. coli, the applicants applied their CRISPR system with recombination engineering using Cas9-induced cell death for selection according to the desired mutations. Plasmid pCas9 was introduced into strain HME63 (31), which was subjected to recombination engineering with the functions Gam, Exo, and Beta of phage n-red. The resulting strain was co-transformed with the plasmid pCRISPR :: rpsL (or control pCRISPR :: ∅) and the oligonucleotide W542 (Figure 27a). The efficiency of recombination engineering, calculated as the fraction of all cells that became resistant to streptomycin, using a control plasmid was 5.3 × 10 ^-5 (Figure 27c). At the same time, transformation with plasmid pCRISPR :: rpsL increased the percentage of mutant cells to 65 ± 14% (figures 2c and 29f). Applicants observed that the number of CFU after transformation with the plasmid pCRISPR :: rpsL was approximately three orders of magnitude lower than after transformation with the control plasmid (4.8 × 10 ⁵ / 5.3 × 10 ² , figure 38a), which indicated that selection is a consequence of CRJSPR-induced death of unedited cells. To measure the level at which Cas9 cleavage, which is an important parameter of the applicants method, was inactivated, the applicants transformed cells either with pCRISPR :: rpsL or with a control plasmid without oligonucleotide for editing W542 (Figure 38a). This background number of CRISPR “escaped cells”, measured as pCRISPR :: rpsL / pCRISPR :: ∅ CFU, was 2.5 × 10 ⁻⁴ (1.2 × 10 ² / 4.8 × 10 ⁵ ). Genotyping of eight of these cells that escaped exposure revealed that a spacer deletion for targeted exposure was present in all cases (Figure 38b). This background amount was higher than the recombination engineering efficiency for the rpsL mutation, 5.3 × 10 ^-5 , which indicated that Cas9 cleavage should induce oligonucleotide recombination to obtain 65% of the edited cells. To confirm this, the applicants compared the number of resistant to kanamycin and streptomycin CFU after transformation pCRISPR :: rpsL or pCRISPR :: ∅ (figure 27d). As in the case of S. pneumoniae, applicants observed a slight induction of recombination, approximately 6.7-fold (2.0 × 10 ^-4 / 3.0 × 10 ^-5 ). Taken together, these results indicate that the CRISPR system provided a method for selecting mutations introduced by recombination engineering.

Applicants have shown that CRISPR-Cas systems can be used for targeted editing of the genome in bacteria by co-introducing a construct for targeted exposure that destroys wild-type cells and editing matrices that jointly excluded CRISPR cleavage and enabled the introduction of the desired mutations. You can create different types of mutations (insertions, deletions or scar-less single nucleotide substitutions). Multiple mutations can be introduced simultaneously. The specificity and versatility of editing using the CRISPR system was based on several unique properties of Cas9 endonuclease: (i) its specificity of targeted exposure can be programmed with small RNAs without the need to construct an enzyme, (ii) specificity of targeted exposure determined using RNA-DNA interaction over 20 p .about. with a low probability of recognition of false targets, it was very high, (iii) almost any sequence can be targeted, the only requirement is the presence of an adjacent NGG sequence, (iv) almost any mutation in the NGG sequence, as well as mutations in the protospacer seed sequence, exclude targeted exposure .

Applicants have shown that constructing a genome using the CRISPR system worked not only in bacteria that have a high recombination rate, such as S. pneumoniae, but also in E. coli. The E. coli results indicated that the method can be applied to other microorganisms to which plasmids can be introduced. In E. coli, this approach complements the recombination engineering of mutagenic oligonucleotides. To use this method in microorganisms where recombination engineering is impossible, the mechanism of homologous recombination of the host can be applied by providing an editing matrix in the plasmid. In addition, since the accumulated data indicate that CRISPR-mediated cleavage leads to the death of cells of many bacteria and archaea, it can be assumed that CRISPR-Cas endogenous systems are used for editing purposes.

In both S. pneumoniae and E. coli, applicants observed that although editing was facilitated by co-selection of transformable cells and a slight induction of recombination at the target site due to Cas9 cleavage, the mechanism that contributed to editing to the greatest extent was selection against unedited cells. Thus, the main limitation of the method was the presence of a background of cells that avoided CRISPR-induced cell death and did not have the desired mutation. Applicants have shown that these “avoidant cells" arose primarily by deletion of the spacer for targeted exposure, presumably after recombination of the repeat sequences that flank the spacer for targeted exposure. Further improvement can be concentrated on the design of flanking sequences to exclude recombination, which can still support the biogenesis of functional crRNAs, but which are quite different from each other. Alternatively, direct transformation of chimeric crRNAs can be investigated. In the particular case of E. coli, the creation of a CRISPR-Cas system was not possible if this organism was also used as a host for cloning. Applicants solved this problem by placing Cas9 and tracrRNA on one plasmid, and a CRISPR array on another. The construction of an inducible system can also circumvent this limitation.

Although new DNA synthesis technologies provide the opportunity to create any sequence with high productivity at optimal costs, the problem of integrating synthetic DNA into living cells to create functional genomes remains. It has recently been shown that the co-selection strategy of MAGE improves the efficiency of recombination engineering mutations by selecting a subpopulation of cells that are more likely to achieve recombination at or near a given locus. In this method, the introduction of breeding mutations is used to increase the likelihood of creating nearby non-breeding mutations. In contrast to the indirect selection provided by this strategy, the use of the CRISPR system makes it possible to perform direct selection for the desired mutation and obtain it with high efficiency. These technologies complement the toolbar of specialists in the field of genetic engineering and, together with DNA synthesis, they can significantly increase both the ability to decrypt gene functions and manipulate organisms for biotechnological purposes. Two other studies also relate to the construction of mammalian genomes using CRISPR. It is expected that these crRNA-directed genome editing technologies can be widely applied in basic and medical sciences.

Strains and cultivation conditions. Strain R6 S. pneumoniae was provided by Dr. Alexander Tomasz. Strain crR6 was obtained in a previous study. Cultures of S. pneumoniae in liquid medium were grown in THYE (30 g / L Todd-Hewitt agar, 5 g / L yeast extract). Cells were plated on tryptic soy agar (TSA) supplemented with 5% defibrinated sheep blood. If necessary, the following antibiotics were added: kanamycin (400 μg / ml), chloramphenicol (5 μg / ml), erythromycin (1 μg / ml) streptomycin (100 μg / ml), or spectinomycin (100 μg / ml). Measurements of β-galactosidase activity were performed using Miller's assay as previously described.

Strains of E. coli MG1655 and HME63 (derived from MG1655, Δ (argF-lac) U169 λ cI857 Δcro-bioA galK tyr 145 UAG mutS <> amp) (31) were provided by Jeff Roberts and Donald Court, respectively. E. coli cultures in liquid medium were grown in LB medium (Difco). If necessary, the following antibiotics were added: chloramphenicol (25 μg / ml), kanamycin (25 μg / ml) and streptomycin (50 μg / ml).

Transformation of S. pneumoniae. Competent cells were obtained as previously described (23). For all transformations for editing the genome, cells were thawed carefully on ice and resuspended in 10 volumes of M2 medium with the addition of 100 ng / ml of competence-stimulating peptide CSP1 (40) and then editing constructs were added (editing constructs were added to the cells at a final concentration from 0.7 ng / μl to 2.5 μg / μl). Cells were incubated for 20 minutes. at 37 ° C before adding 2 μl of constructs for targeted exposure and then incubated for 40 min. at 37 ° C. Serial cell dilutions were plated on an appropriate medium to determine the number of colony forming units (CFU).

Recombination Engineering Lambda-red E. coli. Strain HME63 was used for all recombination engineering experiments. Cells for recombination engineering were obtained and processed in accordance with a previously published protocol (6). Briefly, 2 ml of a 24-hour culture (LB medium) inoculated from one colony obtained from a plate was grown at 30 ° C. The daily culture was diluted 100 times and grown at 30 ° C with shaking (200 rpm) until the OD ₆₀₀ was 0.4-0.5 (approximately 3 hours). For Lambda-red induction, the culture was transferred to a water bath at 42 ° C for shaking at 200 rpm. within 15 minutes Immediately after induction, the culture was mixed in a suspension of ice water and cooled on ice for 5-10 minutes. The cells were then washed and aliquoted in accordance with the protocol. For electrotransformation, 50 μl of cells was mixed with 1 mM desalted oligonucleotides (GOT) or 100-150 ng of plasmid DNA (obtained using QIAprep Spin Miniprep Kit, Qiagen). Cells were electroporated using a 1 mm Gene Pulser cuvette (Bio-rad) at 1.8 kV and immediately resuspended in 1 ml LB medium at room temperature. Cells were harvested at 30 ° C for 1-2 hours before plating on LB agar with appropriate antibiotic resistance and incubated at 32 ° C overnight.

Obtaining genomic DNA of S. pneumoniae. For transformation, S. pneumoniae genomic DNA was isolated using the Wizard genomic DNA purification kit, following the instructions provided by the manufacturer (Promega). For genotyping, 700 μl of diurnal cultures of S. pneumoniae were precipitated by centrifugation, resuspended in 60 μl of lysozyme solution (2 mg / ml) and incubated for 30 min. at 37 ° C. Genomic DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen).

