Engineering of systems, methods and optimized guidance compositions for sequence manipulation

Abstract

The present invention provides systems, methods, and compositions for manipulating the activity of sequences and / or target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for designing and using such vectors. Also provided are methods of directing the formation of CRISPR complexes in eukaryotic cells and methods for screening specific cells by introducing precise mutations using a CRISPR-Cas system.

Description

[0001] This application is a divisional application of an invention patent application with a filing date of December 12, 2013, application number 202110406914.8, and invention name “Systems, methods, and optimized engineering of guiding compositions for sequence manipulation”. This application is a divisional application of an invention patent application with a filing date of December 12, 2013, application number 201380070567.X, and invention name “Systems, methods, and optimized engineering of guiding compositions for sequence manipulation”.

[0002] Related applications and references

[0003] This application claims priority to U.S. Provisional Patent Application No. 61 / 836,127, filed on June 17, 2013, entitled “Engineering Of Systems, Methods And Optimized Compositions For Sequence Manipulation”. This application also claims priority to U.S. Provisional Patent Applications Nos. 61 / 758,468; 61 / 769,046; 61 / 802,174; 61 / 806,375; 61 / 814,263; 61 / 819,803 and 61 / 828,130, each entitled ENGINEERING AND OPTIMIZING SYSTEMS, METHODS, AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013, respectively. Priority is also claimed to U.S. Provisional Patent Applications 61 / 736,527 and 61 / 748,427, both entitled "SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION," filed on December 12, 2012 and January 2, 2013, respectively. Priority is also claimed to U.S. Provisional Patent Applications 61 / 791,409 and 61 / 835,931, both entitled BI-2011 / 008 / 44790.02.2003 and BI-2011 / 008 / 44790.03.2003, filed on March 15, 2013 and June 17, 2013, respectively.

[0004] Reference is also made to U.S. Provisional Patent Applications 61 / 835,936, 61 / 836,101, 61 / 836,080, 61 / 836,123, and 61 / 835,973, each filed on June 17, 2013.

[0005] The foregoing applications, and all documents cited therein or during their prosecution ("application cited documents") and all documents cited or referenced in these application cited documents, and all documents cited or referenced herein ("herein cited documents"), all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or incorporated by reference in any document herein, are hereby incorporated by reference and may be employed in the practice of the present invention. More specifically, all referenced documents are incorporated by reference herein to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference herein. Field of the Invention

[0006] The present invention generally relates to systems, methods, and compositions for controlling gene expression involving sequence targeting, such as genome perturbation or gene editing, which can use vector systems involving clustered regularly interspaced short palindromic repeats (CRISPR) and components thereof.

[0007] Statement Regarding Federally Funded Research

[0008] This invention was made with government support under a NIH Pioneer Award (DP1MH100706) awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background of the Invention

[0010] Recent advances in genome sequencing technologies and analytical methods have significantly accelerated the ability to catalog and map genetic factors associated with a wide range of biological functions and diseases. Accurate genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective interference of individual genetic elements, as well as to advance synthetic biology, biotechnology applications, and medical applications. Although genome editing technologies such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for generating targeted genome interferences, new genome engineering technologies that are affordable, easy to establish, scalable, and convenient for targeting multiple locations within the eukaryotic genome are still needed. SUMMARY OF THE INVENTION

[0012] There is an urgent need for alternative and robust systems and technologies with a series of widely used sequence targeting. The present invention addresses this need and provides related advantages. CRISPR / Cas or CRISPR-Cas systems (two terms are used interchangeably throughout this application) do not require the production of customized proteins targeting specific sequences, but recognize a specific DNA target through a short RNA molecule, and a single Cas enzyme can be programmed, in other words, the short RNA molecule can be used to recruit the Cas enzyme to a specific DNA target. Adding the CRISPR-Cas system to the repertoire of genome sequencing technology and analytical methods can significantly simplify the methodology and improve the ability to catalog and map genetic factors associated with a wide range of biological functions and diseases. In order to effectively and without harmful effects, the CRISPR-Cas system is used for genome editing, it is crucial to understand the engineering and optimization aspects of these genome engineering tools, which are aspects of the present invention for protection.

[0013] 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 tracr pairing sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr pairing sequence, wherein when expressed, the guide sequence guides the CRISPR complex to bind to a sequence-specific target sequence in a cell, such as a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme compounded with the following: (1) a guide sequence hybridized to the target sequence, and (2) a tracr pairing sequence hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme encoding sequence encoding the CRISPR enzyme, the CRISPR enzyme comprising a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr pairing sequence under the control of the first regulatory element. In some embodiments, component (a) further includes two or more guide sequences operably connected to the first regulatory element, wherein when expressed, each of the two or more guide sequences guides the CRISPR complex to bind to a sequence-specific binding of a different target sequence in a eukaryotic cell. In some embodiments, the system includes a tracr sequence under the control of a third regulatory element (such as a polymerase III promoter). In some embodiments, when optimally aligned, along the length of the tracr pairing sequence, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity. In some embodiments, the CRISPR complex includes one or more nuclear localization sequences, which have sufficient strength to drive the CRISPR complex to accumulate in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be limited by theory, it is believed that the nuclear localization sequence is not necessary for the activity of the CRISPR complex in eukaryotic organisms, but including such sequences enhances the activity of the system, especially for nucleic acid molecules in the targeting nucleus. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, 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 eukaryotic cells. In some embodiments, the CRISPR enzyme directs the cutting of one or both strands at the target sequence position. 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 guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides in length, or between 10-30, or between 15-25, or between 15-20 nucleotides. Generally, and throughout this specification, the term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virus-derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced. Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. Moreover, some vectors can direct the expression of the genes that they are operably connected. Such a vector is referred to as an "expression vector" herein. The common expression vector used in recombinant DNA technology is typically a plasmid form.

[0014] Recombinant expression vectors may comprise nucleic acids of the invention in a form suitable for nucleic acid expression in a host cell, which means that these recombinant expression vectors comprise one or more regulatory elements selected based on the host cell to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0015] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). Regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific. In some embodiments, a vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term "regulatory element" are enhancer elements such as WPRE; the CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector may be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by a nucleic acid as described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

[0016] Advantageous vectors include lentiviruses and adeno-associated viruses, and vectors of this type may also be selected to target specific cell types.

[0017] In one aspect, the present invention provides a vector comprising a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, the CRISPR enzyme comprising one or more nuclear localization sequences. In some embodiments, the regulatory element drives transcription of the CRISPR enzyme in a eukaryotic cell such that the CRISPR enzyme accumulates in the nucleus of the eukaryotic cell in a detectable amount. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is a Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme guides the cutting of one or both chains at the target sequence position. In some embodiments, the CRISPR enzyme lacks DNA strand cutting activity.

[0018] In one aspect, the present invention provides a CRISPR enzyme comprising one or more nuclear localization sequences having sufficient strength to drive the CRISPR enzyme to accumulate in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is a Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, and may include a 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 cut one or more chains of the target sequence to which it is bound.

[0019] In one aspect, the present invention provides a eukaryotic host cell comprising: (a) a first regulatory element operably linked to a tracr pairing sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr pairing sequence, wherein when expressed, the guide sequence directs the CRISPR complex to sequence-specific binding to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme compounded with: (1) a guide sequence hybridized to the target sequence, and (2) a tracr pairing sequence hybridized to the tracr sequence; and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding the CRISPR enzyme, the CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr pairing sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences guides the CRISPR complex to sequence-specific binding to a different 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, which is operably linked to the tracr sequence. In some embodiments, when optimally aligned, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pairing sequence. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences having sufficient strength to drive the CRISPR enzyme to accumulate in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, 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 eukaryotic cells. In some embodiments, the CRISPR enzyme directs the cutting of one or both strands at the target sequence location. In some embodiments, the CRISPR enzyme lacks DNA strand cutting 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 guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides in length, or between 10-30, or between 15-25, or between 15-20 nucleotides. In one aspect, the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the embodiments. In other aspects, the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the embodiments. In some embodiments of these aspects, the organism can be an animal; for example a mammal. In addition, the organism can be an arthropod, such as an insect. The organism can also be a plant. Further, the organism can be a fungus.

[0020] 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 tracr pairing sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr pairing sequence, wherein when expressed, the guide sequence guides the CRISPR complex to sequence-specific binding to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme compounded with the following: (1) a guide sequence hybridized to the target sequence, and (2) a tracr pairing sequence hybridized to the tracr sequence; and / or (b) a second regulatory element operably linked to an enzyme encoding sequence encoding the CRISPR enzyme, the CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr pairing sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences guides the CRISPR complex to sequence-specific binding to a different target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element, such as a polymerase III promoter, which is operably linked to the tracr sequence. In some embodiments, when optimally aligned, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pairing sequence. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences having sufficient strength to drive the CRISPR enzyme to accumulate in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, 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 eukaryotic cells. In some embodiments, the CRISPR enzyme directs the cutting of one or both strands at the target sequence location. In some embodiments, the CRISPR enzyme lacks DNA strand cutting 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 guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length.

[0021] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method includes allowing a CRISPR complex to bind to the target polynucleotide to implement the cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex includes a CRISPR enzyme compounded with a guide sequence hybridized to a target sequence within the target polynucleotide, wherein the guide sequence is connected to a tracr pairing sequence, which in turn hybridizes to a tracr sequence. In some embodiments, the cleavage includes cutting one or two chains at the position of the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further includes repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation, including insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors to the eukaryotic cell, wherein the one or more vectors drive expression of one or more of the following: the CRISPR enzyme, the guide sequence connected to the tracr pairing sequence, and the tracr sequence. In some embodiments, the vector is delivered to a eukaryotic cell in a subject. In some embodiments, the modification occurs in the eukaryotic cell in cell culture. In some embodiments, the method further comprises isolating the eukaryotic cell from the subject prior to the modification. In some embodiments, the method further comprises returning the eukaryotic cell and / or cells derived therefrom to the subject.

[0022] In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that the binding results in increased or decreased expression of the polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the polynucleotide, wherein the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to the eukaryotic cell, wherein the one or more vectors drive expression of one or more of the following: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.