The creation of the strain. All primers used in this study are presented in table G. To create S. pneumoniae crR6M, an intermediate strain LAM226 was created. In this strain, the aphA-3 gene (providing resistance to kanamycin) adjacent to the CRISPR array of S. pneumoniae crR6 strain was replaced by the cat gene (providing resistance to chloramphenicol). Briefly, crR6 genomic DNA was amplified using primers L448 / L444 and L447 / L481, respectively. The cat gene was amplified from plasmid pC194 using L445 / L446 primers. Each PCR product was gel purified and all three were fused using PCR with elongation of overlapping fragments (SOEing PCR) with L448 / L481 primers. Competent crR6 S. pneumoniae cells were transformed with the obtained PCR product and chloramphenicol resistant transformants were selected. To create S. pneumoniae crR6M, S. pneumoniae genomic crR6 DNA was amplified by PCR using primers L409 / L488 and L448 / L481, respectively. Each PCR product was gel purified and merged by PCR with elongation of overlapping fragments (SOEing PCR) with primers L409 / L481. The competent LAM226 S. pneumoniae cells were transformed with the obtained PCR product and kanamycin resistant transformants were selected.

To create S. pneumoniae crR6Mc, S. pneumoniae genomic crR6R DNA was amplified by PCR using L430 / W286 primers and S. pneumoniae genomic DNA LAM226 was amplified by PCR using W288 / L481 primers. Each PCR product was gel purified and merged by PCR with elongation of overlapping fragments (SOEing PCR) with L430 / L481 primers. Competent crR6M S. pneumoniae cells were transformed with the obtained PCR product and chloramphenicol resistant transformants were selected.

To create S. pneumoniae crR6Rk, S. pneumoniae genomic crR6M DNA was amplified by PCR using primers L430 / W286 and W287 / L481, respectively. Each PCR product was gel purified and merged by PCR with elongation of overlapping fragments (SOEing PCR) with L430 / L481 primers. Competent crR6Rc S. pneumoniae cells were transformed with the obtained PCR product and kanamycin resistant transformants were selected.

To create JEN37, S. pneumoniae genomic crR6Rk DNA was amplified by PCR using primers L430 / W356 and W357 / L481, respectively. Each PCR product was gel purified and merged by PCR with elongation of overlapping fragments (SOEing PCR) with L430 / L481 primers. Competent crR6Rc S. pneumoniae cells were transformed with the obtained PCR product and kanamycin resistant transformants were selected.

To create JEN38, R6 genomic DNA was amplified using primers L422 / L461 and L459 / L426, respectively. The ermAM gene (determining erythromycin resistance) from plasmid pFW15 ^{43 was} amplified using L457 / L458 primers. Each PCR product was gel purified and all three were fused by PCR with elongation of overlapping fragments (SOEing PCR) with L422 / L426 primers. Competent crR6Rc S. pneumoniae cells were transformed with the obtained PCR product and erythromycin resistant transformants were selected.

JEN53 S. pneumoniae was created in two stages. First, JEN43 was created, as illustrated in Figure 33. JEN53 was created by transforming competent JEN43 cells to JEN25 genomic DNA and selecting both chloramphenicol and erythromycin.

To create S. pneumoniae JEN62, S. pneumoniae genomic crR6Rk DNA was amplified by PCR using primers W256 / W365 and W366 / L403, respectively. Each PCR product was purified and ligated using a Gibson assembly. Competent crR6Rc S. pneumoniae cells were transformed with the assembly product and kanamycin resistant transformants were selected.

Creation of a plasmid. pDB97 was created by phosphorylation and annealing of B296 / B297 oligonucleotides, followed by ligation in pLZ12spec, digested with EcoRI / BamHI. Applicants fully sequenced pLZ12spec and deposited its sequence at Genbank (access number: KC112384).

pDB98 was obtained after cloning the CRISPR leader sequence together with the repeat-spacer-repeat structural unit in pLZ12spec. This was achieved by amplification of crR6Rc DNA with primers B298 / B320 and B299 / B321 followed by PCR with elongation of overlapping fragments (SOEing PCR) for both products and cloning into pLZ12spec with BamHI / EcoRI restriction sites. Thus, the spacer sequence in pDB98 was designed to contain two BsaI restriction sites in opposite directions, which allowed scar-less cloning of new spacers.

From pDB99 to pDB108, they were created by annealing the oligonucleotides B300 / B301 (pDB99), B302 / B303 (pDB100), B304 / B305 (pDB101), B306 / B307 (pDB102), B308 / B309 (pDB103), B3B3 (pDB103), B3103 B312 / B313 (pDB105), B314 / B315 (pDB106), B315 / B317 (pDB107), B318 / B319 (pDB108) followed by ligation in pDB98, cut with BsaI.

Plasmid pCas9 was created as follows. The necessary CRISPR elements were amplified from the genomic DNA of Streptococcus pyogenes SF370 with flanking homologous arms for assembly using the Gibson method. tracrRNA and Cas9 were amplified with oligonucleotides HC008 and HC010. The CRISPR leader and sequences were amplified with HC011 / HC014 and HC015 / HC009, so that two BsaI type IIS sites were inserted between two direct repeats to ensure seamless insertion of spacers.

pCRISPR was created by subcloning an array of CRISPR pCas9 into pZE21-MCS1 by amplification with B298 + B299 oligonucleotides and restriction EcoRI and BamHI. The spacer for targeted rpsL exposure was cloned by annealing the B352 + B353 oligonucleotides and cloned into the cut BsaI pCRISPR to obtain pCRISPR :: rpsL.

Creating designs for focused exposure and editing.

Designs for targeted exposure, used to edit the genome, were obtained using the Gibson assembly of PCR products with right and left primers (table G). Editing constructs were obtained by fusing PCR products A (PCR A), PCR products B (PCR B) and PCR products C (PCR C) using PCR with elongation of overlapping fragments (SOEing PCR), if necessary (table G) . The CRISPR :: ∅ and CRISPR :: ermAM (stop) constructs for targeted exposure were obtained by PCR amplification of genomic DNA JEN62 and crR6, respectively, with oligonucleotides L409 and L481.

Obtaining targets with randomized PAM or protospacer sequences. The 5 nucleotides following the spacer 1 target were randomized by amplification of R68232 5 genomic DNA with W377 / L426 primers. This PCR product was then harvested with the cat gene and the srtA region upstream that were amplified from the same matrix with L422 / W376 primers. 80 ng of collected DNA was used to transform strains R6 and crR6. Samples for randomized targets were obtained using the following primers: B280-B290 / L426 for randomizing the bases 1-10 of the target and B269-B278 / L426 for randomizing the bases 10-20. Primers L422 / B268 and L422 / B279 were used to amplify the cat gene and the upstream srtA region for assembly with the first and last 10 PCR products, respectively. The assembled constructs were combined together and 30 ng transformed R6 and crR6. After transformation, cells were seeded for selection by chloramphenicol. For each sample, more than 2 × 10 ⁵ cells were combined together in 1 ml of THYE and genomic DNA was extracted using the Promega Wizard kit. The B250 / B251 primers were used to amplify the target site. PCR products were labeled and run on a Illurnina MiSeq twin-end sequencing run using 300 cycles.

Analysis of deep sequencing data.

Randomized PAM. For the experiment with randomized PAM, 3429,406 read fragments for crR6 and 3,253,998 for R6 were obtained. It was expected that only half of them would match the PAM target, while the other half would sequence the other end of the PCR product. 1623008 of the read fragments of crR6 and 1537131 of the read fragments of R6 carried an error-free target sequence. The occurrence of each possible PAM among these read fragments is shown in an additional file. To assess the functionality of PAM, its relative share in the crR6 sample was calculated in comparison with the R6 sample and denoted by r _ijklm , where I, j, k, l, m represent one of 4 possible bases. The following statistical model was created:

log (r _ijklm ) = μ + b2 _i + b3 _j + b4 _k + b2b3 _{i, j} + b3b4 _{j, k} + ε _ijklm ,

where ε is the residual error, b2 is the effect of the ^2nd PAM base, b3 is the third, b4 is the fourth, b2b3 is the interaction between the second and third bases, b3b4 is between the third and fourth bases. The analysis of variance was performed:

Analysis of variance table

When b1 or b5 is added to this model, they are insignificant, and other interactions, except those included, can also be excluded. Model selection was made through sequential comparisons of more or less complete models using the analysis of variance in R. The Tukey criterion of true significance was used to determine whether pairwise differences between effects are significant.

NGGNN patterns were significantly different from all other patterns and had the strongest effect (see table below).

In order to show that provisions 1, 4 or 5 do not affect the NGGNN pattern, applicants considered only these sequences. It turns out that their effects are normally distributed (see the quantile-quantile diagram in Figure 71), and comparisons of models using the analysis of variance in R show that the zero model is the best, i.e. there is no significant role for b1, b4 and b5.

Comparison of models using the analysis of variance in R for NGGNN sequences

Partial interference of NAGNN and NNGGN patterns

NAGNN patterns are significantly different from all other patterns, but they have a much smaller effect than NGGNN (see Tukey's true significance criterion below).

Finally, the NTGGN and NCGGN patterns are similar and show significantly greater CRISPR interference than the NTGHN and NCGHN patterns (where H is A, T, or C), as shown using the Bonifronroni paired student criterion.

Paired comparisons of the effect of b4 on NYGNN sequences using t-tests with a total standard deviation (SD)

Data: b4

Taken together, these results suggest that NNGGN patterns generally provide either complete interference, in the case of NGGGN, or partial interference, in the case of NAGGN, NTGGN, or NCGGN.

Multiple Tukey value comparisons: significance level adjusted for the effect of multiple comparisons 95%

5b2: b3

sb3: b4

Random target

For the experiment with randomized targets for crR6, 540,726 read fragments and 753,570 for R6 were obtained. As before, it was expected that only half of the read fragments would sequence the end of the PCR product of interest. After filtering for read fragments that carry a target that is devoid of errors or with a single-point mutation, there were 217656 and 353141 read fragments for crR6 and R6, respectively. The relative fraction of each mutant in the crR6 sample was calculated in comparison with the R6 sample (Figure 24c). All mutations outside the seed sequence (13-20 bases from PAM) showed complete interference. These sequences were used as an identifier to determine whether it can be said that mutations in the seed sequence significantly interfere with the interference. A normal distribution was selected for these sequences using the alignment of the distribution function of the MASS R. packet. A quantile 0.99 of the selected distribution is shown as a dashed line in Figure 24c. Figure 72 shows a histogram of data density with a matched normal distribution (black line) and a quantile of 0.99 (dashed line).

Example 6: RNA Guide Optimization for Cas9 Streptococcus pyogenes (called SpCas9)

Applicants introduced mutations in tracrRNA and direct repeat sequences, or introduced mutations in the chimeric RNA guide to increase RNA expression in cells.

Optimization is based on the observation that thymine (Ts) fragments were present in tracrRNA and RNA guide, which could lead to early termination

transcription by the pol 3 promoter. Thus, the applicants created the following optimized sequences. Optimized tracrRNA and the corresponding optimized direct repeat are presented in pairs. Optimized tracrRNA 1 (mutation underlined):

Optimized direct repeat 1 (mutation underlined):

Optimized tracrRNA 2 (mutation underlined):

Optimized direct repeat 2 (mutation underlined):

Applicants have also optimized a chimeric RNA guide for optimal activity in eukaryotic cells.

Original RNA Guide:

Optimized chimeric guide sequence of RNA 1:

Optimized chimeric guide sequence for RNA 2:

Optimized RNA 3 chimeric guide sequence:

Applicants have shown that an optimized chimeric RNA guide works better, as shown in Figure 3. The experiment was performed by cotransfection of 293FT Cas9 cells and a DNA cassette with a U6 guide RNA to express one of the four RNA forms shown above. The RNA target target is the same target site at the human EMX1 locus: "GTCACCTCCAATGACTAGGG".

Example 7: Cas9 optimization from CRISPR1 LMD-9 Streptococcus thermophiles (called StlCas9)

Applicants have designed guiding chimeric RNAs as shown in Figure 4. StlCas9 guiding RNAs can be optimized in the same way as SpCas9 guiding RNAs by destroying polythymine fragments (Ts).

Example 8: Cas9 diversity and mutations

The CRISPR-Cas system is an adaptive immune mechanism against intruding exogenous DNA, used by a variety of bacteria and archaea species. The CRISPR-Cas9 type II system consists of a set of genes encoding the proteins responsible for the "capture" of foreign DNA into the CRISPR locus, as well as a set of genes encoding the "execution" of the DNA cleavage mechanism; they include DNA nuclease (Cas9), a non-coding trans-activating cr-RNA (tracrRNA) and an array of foreign-derived spacers flanked by direct repeats (crRNA). Upon maturation by the action of Cas9, the duplex of tracRNA and crRNA directs Cas9 nuclease to the target DNA sequence defined by spacer guide sequences and mediates double-stranded breaks in the DNA near the short sequence motif in the target DNA, which are required for cleavage and are specific for each CRISPR-Cas system. CRISPR-Cas type II systems are found everywhere in the realm of bacteria and are very diverse in sequence and size of the Cas9 protein, the sequence of the direct repeat of tracrRNA and crRNA, the organization of these elements in the genome, and the motivation requirements for targeted cleavage. One view may have several separate CRISPR-Cas systems.

Applicants evaluated 207 putative Cas9 from bacterial species identified based on sequence homology with known Cas9 and structures orthologous to known subdomains, including the HNH endonuclease domain and RuvC endonuclease domains [information from Eugene Koonin and Kira Makarova]. Phylogenetic analysis based on the conservatism of the protein sequence in this set revealed five Cas9 families, including three groups of large Cas9 (~ 1400 amino acids) and two small Cas9 (~ 1100 amino acids) (figures 39 and 40A-F).

In this example, the applicants showed that the following mutations can turn SpCas9 into a notching enzyme: D10A, E762A, H840A, N854A, N863A, D986A.

Applicants presented sequences in which it is shown where the mutation points in the SpCas9 gene are located (Figure 41). Applicants also show that nickases can still mediate homologous recombination (assay indicated in Figure 2). Moreover, the applicants showed that SpCas9 with these mutations (separately) did not induce double-stranded cleavage (Figure 47).

Example 9: Supplementing the Specificity of Targeted Exposure to DNA of RNA-Guided Cas9 Nuclease

Cell Culture and Transfection

The human embryo kidney cell line (HEK) 293FT (Life Technologies) was maintained in Eagle's Dulbecco modification (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / ml penicillin and 100 μg / ml of streptomycin, at 37 ° C with incubation at 5% CO2.

293FT cells were seeded either on 6-well plates, 24-well plates, or on 96-well plates (Corning) 24 hours before transfection. Cells were transfected using Lipofectamine 2000 (Life Technologies) at 80-90% confluency following the manufacturer's recommended protocol. A total of 1 μg of Cas9 + sgRNA plasmid was used for each well of the 6-well plate. A total of 500 ng of the Cas9 + sgRNA plasmid was used for each well of the 24-well plate, unless otherwise indicated. 65 ng Cas9 plasmid was used for each well of a 96-well plate at a 1: 1 molar ratio of PCR product U6-sgRNA.

The human embryonic stem cell line HUES 9 (Harvard Stem Cell Institute core) was maintained under conditions without sublayer on GelTrex (Life Technologies) in mTesR medium (Stemcell Technologies) supplemented with 100 μg / ml Normocin (InvivoGen). HUES9 cells were transfected using the Amaxa P3 Primary Cell 4-D Nucleofector Kit (Lonza) following the manufacturer's protocol.

SURVEYOR analysis for genome modification

293FT cells were transfected with plasmid DNA as described above. Cells were incubated at 37 ° C for 72 hours after transfection before genomic DNA extraction. Genomic DNA was extracted with QuickExtract DNA Extraction Solution (Epicentre) following the manufacturer's protocol. Briefly, centrifuged cells were resuspended in a QuickExtract solution and incubated at 65 ° C for 15 minutes and 98 ° C for 10 minutes.

The genomic region flanking the CRISPR target site of each gene was amplified by PCR (primers are listed in Tables J and K) and the products were purified using a QiaQuick Spin Column (Qiagen) column following the manufacturer's protocol. A total of 400 ng of purified PCR products were mixed with 2 μl of 10X Taq DNA Enzymatics PCR buffer and ultra-high purity water to a final volume of 20 μl and subjected to a re-annealing process to ensure heteroduplex formation: 95 ° C for 10 min., linear decrease in temperature from 95 ° C to 85 ° C at a rate of 2 ° C / s, from 85 ° C to 25 ° C at a speed of 0.25 ° C / s and withstand at 25 ° C for 1 minute . After repeated annealing, the products were treated with SURVEYOR nuclease and S SURVEYOR enhancer (Transgenomics), following the protocol recommended by the manufacturer, and analyzed in 4-20% Novex TBE polyacrylamide gels (Life Technologies). The gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and the image was obtained using the Gel Doc gel imaging system (Bio-rad) image processing system. Quantitative analysis was based on the relative intensities of the bands.

Analysis of the expression of tracrRNA in human cells using Northern blotting

Northern blot analyzes were performed as described previously 1. Briefly, RNA molecules were heated to 95 ° C for 5 minutes. before immersion in 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics). After that, the RNA was transferred to a pre-hybridized Hybond N + membrane (GE Healthcare) and crosslinked using a Stratagene UV Crosslinker (Stratagene) automatic crosslinker. The probes were labeled with [gamma-32P] ATP (Perkin Elmer) with T4 polynucleotide kinase (New England Biolabs). After washing, the membrane was exposed on a luminescent screen for one hour and scanned using an image forming apparatus on a phosphor phosphor coating (Typhoon).