[0023] In one aspect, the present invention provides a method for producing a model eukaryotic cell comprising a mutated disease gene. In some embodiments, the disease gene is any gene associated with an increased risk of suffering from or producing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive the expression of one or more of the following: a CRISPR enzyme, a guide sequence connected to a tracr pairing sequence, and a tracr sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to implement the cutting of the target polynucleotide within the disease gene, wherein the CRISPR complex comprises a CRISPR enzyme that is compounded with (1) a guide sequence on the target sequence within the target polynucleotide and (2) a tracr pairing sequence that is hybridized to the tracr, thereby producing a model eukaryotic cell comprising a mutated disease gene. In some embodiments, the cutting comprises cutting one or both chains at the target sequence position by the CRISPR enzyme. In some embodiments, the cutting results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0024] In one aspect, the present invention provides a method for developing a bioactive agent that modulates a cell signaling event associated with a disease gene. In some embodiments, a disease gene is any gene associated with an increased risk of having or producing a disease. In some embodiments, the method includes (a) contacting a test compound with a model cell of any one of the embodiments; and (b) detecting a change in readout that indicates a decrease or increase in a cell signaling event associated with the mutation of the disease gene, thereby developing the bioactive agent that modulates the cell signaling event associated with the disease gene.

[0025] In one aspect, the invention provides a recombinant polynucleotide comprising a guide sequence upstream of a tracr pairing sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a corresponding target sequence present in a 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-oncogene or oncogene.

[0026] In one aspect, the present invention provides a method for selecting one or more prokaryotic cells by introducing one or more mutations in a gene in one or more prokaryotic cells, the method comprising: introducing one or more vectors into one or more prokaryotic cells, wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template; wherein the editing template comprises one or more mutations that eliminate CRISPR enzyme cleavage; allowing the editing template to homologously recombine with a target polynucleotide in the one or more cells to be selected; allowing a CRISPR complex to bind to the target polynucleotide to implement cleavage of the target polynucleotide within the gene, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) a guide sequence on a target sequence within the target polynucleotide, and (2) a tracr mate sequence hybridized to the tracr sequence, wherein the binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing selection of one or more prokaryotic 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 cell to be selected may be a eukaryotic cell. Aspects of the invention allow for the selection of specific cells without the need for selectable markers or a two-step process that may include a counter-selection system.

[0027] In some aspects, the invention provides a non-naturally occurring or engineered composition comprising a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, wherein (a), (b), and (c) are arranged in a 5' to 3' orientation, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence,

[0028] or

[0029] A CRISPR enzyme system, wherein the system is encoded by a vector system, the vector system comprising one or more vectors, the one or more vectors comprising I. a first regulatory element, the first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) one or more guide sequences capable of hybridizing to one or more target sequences in a eukaryotic cell, (b) a tracr mate sequence and (c) one or more tracr sequences, and II. a second regulatory element, the second regulatory element operably linked to a tracr encoding CR The invention relates to an enzyme encoding sequence of an ISPR enzyme, the CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5' to 3' direction, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, or a multiple CR ISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) one or more guide sequences capable of hybridizing to a target sequence in a cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located in the The invention relates to a method for producing a CRISPR complex in a multiplex system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) a guide sequence hybridized to the target sequence, and (2) a tracr mate sequence hybridized to the tracr sequence, and wherein in the multiplex system, multiple guide sequences and a single tracr sequence are used; and wherein one or more of the guide sequence, tracr sequence and tracr mate sequence is modified to improve stability.

[0030] In aspects of the present invention, the modification includes an engineered secondary structure. For example, the modification may include reducing the hybridization region between the tracr pairing sequence and the tracr sequence. For example, the modification may also include fusing the tracr pairing sequence and the tracr sequence through an artificial loop. The modification may include a tracr sequence having a length between 40 and 120 bp. In an embodiment of the present invention, the tracr sequence is between 40 bp and the full length of the tracr. In certain embodiments, the length of the tracRNA includes at least nucleotides 1-67 of the wild-type tracRNA and in some embodiments, at least nucleotides 1-85. In some embodiments, at least nucleotides corresponding to nucleotides 1-67 or 1-85 of the wild-type Streptococcus pyogenes Cas9 tracRNA may be used. In the case where the CRISPR system uses an enzyme other than Cas9 or other than SpCas9, the corresponding nucleotides in the relevant wild-type tracRNA may be present. In some embodiments, the length of the tracRNA includes nucleotides 1-67 or 1-85 not exceeding the wild-type tracRNA. The modification may include sequence optimization. In certain aspects, sequence optimization can include reducing the incidence of polyT sequences in the tracr and / or tracr mate sequence. Sequence optimization can be combined with reducing the hybridization region between the tracr mate sequence and the tracr sequence; for example, reducing the tracr sequence length.

[0031] In one aspect of the invention, a CRISPR-Cas system or CRISPR enzyme system is provided, wherein the modification comprises reducing the polyT sequence in the tracr and / or tracr pairing sequence. In some aspects of the present invention, one or more Ts (that is, a string of more than 3, 4, 5, 6 or more consecutive T bases; in some embodiments, a string of no more than 10, 9, 8, 7, 6 consecutive T bases) present in the poly-T sequence of the relevant wild-type sequence can be replaced by a non-T nucleotide (e.g., A), so that the string is broken into smaller T segments, each of which has 4 or less than 4 (e.g., 3 or 2) consecutive Ts. Bases other than A can be used for substitution, such as C or G, or non-naturally occurring nucleotides or modified nucleotides. If the T string involves the formation of a hairpin (or stem loop), it is advantageous to change the complementary base of the non-T base to complement the non-T nucleotide. For example, if the non-T base is A, its complement can be replaced with T, for example to retain or assist in retaining the secondary structure. For example, 5'-TTTTT can be changed to 5'-TTTAT and the complementary 5'-AAAAA can be changed to 5'-ATAAA.

[0032] In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises adding a polyT terminator sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises adding a polyT terminator sequence in the tracr and / or tracr mate sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises adding a polyT terminator sequence in the guide sequence. The polyT terminator sequence may include 5 or more consecutive T bases.

[0033] In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes changing a loop and / or a hairpin. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes providing a minimum of two hairpins in the guide sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes providing a hairpin formed by complementation between tracr and tracr pairing (direct repeat) sequences. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes providing one or more additional hairpins at the 3' end of the tracrRNA sequence or toward the 3' end of the tracrRNA sequence. For example, a hairpin can be formed in the following manner: a self-complementary sequence connected by a loop is provided within the tracRNA sequence, so that a hairpin is formed when self-folding. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes providing an additional hairpin added to the 3' of the guide sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises extending the 5' end of the guide sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises providing one or more hairpins in the 5' end of the guide sequence. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises appending the sequence (5'-AGGACGAAGTCCTAA) to the 5' end of the guide sequence. Other sequences suitable for forming hairpins should be known to the skilled person and can be used in certain aspects of the present invention. In some aspects of the present invention, at least 2, 3, 4, 5 or more additional hairpins are provided. In some aspects of the present invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises two hairpins. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises three hairpins. In one aspect, the invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises up to five hairpins.

[0034] In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification includes providing crosslinks or providing one or more modified nucleotides in the polynucleotide sequence. Modified nucleotides and / or crosslinks may be provided in any or all sequences in the tracr sequence, tracr pairing sequence and / or guide sequence, and / or in the enzyme encoding sequence, and / or in the vector sequence. Modifications may include at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof. The modified nucleotide may be modified at the ribose, phosphate and / or base moiety. The modified nucleotide may include a 2'-O-methyl analog, a 2'-deoxy analog or a 2'-fluoro analog. The nucleic acid backbone may be modified, for example, a thiophosphate backbone may be used. It is also possible to use a locked nucleic acid (LNA) or a bridged nucleic acid (BNA). Additional examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

[0035] It should be understood that any or all of the above modifications may be provided separately or in combination with a given CRISPR-Cas system or CRISPR enzyme system. Such a system may include one, two, three, four, five or more of the modifications described.

[0036] In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the CRISPR enzyme is a type II CRISPR system enzyme, such as a Cas9 enzyme. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the CRISPR enzyme is composed of less than one thousand amino acids or less than four thousand amino acids. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the Cas9 enzyme is StCas9 or StlCas9, or the Cas9 enzyme is a Cas9 enzyme from an organism selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacterium. In one aspect, the invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the CRISPR enzyme is a nuclease that directs cleavage of both strands at the location of the target sequence.

[0037] In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the first regulatory element is a polymerase III promoter. In one aspect, the present invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the second regulatory element is a polymerase II promoter.

[0038] In one aspect, the invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the guide sequence comprises at least fifteen nucleotides.

[0039] In one aspect, the invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the modification comprises an optimized tracr sequence and / or an optimized guide sequence RNA, and / or a co-folded structure of a tracr sequence and / or one or more tracr pairing sequences, and / or a stabilized secondary structure of a tracr sequence, and / or a tracr sequence with reduced base pairing regions, and / or an RNA element fused to a tracr sequence; and / or, in the multiplex system, there are two RNAs comprising a tracer and comprising multiple guides or one RNA comprising multiple chimeras.

[0040] In aspects of the present invention, the chimeric RNA structure is further optimized according to the results of mutagenesis studies. In a chimeric RNA with two or more hairpins, a mutation in the proximal direct repeat for stabilizing the hairpin can lead to the elimination of CRISPR complex activity. Mutations in the distal direct repeat for shortening or stabilizing the hairpin have no effect on the CRISPR complex activity. Sequence randomization in the protruding region between the proximal and distal repeats can significantly reduce the CRISPR complex activity. Single base pair changes or sequence randomization in the joint region between the hairpins can lead to a complete loss of CRISPR complex activity. The hairpin stabilization of the distal hairpin after the first hairpin after the guide sequence can lead to the maintenance or improvement of CRISPR complex activity. Therefore, in a preferred embodiment of the present invention, the chimeric RNA structure can be further optimized by producing a smaller chimeric RNA, which can be beneficial for therapeutic delivery selection and other uses, and this can be achieved by changing the distal direct repeat to shorten or stabilize the hairpin. In another preferred embodiment of the present invention, the chimeric RNA structure can be further optimized by stabilizing one or more of the distal hairpins. The stabilization of the hairpin can include modifying the sequence suitable for forming the hairpin. In some aspects of the invention, at least 2, 3, 4, 5 or more additional hairpins are provided. In some aspects of the invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. In some aspects of the invention, stabilization can be cross-linking and other modifications. Modification can include comprising at least one non-naturally occurring nucleotide or a modified nucleotide, or its analog. The modified nucleotide can be modified at the ribose, phosphate and / or base moiety. The modified nucleotide can include 2'-O-methyl analogs, 2'-deoxy analogs or 2'-fluoro analogs. The nucleic acid backbone can be modified, for example, a thiophosphate backbone can be used. It is also possible to use locked nucleic acids (LNA) or bridged nucleic acids (BNA). Other examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

[0041] In one aspect, the invention provides a CRISPR-Cas system or a CRISPR enzyme system, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