Bisulfite sequencing to assess DNA methylation status

HEK 293FT cells were transfected with Cas9 as described above. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) and subjected to bisulfite modification using the EZ DNA Methylation-Lightning Kit (Zymo Research). Bisulfite PCR was performed using KAPA2G Robust HotStart DNA polymerase (KAPA Biosystems) with primers designed using Bisulfite Primer Seeker (Zymo Research, Tables J and K). The resulting PCR amplicons were gel purified, digested with EcoRI and Hindlll and ligated into the pUC19 backbone before transformation. Individual clones were then sequenced by Sanger to assess the status of DNA methylation.

In vitro transcription and digestion assay

HEK 293FT cells were transfected with Cas9 as described above. Whole-cell lysates were then prepared using lysis buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1% Triton X-100) with the addition of a mixture of protease inhibitors (Roche). Controlled T7 sgRNAs were transcribed in vitro using special oligonucleotides (Example 10) and the in vitro HiScribe T7 transcription kit (NEB) following the manufacturer's recommended protocol. To obtain methylated target sites, the pUC19 plasmid was methylated using M.SssI and then linearized using NheI. The in vitro digestion assay was performed as follows: for the digestion reaction, 20 μl of 10 μl cell lysate was incubated with 2 μl of digestion buffer (100 mM HEPES, 500 mM KCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol), transcribed in vitro RNA and 300 ng DNA of plasmid pUC 19.

Deep sequencing to assess the specificity of targeted exposure

HEK 293FT cells, which were seeded in 96-well plates, were transfected with a PCR cassette with Cas9 plasmid DNA and single RNA guide (sgRNA) 72 hours before genomic DNA extraction (Fig. 72). The genomic region flanking the CRISPR target site of each gene was amplified (Fig. 74, Fig. 80, (Example 10)) using the PCR method with overlapping primers for attaching Illumina P5 adapters, as well as unique barcodes specific for each sample to the target amplicons ( the schematic depiction described in Fig. 73). PCR products were purified using 96-well EconoSpin filter plates (Epoch Life Sciences) following the manufacturer's recommended protocol.

Purified barcode DNA samples were quantified using the Quant-iT PicoGreen dsDNA or Qubit 2.0 Fluorometer assay kit (Life Technologies) and pooled in an equimolar ratio. The sequencing libraries were then deep sequenced using an Illumina MiSeq Personal Sequencer (Life Technologies) sequencer.

Analysis of sequencing data and detection of inserts / deletions

The read MiSeq fragments were filtered with the required average quality Phred (Q score) of at least 23, as well as full sequence matches to barcodes and direct amplicon primers. The read fragments from the target and non-target loci were analyzed first by performing Smith-Waterman alignments against amplicon sequences that included 50 nucleotides above and below the target site (a total of 120 bp). Alignments were analyzed by inserts / deletions from 5 nucleotides above to 5 nucleotides below the target site (a total of 30 bp). The analyzed target areas were excluded if part of their alignment went beyond the limits of the MiSeq readable fragment itself or if matching base pairs contained less than 85% of their total length.

The negative controls for each sample represented the standard for the inclusion or exclusion of insertions / deletions as expected cutting events. For each sample, insertion / deletion was taken into account only if its quality score exceeded μ-σ, where μ was the average quality score of the negative control corresponding to the sample, and σ was its standard deviation. This gave the ratio of all insertions / deletions of the target sites, both for negative controls and their corresponding samples. Using the error ratios of the negative control on the target-site-to-readable fragment, q, the observed number of insertions / deletions of samples n and its number of readable fragments R, the maximum likelihood estimate for the fraction of readable fragments containing target areas with correct insertions / deletions, p, was detected using the bionormal error model indicated below.

Assuming that the (unknown) number of read fragments in the sample containing the target areas incorrectly calculated as those that have at least 1 insert / deletion is equal to E, we can write (without making any assumptions about the number of correct inserts / deletions)

because R (1-p) represents the number of read fragments containing the target areas without the correct insertions / deletions. Moreover, since the number of readable fragments observed, such as those that contain insertions / deletions, is n, n = E + Rp, in other words, the number of readable fragments containing target sections with errors, but without correct insertions / deletions, plus readable fragments whose target sections contain the correct insertion / deletion. You can rewrite the above:

Assuming that all frequency values of the target sites with the correct insertions / deletions p are a priori equally probable, Prob (n | p) ∝Prob (p | n). The maximum likelihood score (MLE) of the frequency of the target sites with the correct insertions / deletions was thus set as the value of p, which maximized Prob (n | p). This was estimated numerically.

и верхнюю границу^u рассчитывали какTo place error boundaries relative to the frequencies of reading fragments with correct inserts / deletions in their sequencing libraries, Wilson confidence intervals (2) were calculated for each sample, providing an MLE estimate of the target areas with correct inserts / deletions, Rp, and the number of read fragments R. Specifically, lower bound

and the upper boundary ^{u was} calculated as

where z, the standard confidence indicator required for the normal distribution of variance 1, was set to 1.96, which meant a confidence of 95%. The maximum upper bounds and minimum lower bounds for each biological repetition are listed in figures 80-83.

QRT-PCR analysis of relative expression of Cas9 and sgRNA

293FT cells seeded in 24-well plates were transfected as described above. 72 hours after transfection, total RNA was collected using the miRNeasy Micro Kit (Qiagen). The sgRNA negative strand was synthesized using the qScript Flex cDNA kit (VWR) and special primers for the synthesis of the first strand (Tables J and K). Quantitative PCR analysis was performed using Fast SYBR Green Master Mix (Life Technologies) and special primers (Tables J and K) using GAPDH as an endogenous control. Relative quantitative analysis was performed using the ΔΔCT method.

Analysis of qRT-PCR expression of Cas9 and sgRNA

Bisulfite PCR and sequencing

Example 10: additional sequences

All sequences are presented in the direction from 5 'to 3'. For transcription of U6, the trait of the underlined target site (Ts) acts as a transcription terminator. U6 with short tracrRNA (Streptococcus pyogenes SF370):

(the tracrRNA sequence is shown in bold)

> U6-DR guide sequence-DR (SF370 Streptococcus pyogenes)

(a direct repeat of the sequence is highlighted in gray, and the guide sequence N is shown in bold)

> sgRNA containing +48 tracrRNA (SF370 Streptococcus pyogenes)

(the guide sequence is in bold N, and the tracrRNA fragment is in bold)

> sgRNA containing +54 tracrRNA (SF370 Streptococcus pyogenes)

(the guide sequence is in bold N, and the tracrRNA fragment is in bold)

> sgRNA containing +67 tracrRNA (SF370 Streptococcus pyogenes)

(the guide sequence is in bold N, and the tracrRNA fragment is in bold)

> sgRNA containing +85 tracrRNA (SF370 Streptococcus pyogenes)

(the guide sequence is in bold N, and the tracrRNA fragment is in bold)

> CBh-NLS-SpCas9-NLS

(NLS-hSpCas9-NLS in bold)

> Sequencing amplicon for EMX1 1.1, 1-14, 1.17 guides

> Sequencing amplicon for EMX1 1.2, 1.16 guides

> Sequencing amplicon for EMX1 1.3, 1.13, 1.15 guides

> Sequencing amplicon for EMX1 1.6 guides

> Sequencing amplicon for EMX1 1.10 guides

> Sequencing amplicon for EMX1 guides 1.11, 1.12

> Sequencing amplicon for EMX1 1.18, 1.19 guides

> Sequencing amplicon for EMX1 1.20 guides

> T7 promoter primer F for annealing with the target thread

> Oligonucleotide containing target site 1 pUC19 for methylation (reverse T7)

> Oligonucleotide containing pUC19 methylation target site 2 (reverse T7)

Example 11 oligonucleotide-mediated Ca89-induced homologous recombination

The oligonucleotide homologous recombination test is a comparison of the efficacy of all different Cas9 variants and different HR matrices (oligonucleotide compared to plasmid).

Used 293FT cells. SpCas9 = Cas9 wild-type and SpCas9n = Nicase Cas9 (D10A). The chimeric RNA target is the same EMX1 protospacer target 1 as in Examples 5, 9 and 10, and the oligonucleotides were synthesized using IDT using PAGE purification.

Figure 44 shows the structure of the oligonucleotide DNA used as a homologous recombination (HR) matrix in this experiment. Long oligonucleotides contain a homology of 100 bp to the EMX1 locus and the HindIII restriction site. 293FT cells were co-transfected: first, with a plasmid containing the target human EMX1 locus, chimeric RNA and wild-type cas9 protein, and secondly, oligonucleotide DNA as an HR template. Samples were obtained from 293FT cells collected 96 hours after transfection with Lipofectamine 2000. All products were amplified with EMX1 HR Primer, gel purified, followed by HindIII digestion to determine the effectiveness of integrating the HR matrix into the human genome.

Figures 45 and 46 show a comparison of HR efficacy induced using different combinations of Cas9 protein and HR matrix. The Cas9 construct used was either wild-type Cas9, or the Cas9 nickase variant (Cas9n). The HR matrices used were: antisense oligonucleotide DNA (antisense oligonucleotide in the figure above), or sense oligonucleotide DNA (sense oligonucleotide in the figure above), or the plasmid HR matrix (HR matrix in the figure above). The term “sense / antisense” means that an actively transcribed strand with a sequence corresponding to a transcribed mRNA is defined as a semantic strand in the genome. HR efficacy is shown as a percentage of the HindIII cleavage group of the total genome product amplified by PCR (numbers below).

Example 12: an autistic mouse

Recent large-scale sequencing projects have introduced a large number of genes associated with diseases. Identifying genes is only the beginning of understanding what the gene does and how it leads to the phenotype of disease. Existing technologies and approaches for studying candidate genes are slow and time-consuming. Gold standards, targeted effects on the gene and gene knockouts, require a significant investment of time and resources, both monetary and relative to research personnel. Applicants have set the task of using hSpCas9 nuclease for targeted action on many genes and to do this with higher efficiency and lower turnover compared to any other technology. Due to the high efficiency of hSpCas9, applicants can inject RNA into mouse zygotes and immediately receive animals with a modified genome without the need to produce any preliminary genes that target mESC.

The hemodase chromodomain gene of DNA-binding protein 8 (CHD8) is the most important gene involved in the early development of vertebrates and morphogenesis. Mice without CHD8 die during embryonic development. Mutations in CHD8 have been associated with autism spectrum disorder in humans. This association was carried out in three different articles published simultaneously in Nature. In the same three studies, many genes associated with autism spectrum disorder were identified. The applicants' goal was to create knockout mice on four genes that were found in all articles, Chd8, Katna12, Kctd13 and Scn2a. In addition, applicants selected two other genes associated with autism spectrum disorder, schizophrenia and ADHD, GIT1, CACNA1C and CACNB2. Finally, the applicants decided to set MeCP2 as a positive control.

For each gene, applicants have developed three gRNAs that are likely to knock out the genes. Knocking out would occur after the hSpCas9 nuclease has double-stranded, and the error-prone DNA repair path, a non-homologous junction of the ends, clears the gap, creating a mutation. The most likely result is a frameshift mutation that would produce a gene knockout. The targeted exposure strategy included the presence of protospacer in the exons of a gene containing the PAM, NGG sequence, which is unique in the genome. Preference was given to protospacer in the first exon, which would be most damaging to the gene.

Each gRNA was evaluated in a mouse cell line, Neuro-N2a, using liposomal unstable cotransfection of hSpCas9. 72 hours after transfection, genomic DNA was purified using Epicentre QuickExtract DNA. PCR was performed to amplify the locus of interest. This was followed by the use of the Transgenomics SURVEYOR mutation detection kit. SURVEYOR analysis results for each gRNA and corresponding controls are shown in Figure A1. A positive analysis result using SURVEYOR represents one large band corresponding to genomic PCR and two smaller bands, which are products of SURVEYOR nuclease, which produces a double-stranded break at the mutation site. The average cutting efficiency of each gRNA was also determined for each gRNA. The gRNA that was chosen for injection was the highest-performing gRNA that was most unique in the genome.

RNA (hSpCas9 + gRNA RNA) was injected into the zygote pronucleus and later transplanted to the adoptive mother. Mothers were allowed to undergo a full gestation period, and samples were taken from the calves by notching the tail 10 days after birth. DNA was isolated and used as a template for PCR, which was then processed using SURVEYOR. In addition, PCR products were sent for sequencing. The PCR products of the animal genome, which were determined as positive either by SURVEYOR analysis or by PCR sequencing, were cloned into pUC19 vector and sequenced to determine putative mutations of each allele.

To date, pups of mice from the experiment of targeted exposure to Chd8 were completely subjected to treatment, prior to allele sequencing. Surveyor analysis results for 38 live calves (lanes 1-38), 1 dead calf (lane 39) and 1 wild-type calf for comparison (lane 40) are shown in Figure A2. Cubs 1-19 were injected with Chd8.2 gRNA, and cubs 20-38 were injected with CM8.3 gRNA. Of the 38 live pups, 13 were positive for mutation. One dead calf also had a mutation. No mutation was detected in the wild-type sample. PCR genome sequencing was consistent with analysis using SURVEYOR.

Example 13: CRISPR / Cas-mediated Transcription Modulation

The figure 67 shows the structure of CRISPR-TF (transcription factor), with the function of activation of transcription. Chimeric RNA is expressed using the U6 promoter, and the codon-optimized variant of the Cas9 double mutant protein (hSpCas9m), functionally linked to the three-component NLS and VP64 functional domain, is expressed using the EF1a promoter. The double mutations, D10A and H840A, make the Cas9 protein incapable of introducing any cleavage, but support its ability to bind to the target DNA in the direction of chimeric RNA.

Figure 68 shows the activation of transcription of the human SOX2 gene using the CRISPR-TF system (chimeric RNA and Cas9-NLS-VP64 fusion protein). 293FT cells were transfected with plasmids carrying two components: (1) U6-driven different chimeric RNAs that specifically target 20 bp sequences. at or near the locus of the human genome SOX2, and (2) EF1-driven hSpCas9m (double mutant) fusion protein NLS-VP64. 96 hours after transfection, 293FT cells were harvested and the level of activation was measured by induction of mRNA expression using qRT-PCR analysis. All expression levels are normalized compared to the control group (gray column), which represents the results of cells transfected with a CRISPR-TF plasmid without chimeric RNA. QRT-PCR probes were used to detect SOX2 mRNA in the analysis of Taqman Human gene expression (Life Technologies). The data of all experiments of 3 biological replicates, n = 3, are presented; error bars show the standard error of the mean.

Example 14: NLS: Cas9-NLS

293FT cells were transfected with a plasmid containing two components: (1) the EF1 promoter driving the expression of Cas9 (wild-type Sp Cas9, codon-optimized for humans) with different NLS structures, (2) the U6 promoter driving the same human EMX1 locus undergoing targeted exposure to chimeric RNA.

Cells were harvested at 72 hours after transfection and then extracted with 50 μl of QuickExtract genomic DNA extraction solution following the manufacturer's protocol. The target genomic DNA EMX1 was amplified by PCR and then gel purified with 1% agarose gel. The genomic PCR product was annealed a second time and analyzed using SURVEYOR following the manufacturer's protocol. The cleavage efficiency of various genome constructs was measured using SDS-PAGE in a 4-12% TBE-PAGE gel (Life Technologies), analyzed and quantified using ImageLab software (Bio-rad), following the manufacturer's protocol.

69 illustrates the structure of various Cas9-NLS designs. All Cas9s were a human codon-optimized version of Sp Cas9. NLS sequences are linked to the cas9 gene at either the N-terminus or the C-terminus. All Cas9 variants with different NLS structures were cloned into a vector containing the backbone, so that it was driven by the EF1a promoter. In the same vector, there was a chimeric RNA that deliberately acts on the human EMX1 locus, driven by the U6 promoter, which forms a two-component system with it.

Figure 70 depicts the effectiveness of genomic cleavage induced by Cas9 variants bearing different NLS structures. The percentage indicates the portion of the human genomic EMX1 DNA that was digested by each construct. All experiments were obtained from 3 biological replicates. n = 3, error means S.E.M.

Example 15: Design of Microalgae Using Cas9

Shipping Methods Cas9

Method 1: Applicants delivered Cas9 and an RNA guide using a vector that expresses Cas9 under the control of a constitutive promoter, such as the Hsp70A-Rbc S2 promoter or beta-2-tubulin promoter.

Method 2: Applicants delivered Cas9 and T7 polymerase using vectors that express Cas9 and T7 polymerase under the control of a constitutive promoter, such as the Hsp70A-Rbc S2 promoter or beta-2-tubulin promoter. Guide RNA will be delivered using a vector containing the T7 promoter that controls the expression of the guide RNA.

Method 3: Applicants delivered Cas9 mRNA and in vitro transcribed guide RNA in algal cells. RNA can be transcribed in vitro. Cas9 mRNA will consist of a coding region for Cas9, as well as 3 'UTR from Cop1, to provide stabilization of Cas9 mRNA.

For homologous recombination, applicants provided an additional repair matrix involving homologous recombination.

The sequence for the cassette driving the expression of Cas9 under the control of the beta-2-tubulin promoter, followed by 3 'UTR Cop1.

The sequence for the cassette that controls the expression of T7 polymerase under the control of the beta-2-tubulin promoter, followed by 3 'UTR Cop1:

The sequence of the RNA guide driven by the T7 promoter (T7 promoter, N represent the targeting sequence):

Gene delivery

The strains SS-124 and SS-125 Chlamydomonas reinhardtii from the Clilamydomonas Resource Center (Clilamydomonas Resource Center) will be used for electroporation. The electroporation protocol follows the standard recommended protocol for the GeneArt Clilamydomonas Engineering kit.

Applicants also received the Clilamydomonas reinhardtii line, which expresses Cas9 constitutively. This can be accomplished using pChlamyl (linearized using Pvul) and screening for hygromycin-resistant colonies. The sequence for pChlamyl containing Cas9 is shown below. In this way, to achieve gene knockout, you just need to deliver RNA to the RNA guide. For homologous recombination, applicants delivered an RNA guide as well as a linearized matrix for homologous recombination.

pChlamy1-Cas9:

For all modified Chlamydomonas reinhardtii cells, the applicants used PCR, SURVEYOR nuclease analysis and DNA sequencing to confirm successful modification.