[0042] Therefore, in some aspects of the invention, the length of the tracRNA required in the construct (e.g., chimeric construct) of the invention is not necessarily fixed, and in some aspects of the invention, it can be between 40 and 120 bp, and in some aspects of the invention, it is up to the full length of tracr, for example, in some aspects of the invention, until the 3' end of tracr, as interrupted by the transcription termination signal in the bacterial genome. In certain embodiments, the length of tracRNA includes at least nucleotides 1-67 of wild-type tracRNA and in some embodiments, at least nucleotides 1-85. In some embodiments, at least nucleotides corresponding to nucleotides 1-67 or 1-85 of wild-type Streptococcus pyogenes Cas9 tracRNA can be used. In the case where the CRISPR system uses an enzyme other than Cas9 or other than SpCas9, the corresponding nucleotides in the relevant wild-type tracRNA may be present. In some embodiments, the length of tracRNA includes no more than nucleotides 1-67 or 1-85 of wild-type tracRNA. With respect to sequence optimization (e.g., reducing polyT sequences), for example, with respect to the tracr pair (direct repeat) or the internal T string of tracrRNA, in some aspects of the invention, one or more Ts (that is, a string of more than 3, 4, 5, 6 or more consecutive T bases; in some embodiments, a string of no more than 10, 9, 8, 7, 6 consecutive T bases) present in the poly-T sequence of the relevant wild-type sequence can be replaced by a non-T nucleotide (e.g., A), so that the string is broken into smaller T segments, each of which has 4 or less than 4 (e.g., 3 or 2) consecutive Ts. If the T string involves the formation of a hairpin (or stem loop), it is advantageous to change the complementary base of the non-T base to be complementary to the non-T nucleotide. For example, if the non-T base is A, its complement can be replaced with T, for example to preserve or assist in preserving secondary structure. For example, 5'-TTTTT can be changed to 5'-TTTAT and the complementary 5'-AAAAA can be changed to 5'-ATAAA. With respect to the presence of a polyT terminator sequence in a tracr+tracr paired transcript, e.g., a polyT terminator (TTTTT or more), in some aspects of the invention, it is advantageous to add such a terminator sequence to the end of the transcript, whether the transcript is in the form of two RNAs (tracr and tracr paired) or a single guide RNA. With respect to loops and hairpins in tracr and tracr paired transcripts, in some aspects of the invention, it is advantageous that a minimum of two hairpins are present in the chimeric guide RNA. The first hairpin can be a hairpin formed by complementation between the tracr and tracr paired (direct repeat) sequences.The second hairpin can be at the 3' end of the tracrRNA sequence, and this can provide a secondary structure for interacting with Cas9. Additional hairpins can be added to the 3' of the guide RNA, for example, in some aspects of the invention, for increasing the stability of the guide RNA. In addition, in some aspects of the invention, the 5' end of the guide RNA can be extended. In some aspects of the invention, the 20bp in the 5' end can be considered as a guide sequence. The 5' part can be extended. One or more hairpins can be provided in the 5' part, for example, in some aspects of the invention, this can also improve the stability of the guide RNA. In some aspects of the invention, a specific hairpin can be provided by attaching the sequence (5'-AGGACGAAGTCCTAA) to the 5' end of the guide sequence, and in some aspects of the invention, this can help improve stability. Other sequences suitable for forming hairpins should be known to technicians and can be used in some aspects of the present invention. In some aspects of the invention, at least 2, 3, 4, 5 or more additional hairpins are provided. In some aspects of the invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. The foregoing also provides aspects of the invention relating to secondary structures in guide sequences. In some aspects of the invention, crosslinking and other modifications may be present, for example, to improve stability. Modifications may include at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof. The modified nucleotide may be modified at the ribose, phosphate and / or base moiety. The modified nucleotide may include a 2'-O-methyl analog, a 2'-deoxy analog or a 2'-fluoro analog. The nucleic acid backbone may be modified, for example, a thiophosphate backbone may be used. It is also possible to use locked nucleic acids (LNA) or bridged nucleic acids (BNA). Additional examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Such modifications or crosslinking may be present in the guide sequence or other sequences adjacent to the guide sequence.

[0043] It is therefore an object of the present invention that any previously known product, process for making the product, or method for using the product is not covered in the present invention, such that the applicant reserves and hereby discloses the right to disclaim any previously known product, process, or method. It is further noted that within the scope of the present invention, the present invention is not intended to cover any product, process, or method for making the product or using the product that does not meet the written description and enforceability requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that the applicant reserves and hereby discloses the right to disclaim any previously described product, process for making the product, or method for using the product.

[0044] It should be noted that in this disclosure and especially in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. may have the meaning ascribed to it in U.S. patent law; for example, they may mean "includes", "included", "including", etc.; and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements that are found in the prior art or that affect the basic or novel characteristics of the invention. These and other embodiments are disclosed in the following detailed description or are apparent from and encompassed by it.

[0045] The present invention also relates to the following items:

[0046] 1. A non-naturally occurring or engineered composition comprising:

[0047] A) A CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

[0048] (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

[0049] (b) a tracr mate sequence, and

[0050] (c) A tracr sequence

[0051] Among them, (a), (b) and (c) are arranged in the 5' to 3' direction.

[0052] wherein upon transcription, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence,

[0053] wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) a guide sequence hybridized to the target sequence, and (2) a tracr mate sequence hybridized to the tracr sequence,

[0054] or

[0055] B) a CRISPR enzyme system, wherein the system is encoded by a vector system, the vector system comprising one or more vectors, the one or more vectors comprising

[0056] I. A first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises:

[0057] (a) one or more guide sequences that are capable of hybridizing to one or more target sequences in a eukaryotic cell,

[0058] (b) a tracr mate sequence, and

[0059] (c) one or more tracr sequences, and

[0060] II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,

[0061] Among them, (a), (b) and (c) are arranged in the 5' to 3' direction.

[0062] wherein components I and II are located on the same or different carriers of the system,

[0063] wherein upon transcription, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence,

[0064] wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence,

[0065] or

[0066] C) a multiplex CRISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors, wherein the one or more vectors comprise

[0067] I. A first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises:

[0068] (a) one or more guide sequences that are capable of hybridizing to one or more target sequences in a eukaryotic cell,

[0069] (b) a tracr mate sequence, and

[0070] (c) one or more tracr sequences, and

[0071] II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,

[0072] Among them, (a), (b) and (c) are arranged in the 5' to 3' direction.

[0073] wherein components I and II are located on the same or different carriers of the system,

[0074] wherein upon transcription, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence,

[0075] wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence,

[0076] In this multiplex system, multiple chiRNA polynucleotide sequences are used.

[0077] or

[0078] D) a multiplex CRISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors, wherein the one or more vectors comprise

[0079] I. A first regulating element, the first regulating element is operably connected to

[0080] (a) one or more guide sequences capable of hybridizing to a target sequence in a cell, and

[0081] (b) at least one or more tracr mate sequences,

[0082] II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, and

[0083] III. a third regulatory element operably linked to a tracr sequence,

[0084] wherein components I, II and III are located on the same or different vectors of the system,

[0085] wherein upon transcription, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence,

[0086] wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) a guide sequence hybridized to the target sequence, and (2) a tracr mate sequence hybridized to the tracr sequence, and

[0087] wherein in the multiplex system, multiple guide sequences and a single tracr sequence are used;

[0088] and

[0089] Wherein, in the polynucleotide sequence of A), or in the system of B), C) or D), one or more of the guide sequence, tracr sequence and tracr mate sequence is modified to improve stability.

[0090] 2. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 1, wherein the modification comprises an engineered secondary structure.

[0091] 3. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 1 or item 2, wherein the modification comprises reducing the hybridization region between the tracr pairing sequence and the tracr sequence.

[0092] 4. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the modification comprises fusing the tracr mate sequence and the tracr sequence through an artificial loop.

[0093] 5. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the modification comprises a tracr sequence having a length between 40 and 120 bp.

[0094] 6. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the tracr sequence is between 40 bp and the full length of the tracr.

[0095] 7. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the tracr sequence at least includes nucleotides 1-67 of the corresponding wild-type tracRNA.

[0096] 8. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the tracr sequence at least includes nucleotides 1-85 of the corresponding wild-type tracRNA.

[0097] 9. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the tracr sequence comprises nucleotides corresponding to nucleotides 1-67 of wild-type Streptococcus pyogenes Cas9 tracRNA.

[0098] 10. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the tracr sequence comprises nucleotides corresponding to nucleotides 1-85 of wild-type Streptococcus pyogenes Cas9 tracRNA.

[0099] 11. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 9, wherein the tracr sequence consists essentially of nucleotides corresponding to nucleotides 1-67 of wild-type Streptococcus pyogenes Cas9 tracRNA.

[0100] 12. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 10, wherein the tracr sequence consists essentially of nucleotides corresponding to nucleotides 1-85 of wild-type Streptococcus pyogenes Cas9 tracRNA.

[0101] 13. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the modification comprises sequence optimization.

[0102] 14. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 13, wherein the modification comprises reducing the polyT sequence in the tracr and / or tracr pairing sequence.

[0103] 15. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 14, wherein one or more Ts present in the poly-T sequence of the relevant wild-type sequence have been replaced by a non-T nucleotide.

[0104] 16. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 13, 14 or 15, wherein the modified sequence does not include any polyT sequence having more than 4 consecutive Ts.

[0105] 17. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises adding a polyT terminator sequence.

[0106] 18. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 17, wherein the modification comprises adding a polyT terminator sequence to the tracr and / or tracr pairing sequence.

[0107] 19. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 17 or 18, wherein the modification comprises adding a polyT terminator sequence to the guide sequence.

[0108] 20. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the modification comprises changing the loop and / or hairpin.

[0109] 21. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 20, wherein the modification comprises providing a minimum of two hairpins in the guide sequence.

[0110] 22. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 20 or 21, wherein the modification comprises providing a hairpin formed by complementarity between the tracr and tracr pairing sequences.

[0111] 23. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in items 20 to 22, wherein the modification comprises providing one or more additional hairpins at the 3' end of the tracrRNA sequence.

[0112] 24. The CRISPR-Cas system chiRNA or CRISPR enzyme system of items 20 to 23, wherein the modification comprises providing one or more additional hairpins added to the 3' of the guide sequence.

[0113] 25. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises extending the 5' end of the guide sequence.

[0114] 26. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 25, wherein the modification comprises providing one or more hairpins in the 5' end of the guide sequence.

[0115] 27. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 25 or 26, wherein the modification comprises appending the sequence (5'-AGGACGAAGTCCTAA) to the 5' end of the guide sequence.