Example 16: Use of Cas9 as a Repressor of Transcription in Bacteria

The ability to artificially control transcription is important both for studying the function of a gene and for creating synthetic gene networks with the desired properties. Applicants describe in this document the use of Cas9 RNA-directed protein as a programmable transcriptional repressor.

Applicants have previously demonstrated how Cas9 SF370 Streptococcus pyogenes protein can be used for targeted genome editing in Streptococcus pneumoniae. In this study, applicants constructed a crR6Rk strain containing a minimal CRISPR system consisting of cas9, tracrRNA and repeat. In this strain, D10A-H840 mutations were introduced into cas9 to give the crR6Rk ** strain. Four spacers targeting different positions of the bgaA promoter of the β-galactosidase gene were cloned into a CRISPR array transferred by the previously described plasmid pDB98. Applicants observed an X-Y-fold decrease in β-galactosidase activity, depending on the position of the targeted exposure, demonstrating the potential of Cas9 as a programmable repressor (Figure 73).

In order to ensure repression of Cas9 **, a reporter plasmid (pDB127) with green fluorescent protein (GFP) was constructed for Escherichia coli to express the gfpmut2 gene from the constitutive promoter. A promoter was developed that carried several NPP PAMs on both strands to measure the effect of Cas9 ** binding at different positions. Applicants introduced D10A-H840 mutations into pCas9, with the plasmid described as carrying tracrRNA, cas9 and the minimal CRISPR array designed to conveniently clone new spacers. Twenty-two different spacers were designed to target different regions of the gfpmut2 promoter and the open reading frame. An approximately 20-fold decrease in fluorescence was observed with targeted exposure to areas overlapping or adjacent to the -35 and -10 elements of the promoter and to the Shine-Dalgarno sequence. Targets on both strands showed similar levels of repression. These results suggest that the binding of Cas9 ** to any position of the promoter site prevents the initiation of transcription, presumably through steric inhibition of RNAP binding.

To determine whether Cas9 ** can prevent transcription elongation, applicants sent it to the gpfmut2 reading frame. A decrease in fluorescence was observed with a targeted effect on both coding and non-coding strands, which indicated that Cas9 binding is indeed strong enough to constitute an obstacle to the passage of RNAP. However, while a 40% decrease in expression was observed when the coding strand was the target, a 20-fold decrease was observed for the non-coding strand (Fig. 21b, comparison of T9, T10 and T11 with B9, B10 and B11). To directly determine the effects of Cas9 ** binding upon transcription, the applicants isolated RNA from strains carrying either T5, T10, B10 or a control construct that did not specifically target pDB127, and subjected it to Northern blotting using a probe that binds either before (B477 ), or after (B510) target sites B10 and T10. In accordance with the fluorescence methods of the applicants, no gfpmut2 transcription was detected when Cas9 ** was directed to the promoter site (T5 target), and transcription was observed after targeted exposure to the T10 site. Interestingly, a smaller transcript was observed with the B477 probe. This band corresponds to the expected size of the transcript that Cas9 ** will interrupt, and is a direct indication of the transcription termination caused by dgRNA :: Cas9 **, which binds to the coding strand. Surprisingly, the applicants did not find a transcript when they targeted the non-coding strand (B10). Since the binding of Cas9 ** to B1 is unlikely to inhibit transcription initiation, these results suggest that mRNA was destroyed. DgRNA :: Cas9 has been shown to bind ssRNA in vitro. Applicants suggest that binding may trigger mRNA degradation by host nucleases. Indeed, stopping the ribosomes can induce cleavage on the translated mRNA in E. coli.

Some applications require precise regulation of gene expression, rather than its absolute repression. Applicants sought to achieve intermediate levels of repression by introducing mismatches that would weaken the crRNA / target interactions. Applicants created a series of spacers based on constructs B1, T5 and B10 with an increase in the number of mutations at the 5'-end of crRNA. Up to 8 mutations inclusive in B1 and T5 did not affect the level of repression, and a progressive increase in fluorescence was observed for additional mutations.

The observed repression with only 8 nt coincidence between crRNA and its target raises the question of the effects of inappropriate exposure when using Cas9 ** as a transcriptional regulator. Since a suitable PAM (NGG) is also required for Cas9 binding, the number of nucleotides in accordance with obtaining a certain level of restriction is 10. A match of 10 nt occurs randomly each ~ 1 bp, and such sites are thus likely can be found even in small bacterial genomes. However, for effective repression of transcription, it is necessary that such sites are located in the promoter region of the gene, which carries out inappropriate effects with a much lesser probability. Applicants have also shown that gene expression may be impaired if the non-coding strand of the gene is targeted. In order for this to happen, an arbitrary target must be in the correct orientation, but these events occur with a relatively greater probability. In fact, during this study, applicants were unable to construct one of the developed spacers in pCas9 **. Applicants later found this spacer, characterized by a 12 bp match, next to the corresponding PAM in the murC key gene. Such inappropriate effects can easily be avoided by systematically damaging the designed spacers.

Aspects of the present invention are further described in the following numbered paragraphs.

1. A vector system containing one or more vectors, where the system contains

a. a first regulatory element operably linked to a paired traer sequence and one or more embedding sites for embedding a guiding sequence above a paired traer sequence, where upon expression the guiding sequence controls the sequence-specific binding of the CRISPR complex to the target sequence in a eukaryotic cell, where the CRISPR complex contains CRISPR enzyme complexing with (1) a guide sequence that hybridizes to the target sequence lnostyu, and (2) traer-pair sequence that hybridizes to traer-sequence; and

b. a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence;

where components (a) and (b) are in the same or in different vectors of the system.

2. The vector system according to paragraph 1, where component (a) additionally contains a traer sequence below the paired traer sequence under the control of the first regulatory element.

3. The vector system according to paragraph 1, where component (a) further comprises two or more guide sequences operably linked to the first regulatory element, where upon expression each of two or more guide sequences controls the sequence-specific binding of the CRISPR complex to its target sequence in eukaryotic cell.

4. The vector system according to paragraph 1, where the system contains a traer sequence under the control of the third regulatory element.

5. The vector system according to paragraph 1, where the traer sequence is characterized by at least 50% complementarity of the sequence along the length of the paired traer sequence with optimal alignment.

6. The vector system according to paragraph 1, where the CRISPR enzyme contains one or more nuclear localization sequences that are effective enough to control the accumulation of the specified CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

7. The vector system according to paragraph 1, where the CRISPR enzyme is an enzyme of the type II CRISPR system.

8. The vector system according to paragraph 1, where the CRISPR enzyme is a Cas9 enzyme.

9. The vector system according to paragraph 1, where the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

10. The vector system according to paragraph 1, where the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence.

11. The vector system according to paragraph 1, where the CRISPR enzyme lacks activity for cleaving DNA strands.

12. The vector system according to paragraph 1, where the first regulatory element is a promoter of polymerase III.

13. The vector system according to paragraph 1, where the second regulatory element is a promoter of polymerase II.

14. The vector system according to paragraph 4, where the third regulatory element is a promoter of polymerase III.

15. The vector system according to paragraph 1, where the guide sequence is at least 15 nucleotides in length.

16. The vector system according to paragraph 1, where less than 50% of the nucleotides of the guide sequence are involved in self-complementary base pairing with optimal folding.

17. A vector containing a regulatory element operably linked to an enzyme coding sequence for a CRISPR enzyme containing one or more nuclear localization sequences, wherein said regulatory element controls transcription of the CRISPR enzyme in a eukaryotic cell such that the CRISPR enzyme accumulates in a detectable amount in the nucleus eukaryotic cells.

18. The vector according to paragraph 17, where the specified regulatory element is a promoter of polymerase II.

19. The vector according to paragraph 17, wherein said CRISPR enzyme is an enzyme of type II CRISPR system.

20. The vector of paragraph 17, wherein said CRISPR enzyme is a Cas9 enzyme.

21. The vector of paragraph 17, wherein said CRISPR enzyme lacks the ability to cleave one or more strands of the target sequence to which it binds.

22. The CRISPR enzyme, containing one or more nuclear localization sequences, sufficiently effective to control the accumulation of the specified CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

23. The CRISPR enzyme according to paragraph 22, wherein said CRISPR enzyme is an enzyme of the type II CRISPR system.

24. The CRISPR enzyme according to paragraph 22, wherein said CRISPR enzyme is a Cas9 enzyme.

25. The CRISPR enzyme according to paragraph 22, wherein said CRISPR enzyme lacks the ability to cleave one or more strands of the target sequence with which it binds.

26. A eukaryotic host cell containing

a. a first regulatory element operably linked to a paired traer sequence and one or more embedding sites for embedding a guiding sequence above a paired traer sequence, where upon expression the guiding sequence controls the sequence-specific binding of the CRISPR complex to the target sequence in a eukaryotic cell, where the CRISPR complex contains CRISPR enzyme complexing with (1) a guide sequence that hybridizes with the target sequence elnostyu, and (2) traer-pair sequence which gibiridiziruetsya with traer-sequence; and / or

b. a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence.