[0116] 28. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises providing cross-links or providing one or more modified nucleotides in the polynucleotide sequence.

[0117] 29. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 28, wherein modified nucleotides are provided in any or all of the tracr sequence, tracr mate sequence and / or guide sequence, and / or in the enzyme coding sequence, and / or in the vector sequence.

[0118] 30. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 28 or 29, wherein providing a modified nucleotide comprises comprising at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof.

[0119] 31. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 30, wherein the modified nucleotides are modified at the ribose, phosphate and / or base moieties.

[0120] 32. The CRISPR-Cas system chiRNA or CRISPR enzyme system of item 30, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyl analogs, 2'-deoxy analogs or 2'-fluoro analogs.

[0121] 33. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in item 30, wherein the modified nucleotide is selected from the group consisting of 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, and 7-methylguanosine.

[0122] 34. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises two hairpins.

[0123] 35. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises three hairpins.

[0124] 36. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises up to five hairpins.

[0125] 37. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0126] 38. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the CRISPR enzyme is a Cas9 enzyme.

[0127] 39. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the CRISPR enzyme consists of less than one thousand amino acids.

[0128] 40. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any one of items 1 to 38, wherein the CRISPR enzyme consists of less than four thousand amino acids.

[0129] 41. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the Cas9 enzyme is StCas9 or StlCas9.

[0130] 42. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the Cas9 enzyme is a Cas9 enzyme from an organism selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibacter, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacterium.

[0131] 43. The CRISPR-Cas system chiRNA or CRISPR enzyme system as described in any preceding item, wherein the CRISPR enzyme is a nuclease that directs the cleavage of both strands at the location of the target sequence.

[0132] 44. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the first regulatory element is a polymerase III promoter.

[0133] 45. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the second regulatory element is a polymerase II promoter.

[0134] 46. ​​The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the guide sequence comprises at least fifteen nucleotides.

[0135] 47. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any preceding item, wherein the modification comprises an optimized tracr sequence and / or an optimized guide sequence RNA, and / or a co-folded structure of a tracr sequence and / or one or more tracr pairing sequences, and / or a secondary structure that stabilizes the tracr sequence, and / or a tracr sequence with reduced base pairing regions, and / or an RNA element fused to a tracr sequence; and / or, in the multiplex system, there are two RNAs comprising a tracer and comprising multiple guides or one RNA comprising multiple chimeras.

[0136] 48. The CRISPR-Cas system chiRNA or CRISPR enzyme system of any one of items 1-47, wherein the CRISPR enzyme is codon-optimized for expression in eukaryotic cells.

[0137] 49. The composition of any preceding item, wherein the composition comprises a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence.

[0138] 50. The composition of item 49, wherein the composition further comprises a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences.

[0139] 51. A CRISPR enzyme system as described in any preceding item.

[0140] 52. A multiplex CRISPR enzyme system as described in any preceding item.

[0141] 53. The composition of item 49 or 50, the CRISPR enzyme system of item 51, or the transcription or translation product of the multiple CRISPR enzyme system of item 52. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description which describes illustrative embodiments in which the principles of the invention are utilized, and in the accompanying drawings:

[0144] Figure 1A schematic model of the CRISPR system is shown. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA via a synthetic guide RNA (sgRNA) containing a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base pairs with the DNA target (blue), directly upstream of the essential 5'-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-strand break (DSB) approximately 3 bp upstream of the PAM (red triangle).

[0145] Figure 2A -F shows an exemplary CRISPR system, a possible mechanism of action, an exemplary adaptation of expression in eukaryotic cells, and results of experiments assessing nuclear localization and CRISPR activity.

[0146] Figures 3A-C Shown are an exemplary expression cassette for expressing CRISPR system elements in eukaryotic cells, the predicted structures of exemplary guide sequences, and CRISPR system activities as measured in eukaryotic and prokaryotic cells.

[0147] Figures 4A-D show the results of evaluating SpCas9 specificity against exemplary targets.

[0148] Figures 5A-G illustrate an exemplary vector system and results of its use in directing homologous recombination in eukaryotic cells.

[0149] Figure 6A-C shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting.

[0150] 7A-D show an exemplary CRISPR system, an exemplary adaptation of expression in a eukaryotic cell, and results of assays assessing CRISPR activity.

[0151] Figures 8A-C Exemplary manipulation of the CRISPR system for targeting of genomic loci in mammalian cells is demonstrated.

[0152] Figures 9A-B Shown are the results of Northern blot analysis of crRNA processing in mammals.

[0153] Figure 10A -C shows a schematic representation of the chimeric RNA and the results of the SURVEYOR assay for CRISPR system activity in eukaryotic cells.

[0154] Figures 11A-BA graphical representation showing the results of the SURVEYOR assay for CRISPR system activity in eukaryotic cells.

[0155] Figure 12 The predicted secondary structures of exemplary chimeric RNAs including a guide sequence, a tracr mate sequence, and a tracr sequence are shown.

[0156] FIG. 13 is a phylogenetic tree of Cas genes.

[0157] Figure 14A -F shows a phylogenetic analysis revealing five Cas9 families, including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids).

[0158] Figure 15 A graph depicting the functions of different optimized guide RNAs is shown.

[0159] Figure 16 The sequences and structures of different guide chimeric RNAs are shown.

[0160] Figure 17 The co-folded structure of tracrRNA and direct repeats is shown.

[0161] Figure 18A A and B show data from in vitro optimization of St1Cas9 chimeric guide RNA.

[0162] Figures 19A-B Cleavage of unmethylated or methylated targets by SpCas9 cell lysates is shown.

[0163] Figures 20A-GOptimization of guide RNA structure for SpCas9-mediated mammalian genome editing is shown. (a) Schematic diagram of a bicistronic expression vector (PX330) of a single guide RNA (sgRNA) driven by the U6 promoter and a human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9) driven by the CBh promoter for all subsequent experiments. The sgRNA consists of a 20-nt guide sequence (blue) and a scaffold (red), which is truncated at different positions as indicated. (b) SURVEYOR assay for SpCas9-mediated insertion and deletion (indel) at human EMX1 and PVALB seats. Arrows indicate expected SURVEYOR fragments (n=3). (c) Northern blot analysis of four sgRNA truncated structures, with U1 as a loading control. (d) Both wild-type (wt) or nickase mutants (D10A) of SpCas9 promote insertion of the HindIII site into the human EMX1 gene. Single-stranded oligonucleotides (ssODNs) directed in the sense or antisense direction relative to the genomic sequence were used as homologous recombination templates. (e) Schematic diagram of the human SERPINB5 locus. sgRNA and PAM are indicated by colored bars on the sequence; methylcytosine (Me) is highlighted (pink) and numbered relative to the transcription start site (TSS, +1). (f) Methylation status of SERPINB5 determined by bisulfite sequencing of 16 clones. Solid circles, methylated CpGs; open circles, unmethylated CpGs. (g) Efficiency of modification by three sgRNAs targeting the methylated regions of SERPINB5, determined by deep sequencing (n=2). Error bars indicate Wilson intervals (Online Methods).

[0164] Figures 21A-B Further optimization of CRISPR-Cas sgRNA construction is shown. (a) Schematic diagram of four additional sgRNA constructions I-IV. Each construction consists of a 20-nt guide sequence (blue) connected to a direct repeat (DR, gray), which hybridizes to tracrRNA (red). The DR-tracrRNA hybrid is truncated by an artificial GAAA stem loop at +12 or +22 as indicated. The tracrRNA truncation positions are numbered according to the transcription start sites previously reported for tracrRNA. sgRNA constructions II and IV carry mutations in their poly-U bundles, which can act as early transcription terminators. (b) SURVEYOR assay for SpCas9-mediated indels at target sites 1-3 at the human EMX1 locus. Arrows indicate expected SURVEYOR fragments (n=3).

[0165] Figure 22Visualization of some target sites in the human genome is shown.

[0166] Figures 23A-B Shown are (A) schematic diagram of sgRNA and (B) SURVEYOR analysis of five sgRNA variants of SaCas9 for the optimized truncation construct with the highest cleavage efficiency.

[0167] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0168] Detailed description of the invention

[0169] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (a locus) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure may be performed before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeled component.

[0170] In various aspects of the invention, the terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence, a tracr sequence and a tracr mate sequence. The term "guide sequence" refers to a sequence of about 20 bp within a guide RNA that specifies a target site and can be used interchangeably with the terms "guide" or "spacer". The term "tracr mate sequence" can also be used interchangeably with the term "(one or more) direct repeats".

[0171] The term "wild type" as used herein is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene, or characteristic that distinguishes it from mutant or variant forms as it occurs in nature.

[0172] The term "variant" as used herein should be understood to mean a display having properties derived from a pattern found in nature.

[0173] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human effort. These terms, when referring to nucleic acid molecules or polypeptides, indicate that the nucleic acid molecules or polypeptides are at least substantially free from at least one other component with which they are associated in nature or as found in nature.

[0174] "Complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick base pairing or other non-traditional types. The percentage of complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region 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 to two nucleic acids that hybridize under stringent conditions.

[0175] As used herein, "stringent conditions" for hybridization refer to a nucleic acid with complementarity to a target sequence that primarily hybridizes with the target sequence and does not substantially hybridize to a condition on a non-target sequence. Stringent conditions are generally sequence-dependent and vary depending on many factors. Generally speaking, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in Tijssen (1993) Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes, Part I, Chapter II, "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, New York.

[0176] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding of bases between the nucleotide residues. Hydrogen bonding can occur by means of Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may contain two chains forming a duplex, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction may constitute a step in a broader process such as the start of PCR, or the cleavage of a polynucleotide via an enzyme. A sequence that is capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.

[0177] As used herein, "stabilization" or "increasing stability" refers to fixing or stabilizing the structure of a molecule relative to a component of a CRISPR system. This can be accomplished by introducing one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, crosslinking, disconnecting specific nucleotide segments, and other modifications. Modifications may include the inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof. The modified nucleotide may be modified at the ribose, phosphate, and / or base moiety. The modified nucleotide may include a 2'-O-methyl analog, a 2'-deoxy analog, or a 2'-fluoro analog. The nucleic acid backbone may be modified, for example, a thiophosphate backbone may be used. It is also possible to use locked nucleic acids (LNA) or bridged nucleic acids (BNA). Additional examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. These modifications may be applied to any component of the CRISPR system. In a preferred embodiment, these modifications may be made to an RNA component (e.g., the guide RNA or a chimeric polynucleotide sequence).

[0178] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0179] The terms "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 it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; such modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling component. The term "amino acid" as used herein includes natural and / or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptide mimetics.