27. The eukaryotic host cell according to paragraph 26, wherein said host cell contains components (a) and (b).

28. The eukaryotic host cell according to paragraph 26, where component (a), component (b), or components (a) and (b) are stably integrated into the genome of the eukaryotic host cell.

29. The eukaryotic host cell according to paragraph 26, where component (a) further comprises a traer sequence below the paired traer sequence under the control of the first regulatory element.

30. The eukaryotic host cell according to paragraph 26, where component (a) further comprises two or more guide sequences operably linked to the first regulatory element, where upon expression each of two or more guide sequences controls the sequence-specific binding of the CRISPR complex to its target sequence in a eukaryotic cell.

31. The eukaryotic host cell according to paragraph 26, further comprising a third regulatory element operably linked to said traer sequence.

32. The eukaryotic host cell according to paragraph 26, where the traer sequence is characterized by at least 50% complementarity of the sequence along the length of the paired traer sequence with optimal alignment.

33. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme contains one or more nuclear localization sequences that are effective enough to control the accumulation of the specified CRISPR enzyme in a detectable amount in the nucleus of the eukaryotic cell.

34. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme is an enzyme of the type II CRISPR system.

35. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme is a Cas9 enzyme.

36. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

37. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence.

38. The eukaryotic host cell according to paragraph 26, where the CRISPR enzyme lacks activity for cleaving DNA strands.

39. The eukaryotic host cell according to paragraph 26, where the first regulatory element is a promoter of polymerase III.

40. The eukaryotic host cell according to paragraph 26, where the second regulatory element is a promoter of polymerase II.

41. The eukaryotic host cell according to paragraph 31, where the third regulatory element is a promoter of polymerase III.

42. The eukaryotic host cell according to paragraph 26, where the guide sequence is at least 15 nucleotides in length.

43. The eukaryotic host cell according to paragraph 26, where less than 50% of the nucleotides of the guide sequence is involved in self-complementary base pairing with optimal folding.

44. A non-human animal containing a eukaryotic host cell according to any one of paragraphs 26-43.

45. A kit containing a vector system and instructions for using the specified kit, while the vector system contains

a. a first regulatory element operably linked to a paired traer sequence and one or more embedding sites for embedding a guiding sequence above a paired traer sequence, where upon expression the guiding sequence controls the sequence-specific binding of the CRISPR complex to the target sequence in a eukaryotic cell, where the CRISPR complex contains CRISPR enzyme complexing with (1) a guide sequence that hybridizes with the target sequence elnostyu, and (2) tracr-pair sequence which gibiridiziruetsya with traer-sequence; and / or

b. a second regulatory element operably linked to an enzyme coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence.

46. The set according to paragraph 45, where the specified set contains components (a) and (b) located in the same or in different vectors of the system.

47. The kit of paragraph 45, wherein component (a) further comprises a traer sequence below the paired traer sequence under the control of the first regulatory element.

48. The kit of paragraph 45, wherein component (a) further comprises two or more guide sequences operably linked to the first regulatory element, where upon expression each of two or more guide sequences controls sequence specific binding of the CRISPR complex to its target sequence in eukaryotic the cage.

49. The kit according to paragraph 45, where the system contains a traer sequence under the control of the third regulatory element.

50. The kit of paragraph 45, wherein the traer sequence is characterized by at least 50% complementarity of the sequence along the length of the paired traer sequence with optimal alignment.

51. The kit of paragraph 45, wherein the CRISPR enzyme contains one or more nuclear localization sequences that are effective enough to control the accumulation of the specified CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

52. The kit according to paragraph 45, where the CRISPR enzyme is an enzyme of the CRISPR type II system.

53. The kit according to paragraph 45, where the CRISPR enzyme is a Cas9 enzyme.

54. The kit of paragraph 45, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

55. The kit of paragraph 45, wherein the CRISPR enzyme controls the cleavage of one or two strands at a specific point in the target sequence.

56. The kit of paragraph 45, wherein the CRISPR enzyme lacks activity for cleaving DNA strands.

57. The kit of paragraph 45, wherein the first regulatory element is a polymerase III promoter.

58. The kit according to paragraph 45, where the second regulatory element is a promoter of polymerase II.

59. The kit according to paragraph 49, where the third regulatory element is a promoter of polymerase III.

60. The kit of paragraph 45, wherein the guide sequence is at least 15 nucleotides in length.

61. The kit according to paragraph 45, where less than 50% of the nucleotides of the guide sequence is involved in self-complementary base pairing with optimal folding.

62. A computer system for selecting a candidate target sequence within a nucleic acid sequence in a eukaryotic cell for targeted exposure to the CRISPR complex, the system comprising

a. a memory unit configured to receive and / or store said nucleic acid sequence; and

b. one or more processors, individually or in combination, programmed to (i) locate a CRISPR motif sequence within a specified nucleic acid sequence and (ii) select a sequence adjacent to a specified locally defined CRISPR motif sequence as a candidate target sequence, with which binds CRISPR complex.

63. The computer system of paragraph 62, wherein said locating step comprises identifying a CRISPR motif sequence located less than about 500 nucleotides from the specified target sequence.

64. The computer system of paragraph 62, wherein said candidate target sequence is at least 10 nucleotides in length.

65. The computer system of paragraph 62, wherein the nucleotide at the 3'-end of the candidate target sequence is located no more than about 10 nucleotides above the CRISPR motif sequence.

66. The computer system of paragraph 62, wherein the nucleic acid sequence in a eukaryotic cell is endogenous to the eukaryotic genome.

67. The computer system of paragraph 62, wherein the nucleic acid sequence in the eukaryotic cell is exogenous to the eukaryotic genome.

68. A computer-readable medium containing codes that, when executed by one or more processors, implement a method for selecting a candidate target sequence within a nucleic acid sequence in a eukaryotic cell for targeted exposure to the CRISPR complex, the method comprising (a) locating the CRISPR motif sequence within said nucleic acid sequence; and (b) selecting a sequence adjacent to said CRISPR motif sequence with op edelennym located, as a candidate of a target sequence with which the complex binds CRISPR.

69. The computer-readable medium of paragraph 68, wherein said locating includes locating a CRISPR motif sequence that is less than about 500 nucleotides from the specified target sequence.

70. Machine-readable according to paragraph 68, wherein said candidate target sequence is at least 10 nucleotides in length.

71. Machine-readable according to paragraph 68, where the nucleotide at the 3'-end of the candidate target sequence is located no more than approximately 10 nucleotides above the sequence of the CRISPR motif.

72. Machine-readable according to paragraph 68, where the nucleic acid sequence in a eukaryotic cell is endogenous to the eukaryotic genome.

73. Machine-readable according to paragraph 68, where the nucleic acid sequence in a eukaryotic cell is exogenous to the eukaryotic genome.

74. A method for modifying a target polynucleotide in a eukaryotic cell, the method comprising binding a CRISPR complex to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, where the CRISPR complex contains a CRISPR enzyme that is complexed with a guide sequence that hybridizes with the target sequence in the specified target polynucleotide, where the specified guide sequence is associated with a paired traer sequence consistently, which, in turn, hybridize to Traer-sequence.

75. The method of paragraph 74, wherein said cleavage comprises cleaving one or two strands at a specific point in the target sequence with said CRISPR enzyme.

76. The method of paragraph 74, wherein said cleavage results in reduced transcription of the target gene.

77. The method of paragraph 74, further comprising repairing said cleaved target polynucleotide by homologous recombination with an exogenous matrix polynucleotide, wherein said repair leads to a mutation comprising the insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.

78. The method of paragraph 77, wherein said mutation results in one or more amino acid substitutions in a protein expressed from a gene containing the target sequence.

79. The method of paragraph 74, further comprising delivering one or more vectors to the indicated eukaryotic cell, where one or more vectors control the expression of one or more of the following: CRISPR enzyme, a guide sequence associated with a paired traer sequence, and a traer sequence.

80. The method of paragraph 79, wherein said vectors are delivered to a eukaryotic cell in a subject.

81. The method of paragraph 74, wherein said modification takes place in said eukaryotic cell in cell culture.

82. The method of paragraph 74, further comprising isolating said eukaryotic cell from a subject prior to said modification.

83. The method of paragraph 82, further comprising returning said eukaryotic cell and / or cells obtained from a subject to said subject.

84. A method of modifying expression of a polynucleotide in a eukaryotic cell, the method comprising: binding the CRISPR complex to the polynucleotide so that said binding results in increased or decreased expression of the indicated polynucleotide; where the CRISPR complex contains the CRISPR enzyme, which forms a complex with a guide sequence that hybridizes to the target sequence in the specified polynucleotide, where the specified guide sequence is associated with a paired traer sequence, which, in turn, hybridizes with the traer sequence.

85. The method of paragraph 74, further comprising delivering one or more vectors to said eukaryotic cells, wherein one or more vectors control the expression of one or more of the following: a CRISPR enzyme, a guide sequence associated with a paired traer sequence, and a traer sequence.