[0180] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sports animals and pets. Also included are tissues, cells and progeny of a biological entity obtained in vivo or cultured in vitro. In some embodiments, the subject may be an invertebrate, such as an insect or nematode; while in other embodiments, the subject may be a plant or fungus.

[0181] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. The beneficial effects include achievement of a diagnostic determination; improvement of a disease, symptom, disorder, or pathological condition; reduction or prevention of the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0182] As used herein, "treatment" or "treating" or "alleviate" or "ameliorate" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit means any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the composition can be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

[0183] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that provides an image for detection by any of the imaging methods described herein. The specific dose may vary depending on one or more of the following: the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system that carries it.

[0184] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); ANTIBODIES, A LABORATORY MANUAL, 2nd ed. (1988); MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0185] Several aspects of the present invention relate to vector systems including one or more vectors, or the vector itself. The vector can be designed to express CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as 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, California (Calif.) (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0186] Vectors can be introduced into prokaryotes and propagated therein. In some embodiments, prokaryotes are used to amplify multiple copies of vectors to be introduced into eukaryotic cells, or as intermediate vectors in the production of vectors to be introduced into eukaryotic cells (e.g., amplification plasmids as part of a viral vector packaging system). In some embodiments, prokaryotes are used to amplify multiple copies of a vector and express one or more nucleic acids, such as providing a source for one or more proteins delivered to a host cell or host organism. The expression of proteins in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion proteins. Fusion vectors add multiple amino acids to the protein encoded therein, such as the amino terminus of the recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of recombinant proteins; (ii) increasing the solubility of recombinant proteins; and (iii) assisting the purification of recombinant proteins by acting as ligands in affinity purification. Typically, in fusion expression vectors, protein cleavage sites are introduced into the junction of the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety after the fusion protein is purified. Such enzymes and their homologous recognition sequences include factor Xa, thrombin and enterokinase. Exemplary 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), which fuse glutathione S-transferase (GST), maltose E binding protein or protein A to the target recombinant protein, respectively.

[0187] Examples of suitable inducible non-fusion E. coli expression vectors 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.

[0188] In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari et al., 1987. EMBO J 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (Invitrogen Corp, San Diego, Calif).

[0189] In some embodiments, a baculovirus vector is used, and the vector drives protein expression in insect cells. Baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0190] In some embodiments, a mammalian expression vector is used, and the vector is capable of driving one or more sequences to be expressed in mammalian cells. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the control function of the expression vector is typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and other viruses described herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook et al., Molecular Cloning: A Laboratory Manual. (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.

[0191] In some embodiments, the recombinant mammalian expression vector is capable of directing the nucleic acid to be preferentially expressed in a particular cell type (e.g., using tissue-specific regulatory elements to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol 43: 235-275), in particular the promoter of the T cell receptor (Winoto and Baltimore, 1989. EMBO Journal). 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 promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, for example, the murine homeobox protein (hox) promoters Kessel and Gruss (1990. Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546).

[0192] In some embodiments, the regulatory element is operably connected to one or more elements of the CRISPR system, thereby driving the expression of the one or more elements of the CRISPR system. In general, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDR (SPacer Interspaced Direct Repeats), constitutes a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus includes a different class of short sequence repeats (SSRs) that are spaced apart and identified in Escherichia coli (Ishino et al., J. Bacteriol., 169: 5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171: 3553-3556

[1989] ) and related genes. Similar interspersed 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., 5:254-263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). These CRISPR loci are typically distinct from other SSRs in their repeat structure, which has been termed short regularly interspaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] ; and Mojica et al., Mol. Microbiol., 36:244-246

[2000] ). In general, these repeats are short elements that occur in clusters and are regularly spaced apart by unique intervening sequences of substantially constant length (Mojica et al.,

[2000] supra). Although the repeat sequences are highly conserved between strains, the sequences of many of the intervening repeats and these spacers generally differ from strain to strain (van Embden et al., J. Bacteriol.), 182:2393-2401

[2000] ). CRISPR loci have been identified in more than 40 prokaryotes (see, e.g., Janssen et al., Mol. Microbiol.), 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ), including but not limited to: Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0193] In general, "CRISPR system" refers collectively to transcripts and other elements related to the expression of CRISPR-related ("Cas") genes or directing their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr pairing sequences (covering "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. In some embodiments, one or more elements of the CRISPR system are derived from type I, type II, or type III CRISPR systems. In some embodiments, one or more elements of the CRISPR system are derived from special organisms comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, the CRISPR system is characterized by elements that promote the formation of a CRISPR complex (also referred to as a prototype spacer in the context of an endogenous CRISPR system) at the site of the target sequence. In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not required, provided that there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus including the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In aspects of the present invention, an exogenous template polynucleotide may be referred to as an editing template. In one aspect of the present invention, the recombination is homologous recombination.

[0194] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of) the target sequence. Without wishing to be bound by theory, the tracr sequence (which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence)) may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in forming a CRISPR complex. With respect to the target sequence, it is believed that full complementarity is not required, as long as it is sufficient to be functional. In some embodiments, when optimally aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pairing sequence. In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell so that the expression of these elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence connected to a tracr pairing sequence, and a tracr sequence can each be operably connected to a separate regulatory element on a separate vector. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, wherein one or more additional vectors providing any component of the CRISPR system are not included in the first vector. The CRISPR system elements combined in a single vector can be arranged in any suitable orientation, such as one element is located 5' ("upstream") or 3' ("downstream") relative to a second element. The coding sequence of an element can be located on the same strand or opposite strand of the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

[0195] In some embodiments, a vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr pairing sequence and optionally downstream of a regulatory element operably connected to the tracr pairing sequence, so that after the guide sequence is inserted into the insertion site and when expressed, the guide sequence guides the CRISPR complex to bind to the sequence-specific target sequence in a eukaryotic cell. In some embodiments, a vector comprises one or more insertion sites, each of which is located between two tracr pairing sequences, thereby allowing the insertion of a guide sequence at each site. In such an arrangement, two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or a combination of these. When a plurality of different guide sequences are used, a single expression construct may be used to target CRISPR activity to a plurality of different corresponding target sequences within a cell. For example, a single vector can 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 such target sequence-containing vectors can be provided and, optionally, delivered to a cell.

[0196] In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme (such as 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, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database accession number Q99ZW2. In some embodiments, an unmodified CRISPR enzyme such as Cas9 has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9, and can be Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae. In some embodiments, the CRISPR enzyme guides the cutting of one or both chains at the position of the target sequence (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the CRISPR enzyme guides the cutting of one or both chains 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, a vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cut one and both chains of a target polynucleotide containing a target sequence. For example, aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single chain). Other mutation examples that cause Cas9 to be a nickase include but are not limited to H840A, N854A, and N863A. In certain embodiments, a Cas9 nickase can be used in combination with one or more guide sequences, for example, two guide sequences that target the sense and antisense strands of the DNA target, respectively. This combination allows two chains to be nicked and used to induce NHEJ. Applicants have demonstrated (data not shown) the effectiveness of two nickase targets (i.e., sgRNAs targeting different chains of DNA at the same position) in inducing mutagenesis NHEJ.A single nickase (Cas9-D10A with a single sgRNA) is not able to induce NHEJ and create indels, but Applicants have shown that a dual nickase (Cas9-D10A and two sgRNAs targeting different strands at the same position) can do so in human embryonic stem cells (hESCs). The efficiency is about 50% of the nuclease (i.e., conventional Cas9 without the D10 mutation) in hESCs.

[0197] As an additional example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvCIII) can be mutated to produce a mutated Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, when the DNA cleavage activity of the mutated CRISPR enzyme is about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less than that of its non-mutated form, the enzyme is considered to be substantially lacking all DNA cleavage activity. Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in the corresponding amino acids may be produced to achieve similar effects.

[0198] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. These eukaryotic cells can be those of or derived from specific organisms, such as mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons with specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on (among other things) the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database, and these tables can be adapted for use in different ways. See, Nakamura Y. 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 for codon optimization of specific sequences for expression in specific host cells are also available, such as GeneForge (Aptagen; Jacobus, PA). 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 frequently used codons for a particular amino acid.

[0199] In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxyl terminus, or a combination of these (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxyl terminus). When more than one NLS is present, each can be selected to be independent of the other NLSs, such that a single NLS can be present in more than one copy and / or combined with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the CRISPR enzyme comprises up to 6 NLSs. In some embodiments, an NLS can be considered to be close to the N-terminus or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of the protein, but other types of NLS are known. Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, which has the amino acid sequence PKKKRKV; an NLS from a nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); a c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; an hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of importin-α; the sequences VSRKRPRP and PPKKARED of myoma T protein; the sequence POPKKKPL of human p53; the sequence of mouse c-abl The sequence of IV is SALIKKKKKMAP; the sequences of influenza virus NS1 are DRLRR and PKQKKRK; the sequence of hepatitis virus delta antigen is RKLKKKIKKL; the sequence of mouse Mx1 protein is REKKKFLKRR; the sequence of human poly (ADP-ribose) polymerase is KRKGDEVDGVDEVAKKKSKK; and the sequence of steroid hormone receptor (human) glucocorticoid is RKCLQAGMNLEARKTKK.

[0200] In general, the one or more NLSs have sufficient strength to drive the CRISPR complex to accumulate in a detectable amount in the nucleus of a eukaryotic cell. In general, the intensity of nuclear localization activity can drive the number of NLSs in the CRISPR enzyme, the one or more specific NLSs used, or a combination of these factors. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detection marker can be fused to the CRISPR enzyme so that the position in the cell can be visualized, such as in combination with a means for detecting the position of the nucleus (e.g., a dye specific for the nucleus, such as DAPI). Examples of detectable markers include fluorescent proteins (e.g., green fluorescent protein, or GFP; RFP; CFP), and epitope tags (HA tags, flag tags, SNAP tags). The nucleus can also be separated from the cell, and its contents can then be analyzed by any suitable method for detecting proteins, such as immunohistochemistry, Western blotting, or enzyme activity assays. Accumulation in the nucleus can also be determined indirectly, such as by assaying the effect of CRISPR complex formation (e.g., assaying DNA cleavage or mutation at the target sequence, or assaying gene expression activity that is altered due to the effect of CRISPR complex formation and / or CRISPR enzyme activity), compared to a control not exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.

[0201] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, Inc.), 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 fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, for example by transfection with a vector encoding the components of the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence can be assessed in a test tube by providing the target sequence, components of a CRISPR complex including the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence and the control guide sequence reactions. Other assays are possible and will occur to those skilled in the art.