86. A method for producing a model eukaryotic cell containing a mutant gene responsible for the development of a disease, the method comprising

a. introducing one or more vectors into a eukaryotic cell, where one or more vectors control the expression of one or more of: the CRISPR enzyme, the guide sequence associated with the paired traer sequence, and the traer sequence; and

b. providing binding of the CRISPR complex to the target polynucleotide to carry out the cleavage of the target polynucleotide in the indicated gene responsible for the development of the disease, where the CRISPR complex contains the CRISPR enzyme, complexing with (1) the guide sequence that hybridizes with the target sequence in the target polynucleotide, and (2) paired traer sequence that hybridizes with the traer sequence, thus obtaining a model eukaryotic cell containing the mutant gene, corresponding for developing the disease.

87. The method of paragraph 86, wherein said cleavage comprises cleaving one or two strands at a specific point in the target sequence with said CRISPR enzyme.

88. The method of paragraph 86, wherein said cleavage results in reduced transcription of the target gene.

89. The method of paragraph 86, further comprising repairing said cleaved target polynucleotide by homologous recombination with an exogenous matrix polynucleotide, wherein said repair leads to a mutation comprising the insertion, deletion, or replacement of one or more nucleotides of said target polynucleotide.

90. The method of paragraph 89, wherein said mutation results in one or more amino acid substitutions in a protein expressed from a gene containing the target sequence.

91. A method of obtaining a biologically active agent that modulates the signal transmission process in the cell associated with the gene responsible for the development of the disease, including

a. bringing the test compound into contact with the model cell according to any one of paragraphs 86-90 and

b. detecting a change in reading, which indicates a decrease or increase in the process of signal transmission in the cell associated with the specified mutation in the specified gene responsible for the development of the disease, thus obtaining the specified biologically active agent that modulates the specified process of signal transmission in the cell, associated with the specified gene responsible for the development of the disease.

92. A recombinant polynucleotide containing a guide sequence above a paired traer sequence, where the guide sequence upon expression controls the sequence-specific binding of the CRISPR complex to the corresponding target sequence present in the eukaryotic cell.

93. The recombinant polynucleotide of paragraph 89, wherein the target sequence is a viral sequence present in a eukaryotic cell.

94. The recombinant polynucleotide of paragraph 89, wherein the target sequence is a proto-oncogen or oncogen.

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. Numerous variations, changes and substitutions will now be apparent to those skilled in the art without departing from the gist of the present invention. It should be understood that various alternative embodiments of the present invention disclosed herein can be used in the practical implementation of the present invention. The following claims are intended to define the scope of the present invention, and that thus, methods and structures within the scope of this claims and their equivalents are encompassed.

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Claims (59)

1. A non-naturally occurring or engineered composition for modifying one or more genomic loci of interest in a eukaryotic cell, comprising a vector system that contains one or more vectors containing I) a first promoter operably linked to a nucleotide sequence encoding a chimeric RNA (chiRNA) of the CRISPR-CAS system, where chiRNA contains (a) a guide sequence capable of hybridizing with a target sequence in a eukaryotic cell and directing sequence specific binding of the CRISPR complex to that target sequence, (b) a paired tracr sequence capable of hybridizing with the tracr sequence, and (c) a tracr sequence, and Ii) a second promoter operably linked to an enzyme coding sequence for Cas9, containing at least one or more nuclear localization sequences (NLS) near the end of Cas9, where (a), (b) and (c) are located in a 5’-3 ’orientation, where the components I) and II) are in the same or in different vectors of the system, where the chimeric RNA contains two or more hairpins, where the eukaryotic cell is not a human germline cell. 2. The composition according to p. 1, characterized in that it uses a lot of chiRNA to obtain a multiplex system. 3. The CRISPR-Cas system for changing one or more genomic loci of interest in a eukaryotic cell, characterized in that it contains one or more vectors containing I) a first promoter operably linked to a nucleotide sequence encoding a chimeric RNA (chiRNA) of the CRISPR-CAS system, where chiRNA contains (a) a guide sequence capable of hybridizing with a target sequence in a eukaryotic cell and directing sequence specific binding of the CRISPR complex to that target sequence, (b) a paired tracr sequence capable of hybridizing with the tracr sequence, and (c) a tracr sequence, and II) a second promoter operably linked to an enzyme-coding sequence encoding a Cas9 enzyme comprising at least one or more nuclear localization (NLS) sequences near the end of Cas9, where (a), (b) and (c) are located in a 5’-3 ’orientation, where the components I) and II) are in the same or in different vectors of the system, where chiRNA contains two or more hairpins and where one or more vectors encode multiple chiRNA, where the eukaryotic cell is not a human germline cell. 4. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the first promoter is a polymerase III promoter. 5. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the second promoter is a promoter of polymerase II. 6. A composition according to claim 1 or 2 or a system according to claim 3, characterized in that Cas9 contains one or more NLS effective enough to control the accumulation of said Cas9 in a detectable amount in the nucleus of a eukaryotic cell. 7. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the tracr sequence is characterized by at least 50% complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. 8. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the Cas9 enzyme is Cas9 from Streptococcus pyogenes. 9. The composition according to claim 1 or 2 or the system according to claim 3, wherein the Cas9 enzyme is a Cas9 enzyme from Staphylococcus aureus or Streptococcus thermophilus. 10. The composition of claim 1 or 2 or the system of claim 3, wherein the enzyme coding sequence encoding the Cas9 enzyme is codon-optimized for expression in a eukaryotic cell. 11. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the guide sequence is at least 15 nucleotides in length. 12. The composition according to p. 1 or 2 or the system according to p. 3, characterized in that the polynucleotide sequence of the chimeric RNA contains two, three, four or five "hairpins". 13. A non-naturally occurring or engineered composition for modifying one or more genomic loci of interest in a eukaryotic cell, comprising a vector system that contains one or more vectors containing I) a first promoter operably linked to a nucleotide sequence encoding a RIS guide of a CRISPR-Cas system containing (a) a guide sequence capable of hybridizing with a target sequence in a eukaryotic cell and directing sequence specific binding of the CRISPR complex to that target sequence, and (b) a paired tracr sequence capable of hybridizing with a tracr sequence, II) a second promoter operably linked to an enzyme coding sequence encoding Cas9 containing at least one or more nuclear localization sequences (NLS) near the end of Cas9, and III) a third promoter operably linked to a nucleotide sequence encoding a tracrRNA containing the tracr sequence, where the components I), II) and III) are in the same or in different vectors of the system, where the eukaryotic cell is not a human germline cell. 14. The composition according to p. 13, characterized in that a plurality of guide sequences and one tracr sequence are used to obtain a multiplex system. 15. The CRISPR-Cas system for changing one or more genomic loci of interest in a eukaryotic cell, characterized in that it contains one or more vectors, including I) a first promoter operably linked to a nucleotide sequence encoding a RIS guide of a CRISPR-Cas system containing (a) a guide sequence capable of hybridizing with a target sequence in a eukaryotic cell and directing sequence specific binding of the CRISPR complex to that target sequence, and (b) a paired tracr sequence capable of hybridizing with a tracr sequence, II) a second promoter operably linked to an enzyme coding sequence encoding Cas9 containing at least one or more nuclear localization sequences (NLS) near the end of Cas9, and III) a third promoter operably linked to a nucleotide sequence encoding a tracrRNA containing the tracr sequence, where the components I), II) and III) are in the same or in different vectors of the system, where one or more vectors encode multiple guide sequences and one tracr sequence, where the eukaryotic cell is not a human germline cell. 16. The composition according to p. 13 or 14 or the system according to p. 15, characterized in that the first promoter is a polymerase III promoter. 17. The composition according to p. 13 or 14 or the system according to p. 15, characterized in that the second promoter is a polymerase II promoter. 18. The composition according to p. 13 or 14 or the system according to p. 15, characterized in that the third promoter is a polymerase III promoter. 19. A composition according to claim 13 or 14 or a system according to claim 15, characterized in that Cas9 contains one or more NLS effective enough to control the accumulation of said Cas9 in a detectable amount in the nucleus of a eukaryotic cell. 20. The composition according to p. 13 or 14 or the system according to p. 15, wherein the tracr sequence is characterized by at least 50% complementarity of the sequence along the length of the paired tracr sequence with optimal alignment. 21. The composition of claim 13 or 14 or the system of claim 15, wherein Cas9 is Cas9 from Streptococcus pyogenes. 22. The composition of claim 13 or 14 or the system of claim 15, wherein Cas9 is a Cas9 enzyme from Staphylococcus aureus or Streptococcus thermophilus. 23. The composition of claim 13 or 14 or the system of claim 15, wherein the enzyme coding sequence encoding Cas9 is codon-optimized for expression in a eukaryotic cell. 24. The composition according to p. 13 or 14 or the system according to p. 15, characterized in that the guide sequence is at least 15 nucleotides in length. 25. A kit containing a composition according to any one of paragraphs. 1, 2, 13 and 14 or a system according to any one of paragraphs. 3 and 15 and instructions for using the kit to modify one or more genomic loci of interest in a eukaryotic cell, where the eukaryotic cell is not a human germline cell.

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