[0202] The guide sequence can be selected to target any target sequence. In certain embodiments, the target sequence is a sequence in the genome of the cell. Exemplary target sequences are included in those that are unique in the target genome. For example, for Streptococcus pyogenes Cas9, the unique target sequence in the genome can include the Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG, wherein NNNNNNNNNNNNNXGG (N is A, G, T or C; and X can be anything) single occurrence in the genome. The unique target sequence in the genome can include the Streptococcus pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, wherein NNNNNNNNNNNNXGG (N is A, G, T or C; and X can be anything) single occurrence in the genome. For S. thermophilus CRISPR1 Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) occurs single in the genome. A unique target sequence in a genome may include a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) occurs single in the genome. For S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) occurs single in the genome. A unique target sequence in a genome can include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) occurs once in the genome. In each of these sequences, "M" can be A, G, T, or C, and need not be considered unique in sequence identification.

[0203] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some algorithms are based on calculating the minimum Gibbs (Gibbs) free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler "Nucleic Acids Res." 9 (1981), 133-148). Another example of a folding algorithm is the use of an online web server RNAfold developed by the Institute for Theoretical Chemistry of the University of Vienna (University of Vienna) (see, for example, AR Gruber et al., 2008, "Cell" (Cell) 106 (1): 23-24; and PA Carr (PA Carr) and GM Church (GM Church), 2009, "Nature Biotechnology" (Nature Biotechnology) 27 (12): 1151-62). Additional algorithms can be found in U.S. Application Serial No. TBA (Broad Reference No. BI-2012 / 08444790.11.2022), incorporated herein by reference.

[0204] In general, a tracr mate sequence includes any sequence that has sufficient complementarity to a tracr sequence to promote one or more of the following: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. Typically, the degree of complementarity is with respect to optimal alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm, and can further account for secondary structure, such as self-complementarity within the tracr sequence or tracr mate sequence. In some embodiments, when optimally aligned, the degree of complementarity between the tracr sequence and the tracr mate sequence along the length of the shorter of the two is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more. An example of an optimal alignment between a tracr sequence and a tracr pairing sequence is provided in Figures 7B and 13B. In some embodiments, the tracr sequence is about or more than about 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 tracr pairing sequence are contained in a single transcript so that hybridization between the two produces a transcript with a secondary structure (such as a hairpin). The preferred loop-forming sequence for use in a hairpin structure is four nucleotides in length, and most preferably has the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. These sequences preferably include triplets (e.g., AAA), and additional nucleotides (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In one embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has up to five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence; preferably this is a polyT sequence, such as six T nucleotides. Figure 13BAn example illustration of such a hairpin structure is provided in the following section in which the portion of the sequence 5' to the final "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' to the loop corresponds to the tracr sequence. Additional non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), wherein "N" represents a base of the guide sequence, the first block in lower case letters represents the tracr mate sequence, and the second block in lower case letters represents the tracr sequence, and the last poly-T sequence represents a transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTT TT; (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcatttta tggcagggtgtTTTTTT; (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaa gtggcaccgagtcggtgcTTTTTT; (5)NNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaact tgaaaaagtgTTTTTTTT; and (6)NNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT. In some embodiments, sequences (1) to (3) are used in combination with Cas9 from CRISPR1 of Streptococcus thermophilus. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from CRISPR1 of Streptococcus pyogenes.In some embodiments, the tracr sequence is a separate transcript from (e.g., in) a transcript comprising the tracr mate sequence. Figure 13B ).

[0205] In some embodiments, a recombinant template is also provided. The recombinant template can be a component of another vector as described herein, which is contained in a separate vector, or is provided as a separate polynucleotide. In some embodiments, the recombinant template is designed to be used as a template in homologous recombination, such as within or near a target sequence cut or cut by a CRISPR enzyme as a part of a CRISPR complex. The template polynucleotide can have any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotide lengths. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When performing optimal comparison, the template polynucleotide can overlap with one or more nucleotides (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides) of the target sequence. In some embodiments, when a template sequence is optimally aligned with a polynucleotide comprising a target sequence, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides of the target sequence.

[0206] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may comprise any other protein, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) 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), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4 DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, which is incorporated herein by reference. In some embodiments, a labeled CRISPR enzyme is used to identify the location of a target sequence.

[0207] In some aspects, the present invention provides the following methods, which include delivering one or more polynucleotides to a host cell, such as or as one or more vectors described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom. In some aspects, the present invention further provides cells produced by such methods and organisms (e.g., animals, plants, or fungi) including such cells or produced by such cells. In some embodiments, the CRISPR enzyme combined with (and optionally compounded with) a guide sequence is delivered to a 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 give nucleic acids encoding components of the CRISPR system to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids compounded with delivery excipients (e.g., liposomes). Viral vector delivery systems include DNA and RNA viruses, which have free or integrated genomes after being delivered to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, 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. Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., Current Topics in Microbiology and Immunology; Doerfler and Bohm, ed. (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0208] Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agents that enhance the uptake of DNA. Lipofection is described in, for example, U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424; WO 91 / 16024. Delivery can be directed to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration).

[0209] Preparation of lipid:nucleic acid complexes (including targeted liposomes such as immunolipid complexes) is well known to those skilled in the art (see, e.g., 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. 2:414-415 (1996); Res.) 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787).

[0210] The system delivery nucleic acid using RNA or DNA virus base utilizes the specific cells in the virus targeting body and the highly evolved process that the virus payload (payload) is transported to the nucleus. The viral vector can be directly given to the patient (in vivo) or they can be used to treat cells in vitro, and optionally, the modified cells can be given to the patient (ex vivo). Conventional virus-based system can include retroviral vectors, slow virus vectors, adenovirus vectors, adeno-associated virus vectors and herpes simplex virus vectors for gene transfer. It is possible to integrate into the host genome with retrovirus, slow virus and adeno-associated virus gene transfer methods, which usually results in the long-term expression of the insertion transgene. In addition, high transduction efficiency has been observed in different cell types and target tissues.

[0211] The tropism of retrovirus can be changed by incorporating exogenous envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce higher viral titers. Therefore, the selection of retroviral gene transfer systems will depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats, which have the ability to package up to 6-10kb of exogenous sequences. The minimum amount of cis-acting LTR is sufficient for the replication and packaging of the vector, and then these vectors are used to integrate the therapeutic gene into the target cell to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., 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).

[0212] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors can have very high transduction effects in many cell types and do not require cell division. With such vectors, higher titers and expression levels have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, for example, nucleic acids and peptides are produced in vitro, and for in vivo and ex vivo gene therapy procedures (see, for example, West et al., Virology 160: 38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994)). The construction of recombinant AAV vectors is described in multiple publications, including U.S. 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. 63:03822-3828 (1989).

[0213] Typically, packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells that package adenovirus and ψ2 cells or PA317 cells that contain retroviruses. The viral vectors used in gene therapy are usually produced by cell lines that package nucleic acid vectors into viral particles. These vectors typically contain the minimum amount of sequences required for packaging and subsequent integration into the host, and other viral sequences are replaced by expression cassettes for one or more polynucleotides to be expressed. The lost viral function is typically provided in trans by the packaging cell line. For example, the AAV vector used in gene therapy typically has only ITR sequences from the AAV genome, which are required for packaging and integration into the host genome. The viral DNA is packaged into the following cell line, which contains other AAV genes encoding auxiliary plasmids, i.e., rep and cap, but lacks ITR sequences. The cell line can also be infected by adenovirus as an assistant. The helper virus promotes the replication of the AAV vector and expresses the AAV gene from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in a significant amount. The contamination of adenovirus can be reduced by, for example, heat treatment that adenovirus is more sensitive to 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.

[0214] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected when they occur naturally in a subject. In some embodiments, the transfected cells are taken from a subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 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, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, 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, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero (Vero) cells, WM39, WT-49, X63, YAC-1, YAR and transgenic variants thereof. Cell lines can be obtained from a variety of sources known to those skilled in the art (see, e.g., American Type Culture Collection (ATCC) (Manassas, Virginia)). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines, the new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transfected with components of a CRISPR system as described herein (such as by transient transfection with one or more vectors or transfection with RNA) and modified by the activity of a CRISPR complex are used to establish new cell lines, the new cell lines comprising cells that contain the molecule but lack any other exogenous sequences. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein or cell lines derived from such cells are used in evaluating one or more test compounds. .

[0215] In some embodiments, one or more vectors described herein are used to produce non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, such as 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 algae. Methods for producing transgenic plants and animals are known in the art, and usually start with cell transfection methods as described herein. Transgenic animals are also provided, just like transgenic plants, especially crops and algae. Transgenic animals or plants can be used in applications other than providing disease models. By expressing, for example, higher protein, carbohydrate, nutrient or vitamin levels than are usually visible in the wild type, these applications may include food or feed production. In this regard, transgenic plants, especially beans and tubers, and animals, especially mammals, such as livestock (cows, sheep, goats and pigs), and also poultry and edible insects, are preferred.

[0216] Transgenic algae or other plants, such as rapeseed, can be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol). These can be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industry.

[0217] In one aspect, the invention provides methods for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

[0218] In one aspect, the invention provides methods of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.

[0219] With recent advances in crop genomics, the ability to perform efficient and cost-effective gene editing and manipulation using CRISPR-Cas systems will allow rapid selection and comparison of single and multiple genetic manipulations to transform such genomes for improved production and enhanced traits. In this regard, reference is made to U.S. patents and publications: U.S. Patent No. 6,603,061 - Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149 - Plant Genome Sequences and Uses Thereof and US2009 / 0100536 - Transgenic Plants with Enhanced Agronomic Traits, all contents and disclosures of each of which are incorporated herein by reference in their entirety. In the practice of the present invention, the contents and disclosures of Morrell et al. "Cropgenomics: advances and applications" Nat Rev Genet. Dec. 29, 2011; 13(2):85-96 are also incorporated herein by reference in their entirety. In an advantageous embodiment of the present invention, the CRISPR / Cas9 system is used to engineer microalgae (Example 14). Therefore, mutatis mutandis, references to animal cells herein also apply to plant cells unless otherwise apparent.

[0220] In one aspect, the invention provides methods for modifying target polynucleotides in eukaryotic cells, which methods can be in vivo, in vitro or in vitro. In certain embodiments, the method includes sampling cells or cell groups from humans or non-human animals or plants (including microalgae), and modifying the cells or cells. Cultivation can occur at any stage in vitro. The cells or cells can even be reintroduced into the non-human animal or plant (including microalgae).

[0221] In one aspect, the present invention provides the following kits, which include any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit includes a vector system and instructions for using the kit. In some embodiments, the vector system includes: (a) a first regulatory element, the first regulatory element is operably linked to a tracr pairing sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr pairing sequence, wherein when expressed, the guide sequence guides the CRISPR complex to bind to a sequence-specific target sequence in a eukaryotic cell, wherein the CRISPR complex includes a CRISPR enzyme compounded with the following: (1) a guide sequence that hybridizes to the target sequence, and (2) a tracr pairing sequence that hybridizes to the tracr sequence; and / or (b) a second regulatory element, the second regulatory element is operably linked to an enzyme coding sequence encoding the CRISPR enzyme including a nuclear localization sequence. Elements can be provided individually or in combination and can be provided in any suitable container, such as a vial, a bottle or a tube. In some embodiments, the kit includes instructions in one or more languages, such as more than one language.

[0222] In some embodiments, the kit includes one or more reagents for use in the method utilizing one or more of the elements described herein. The reagent may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagent may be provided in a form available in a specific assay or in a form (e.g., in a concentrated or lyophilized form) in which one or more other components need to be added before use. 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 buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably connect the guide sequence and a regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0223] 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 for modifying a target polynucleotide. The CRISPR complex of the present invention has a variety of practicability, including modification (e.g., deletion, insertion, translocation, inactivation, activation) of target polynucleotides in a variety of cell types. Because of this, the CRISPR complex of the present invention has a broad spectrum of applications in, for example, gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex includes a CRISPR enzyme compounded with a guide sequence that hybridizes with a target sequence within a target polynucleotide. The guide sequence is connected to a tracr pairing sequence, which in turn hybridizes with a tracr sequence.

[0224] The target polynucleotide of the CRISPR complex can be any polynucleotide that is endogenous or exogenous to a eukaryotic cell. For example, the target polynucleotide can be a polynucleotide that resides in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a useless DNA). Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, associated with a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for PAM vary depending on the CRISPR enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the protospacer (that is, the target sequence). Examples of PAM sequences are given in the following example section, and a skilled person will be able to identify additional PAM sequences for use with a given CRISPR enzyme.

[0225] The target polynucleotides of the CRISPR complex can include a plurality of disease-associated genes and polynucleotides and signaling biochemical pathway-associated genes and polynucleotides, as listed in U.S. Provisional Patent Applications 61 / 736,527 and 61 / 748,427, filed on December 12, 2012 and January 2, 2013, respectively, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, with Broad Reference Nos. BI-2011 / 008 / WSGR Docket No. 44063-701.101 and BI-2011 / 008 / WSGR Docket No. 44063-701.102, the contents of all of which are incorporated herein by reference in their entirety.

[0226] Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. "Disease-related" genes or polynucleotides refer to any genes or polynucleotides that produce transcription or translation products at abnormal levels or in abnormal forms in cells derived from disease-affected tissues compared to tissues or cells of non-disease controls. In the case where the altered expression is associated with the appearance and / or progression of the disease, it can be a gene expressed at an abnormally high level; it can be a gene expressed at an abnormally low level. Disease-related genes also refer to genes with one or more mutations or genetic variations that are directly responsible for or are disequilibrium with one or more genes responsible for the etiology of the disease. The transcribed or translated products can be known or unknown and can be at normal or abnormal levels.

[0227] Examples of disease-associated genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Maryland) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Maryland).

[0228] Examples of disease-associated genes and polynucleotides are listed in Tables A and B. Disease-specific information available on the World Wide Web can be obtained from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Maryland) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Maryland). Examples of signal transduction biochemical pathway-related genes and polynucleotides are listed in Table C.

[0229] Mutations in these genes and pathways can result in the production of inappropriate proteins or proteins that affect function in inappropriate amounts. Additional examples of genes, diseases, and proteins from U.S. provisional applications 61 / 736,527 and 61 / 748,427 are hereby incorporated by reference. Such genes, proteins, and pathways can be target polynucleotides for CRISPR complexes.

[0230] Table A

[0231]

[0232]

[0233] Table B:

[0234]

[0235]

[0236]

[0237] Table C:

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] Embodiments of the present invention also relate to methods and compositions for 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, 2nd ed., Academic Press, October 13, 2011 - Medical). Specific aspects of tandem repeat sequences have been found to be responsible for more than twenty human diseases (New insights into repeat instability: role of RNA·DNA hybrids. McIvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct; 7(5):551-8). CRISPR-Cas systems can be used to correct genomic instability defects.

[0251] Another aspect of the present invention relates to the use of CRISPR-Cas systems to correct EMP2A and EMP2B gene defects, which have been identified as being associated with Lafora disease. Lafora disease is an autosomal recessive disorder characterized by progressive myoclonic epilepsy that can begin as adolescence. A few cases of the disease can be caused by mutations in genes that have not yet been identified. The disease causes seizures, muscle spasms, difficulty walking, dementia, and ultimately causes death. Currently no therapy has been proven to be effective against disease progression. Other genetic abnormalities associated with epilepsy can also target the CRISPR-Cas system and basic genetics are further described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini and Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology: 20; 2009).

[0252] In yet another aspect of the invention, the CRISPR-Cas system can be used to correct eye defects caused by several genetic mutations, which are further described in Genetic Diseases of the Eye, 2nd ed., edited by Elias I. Traboulsi, Oxford University Press, 2012.

[0253] Several additional aspects of the invention relate to correcting defects associated with a wide range of genetic diseases, which are further described on the National Institutes of Health website under the topical subsection Genetic Disorders. Genetic brain diseases can include, but are not limited to, adrenoleukodystrophy, corpus callosum dysgenesis, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease, Barth Syndrome, Batten Disease, CADASIL, cerebellar degeneration, Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington's disease and other triplet repeat disorders, Leigh's Disease, Lesch-Nyne Syndrome, Menkes Disease, mitochondrial myopathy, and NINDS Colpocephaly. These disorders are further described on the National Institutes of Health website under the section Genetic Brain Disorders.

[0254] In some embodiments, the disease can be a neoplasia. In some embodiments, in the case where the disease is a neoplasia, the gene to be targeted is any one of those genes listed in Table A (PTEN, etc. in this case). In some embodiments, the disease can be age-related macular degeneration. In some embodiments, the disease can be a schizophrenia disorder. In some embodiments, the disease can be a trinucleotide repeat disorder. In some embodiments, the disease can be fragile X syndrome. In some embodiments, the disease can be a secretase-related disorder. In some embodiments, the disease can be a prion-related disorder. In some embodiments, the disease can be ALS. In some embodiments, the disease can be a drug addiction. In some embodiments, the disease can be autism. In some embodiments, the disease can be Alzheimer's disease. In some embodiments, the disease can be inflammation. In some embodiments, the disease can be Parkinson's disease.

[0255] Examples of proteins associated with Parkinson's disease include, but are not limited to, α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1.

[0256] Examples of addiction-related proteins may include, for example, ABAT.

[0257] 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 (FCGR2b, also known as CD32) encoded by the Fcgr2b gene, or FcεR1g (FCER1g) protein encoded by the Fcer1g gene.

[0258] Examples of cardiovascular disease-related proteins may include, for example, IL1B (interleukin 1, β), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, subfamily G (WHITE), member 8) or CTSK (cathepsin K).

[0259] Examples of Alzheimer's disease-associated proteins may include, for example, very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene.

[0260] Examples of proteins associated with autism spectrum disorders may include, for example, benzodiazepine receptor (peripheral)-associated protein 1 (BZRAP1) encoded by the BZRAP1 gene, AF4 / FMR2 family member 2 protein (AFF2) (also known as MFR2) encoded by the AFF2 gene, fragile X mental retardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or fragile X mental retardation autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene.

[0261] Examples of proteins associated with macular degeneration may include, for example, the ATP-binding cassette, subfamily A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded by the APOE gene, or the chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene.

[0262] Examples of proteins associated with schizophrenia may include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

[0263] Examples of proteins involved in tumor suppression can include, for example, ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroleukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroleukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroleukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3 or Notch 4.

[0264] Examples of proteins associated with secretase disorders may include, for example, PSENEN (presenilin enhancer 2 homolog (Caenorhabditis elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B (anterior pharyngeal defect 1 homolog B (Caenorhabditis elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1).

[0265] Examples of proteins associated with amyotrophic lateral sclerosis may include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

[0266] Examples of proteins associated with prion diseases may include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

[0267] Examples of proteins associated with neurodegenerative diseases in prion disorders may include, for example, A2M (alpha-2-macroglobulin), AATF (apoptosis antagonist transcription factor), ACPP (acid phosphatase of the prostate), ACTA2 (actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidase domain), ADORA3 (adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergic receptor or Alpha-1D adrenoreceptor).

[0268] Examples of proteins associated with immunodeficiency may include, for example, A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette, subfamily A (ABC1), member 1]; ABCA2 [ATP-binding cassette, subfamily A (ABC1), member 2]; or ABCA3 [ATP-binding cassette, subfamily A (ABC1), member 3].

[0269] Examples of proteins associated with trinucleotide repeat disorders include, for example, AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin) or DMPK (myotonic dystrophy protein kinase), FXN (frataxin), ATXN2 (ataxin 2).

[0270] Examples of proteins associated with neurotransmission disorders include, for example, SST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A (adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-, receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C).

[0271] Examples of neurodevelopment-associated sequences include, for example, A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrate aminotransferase], ABCA1 [ATP-binding cassette, subfamily A (ABC1), member 1] or ABCA13 [ATP-binding cassette, subfamily A (ABC1), member 13].

[0272] Additional examples of preferred disorders that can be treated with the system of the present invention can be selected from: Aicardi-Goutières Syndrome; Alexander disease; Allan-Herndon-Dudley Syndrome; POLG-related disorders; α-mannosidosis (types II and III); Alström's syndrome ( Syndrome; Angelman; Syndrome; Ataxia-telangiectasia; Neuronal ceroid-lipofuscinosis; Beta-thalassemia; Bilateral optic atrophy and type 1 (infantile) optic atrophy; Retinoblastoma (bilateral); Canavan Disease; Cerebro-oculofacioskeletal Syndrome 1 [COFS1]; Cerebrotendinous xanthomatosis; Cornelia de Lange Syndrome; MAPT-related disorders; Hereditary prion diseases; Dravet Syndrome; Early-onset familial Alzheimer's disease; Friedreich's ataxia [FRDA]; Multiple malformations; Fucosidosis; Fukuyama congenital muscular dystrophy; Galactosialidosis; Gaucher disease Disease; organic acidemias; hemophagocytic lymphohistiocytosis; progeria; mucolipidosis II; infantile free sialic acid storage disease; PLA2G6-related neurodegeneration; Jervell and Lange-Nielsen Syndrome; epidermolysis bullosa, conjunctival; Huntington disease; Krabbe disease (infantile); mitochondrial DNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan syndrome; LIS1-related lissencephaly; Lowe syndrome; maple syrup urine disease; MECP2 duplication syndrome; ATP7A-related copper transport disorder; LAMA2-related muscular dystrophy; arylsulfatase A deficiency; mucopolysaccharidosis type I, II, or III; peroxisome biogenesis disorders, Zellweger spectrum syndrome Spectrum; Neurodegeneration with brain iron accumulation disorder; Acid sphingomyelinase deficiency; Niemann-Pick disease type C; Glycine encephalopathy; ARX-related disorders; Urea cycle disorders; COL1A1 / 2-related osteogenesis imperfecta; Mitochondrial DNA deletion syndrome; PLP1-related disorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen storage disease type II (Pompe Disease) (infantile); MAPT-related disorders; MECP2-related disorders; Rhizomelic Chondrodysplasia Punctata Type 1; Roberts Syndrome;Sandhoff Disease; Schindler Disease, Type 1; Adenosine Deaminase Deficiency; Stevens-Lemme-Ossen Syndrome; Spinal Muscular Atrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase A Deficiency; Thanatosyl Dysplasia Type 1; Collagen Type VI-Related Disorders; Usher Syndrome Type 1; Congenital Muscular Dystrophy; Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; and Xeroderma Pigmentosum.

[0273] Long-term administration of protein therapeutics can elicit unacceptable immune responses against the particular protein. The immunogenicity of protein drugs can be attributed to a few immunodominant helper T lymphocyte (HTL) epitopes. Reducing the MHC binding affinity of these HTL epitopes contained in these proteins can produce drugs with lower immunogenicity (Tangri S et al. "Rationally engineered therapeutic proteins with reduced immunogenicity" Journal of Immunology (J Immunol.) March 15, 2005; 174 (6): 3187-96.) In the present invention, the immunogenicity of the CRISPR enzyme can be reduced by specifically following the method first proposed by Tangri et al. for erythropoietin and subsequently developed. Therefore, directed evolution or rational design can be used to reduce the immunogenicity of CRISPR enzymes (e.g., Cas9) in host species (human or other species).

[0274] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f.sp.lycopersici causes tomato wilt, and only attacks tomatoes, and F.oxysporum f.dianthii Puccinia graminis f.sp.tritici attacks wheat only. Plants have existing and inductive defenses against most pathogens. Mutations and recombination events across plant generations lead to genetic variability that produces sensitivity, particularly when pathogens are bred at a higher frequency than plants. Non-host resistance can exist in plants, for example, the host and pathogen are incompatible. Horizontal resistance can also exist, for example, partial resistance to pathogens of all kinds, typically controlled by many genes, and vertical resistance, for example, complete resistance to some species of pathogens, rather than other species, typically controlled by very few genes. In gene-to-gene level, plants and pathogens evolve together, and the genetic changes in one balance the changes in the other. Thus, using natural variation, breeders combine the most useful genes for yield, quality, uniformity, tolerance, and resistance. Sources of resistance genes include natural or exotic varieties, heirloom varieties, closely related wild plants, and induced mutations, such as treatment of plant materials with mutagens. Using the present invention, a new tool for inducing mutations is provided to plant breeders. Thus, those skilled in the art can analyze the genome of the source of resistance genes and use the present invention to induce the occurrence of resistance genes in varieties with desired characteristics or traits, which has better accuracy than previous mutagens, and thus accelerates and improves plant breeding programs.

[0275] As will be apparent, it is envisioned that any interested polynucleotide sequence can be targeted using the system of the present invention. Some examples of disorders or diseases that may be useful for treatment using the system of the present invention are included in the above table and examples of genes currently associated with those disorders are also provided herein. However, the genes of the examples are not exhaustive.

[0276] This application contains the following items:

[0277] 1. An engineered, non-naturally occurring CRISPR-Cas system comprising:

[0278] (a) a protein comprising a Cas9 nickase or a polynucleotide encoding the protein, wherein the Cas9 nickase comprises at least one mutation in the catalytic domain and lacks the ability to cleave one DNA strand, wherein the Cas9 nickase is linked to at least two nuclear localization signals (NLS);

[0279] (b) A CRISPR-Cas system chimeric guide sequence, comprising a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a nucleus of a eukaryotic cell and directing a CRISPR-Cas complex to sequence-specifically bind to the target sequence, a tracr mate sequence capable of hybridizing to a tracr sequence, and a tracr sequence of at least 30 nucleotides in length.

[0280] 2. An engineered, non-naturally occurring CRISPR-Cas vector system comprising one or more vectors comprising:

[0281] (a) a first regulatory element operably linked to a nucleotide sequence encoding a protein comprising a Cas9 nickase, the Cas9 nickase comprising at least one mutation in the catalytic domain and lacking the ability to cleave one DNA strand, wherein the Cas9 nickase is linked to at least two nuclear localization signals (NLS);

[0282] (b) a second regulatory element operably linked to a nucleotide sequence encoding a CRISPR-Cas system chimeric guide sequence, the CRISPR-Cas system chimeric guide sequence comprising a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a nucleus of a eukaryotic cell and directing sequence-specific binding of a CRISPR-Cas complex to the target sequence, a tracr mate sequence capable of hybridizing to a tracr sequence, and a tracr sequence of at least 30 nucleotides in length;

[0283] Wherein components (a) and (b) are located on the same or different carriers of the system.

[0284] 3. The system according to item 2, wherein the vector is a viral vector.

[0285] 4. A system according to item 3, wherein the viral vector is an adeno-associated vector.

[0286] 5. The system of item 1 or 2, wherein the tracr sequence is at least 40 nucleotides in length.

[0287] 6. The system of item 1 or 2, wherein the tracr sequence is at least 50 nucleotides in length.

[0288] 7. The system according to item 1 or 2, wherein the guide sequence has a length of 15-30 nucleotides.

[0289] 8. A system according to item 1 or 2, wherein the guide sequence is at least 20 nucleotides in length.

[0290] 9. A system according to item 1 or 2, wherein the Cas9 comprises at least one mutation in the RuvC domain.

[0291] 10. A system according to item 1 or 2, wherein the Cas9 comprises a mutation corresponding to D10A, H840A, N854A or N863A of Streptococcus pyogenes Cas9.

[0292] 11. A system according to item 1 or 2, wherein the Cas9 is Streptococcus pyogenes Cas9 and the PAM is NGG; or wherein the Cas9 is Streptococcus thermophilus Cas9 and the PAM is NAGAAW.

[0293] 12. A system according to item 1 or 2, wherein the protein comprises at least one heterologous protein domain linked to the Cas9.

[0294] 13. A system according to item 12, wherein the heterologous protein domain has one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.

[0295] 14. The system according to item 12, wherein the heterologous protein domain is GFP.

[0296] 15. A system according to item 1 or 2, wherein the protein comprises at least one SV40 virus large T antigen NLS (PKKKRKV) linked to the Cas9.

[0297] 16. The system according to item 1 or 2, wherein the protein comprises at least one N-terminal NLS and at least one C-terminal NLS.

[0298] 17. The system according to item 1, wherein the polynucleotide encoding the protein is codon-optimized for expression in mammalian cells or human cells.

[0299] 18. The system according to item 1, wherein the polynucleotide encoding the protein is mRNA.

[0300] 19. The system according to item 1, wherein the system further comprises a template polynucleotide, wherein the template polynucleotide overlaps with at least 5 nucleotides of the target sequence.

[0301] 20. Use of the system according to item 1 or 2 in the manufacture of a drug for gene therapy.

[0302] 21. Use of the system according to item 1 or 2 for modifying eukaryotic cells in vitro or in vitro.

[0303] 22. A eukaryotic cell comprising the system according to item 1 or 2.

[0304] 23. A non-human organism comprising the eukaryotic cell of item 22, wherein the non-human organism is not an animal or plant species.

[0305] 24. A Cas9 enzyme derived from Streptococcus pyogenes.

[0306] 25. A Cas9 enzyme derived from Streptococcus thermophilus.

[0307] 26. A kit comprising a Cas9 enzyme.

[0308] 27. A fusion protein comprising RNAse III, mCherry and a nuclear localization signal (NLS).

[0309] 28. A composition comprising one or more PCR primers selected from the group consisting of AAAACCACCCTTCTCTCTGGC, GGAGATTGGAGACACGGAGAG, CTGGAAAGCCAATGCCTGAC, and GGCAGCAAACTCCTTGTCCT.

[0310] Examples

[0311] The following examples are given for the purpose of illustrating different embodiments of the present invention and are not intended to limit the present invention in any way. The present examples, together with the methods described herein, currently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Variations herein and other uses encompassed within the spirit of the present invention as defined by the scope of the claims are within the purview of those of ordinary skill in the art.

[0312] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[0313] An exemplary type II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA, and a characteristic array of repetitive sequences (direct repeats) separated by short stretches of non-repetitive sequences (spacers, approximately 30 bp each). In this system, targeted DNA double-strand breaks (DSBs) are generated in four sequential steps ( Figure 2A). In the first step, two non-coding RNAs, the pre-crRNA array and the tracrRNA, are transcribed from the CRISPR locus. In the second step, the tracrRNA is hybridized to the direct repeats of the pre-crRNA, which is then processed into mature crRNA containing a separate spacer sequence. In the third step, the mature crRNA:tracrRNA complex directs Cas9 to a DNA target consisting of a protospacer and a corresponding PAM via the formation of a heteroduplex between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates the cleavage of the target DNA upstream of the PAM to generate a DSB within the protospacer ( Figure 2A ). This example describes an exemplary process for adapting this RNA-programmable nuclease system to direct CRISPR complex activity in the nucleus of a eukaryotic cell.

[0314] To improve the expression of CRISPR components in mammalian cells, two genes from the SF370 locus of Streptococcus pyogenes (S. pyogenes) can be codon optimized, namely Cas9 (SpCas9) and RNase III (SpRNase III). To promote nuclear localization, a nuclear localization signal (NLS) is included at the amino (N)- or carboxyl (C)-terminus of both SpCas9 and SpRNase III ( Figure 2B To facilitate visualization of protein expression, a fluorescent protein marker was also included at the N- or C-terminus of both proteins ( Figure 2B ). A version of SpCas9 with an NLS attached to both the N- and C-termini (2xNLS-SpCas9) was also produced. Constructs containing NLS-fused SpCas9 and SpRNase III were transfected into 293FT human embryonic kidney (HEK) cells, and it was found that the relative positioning of the NLS to SpCas9 and SpRNase III affected its nuclear localization efficiency. While the C-terminal NLS is sufficient to target SpRNase III to the nucleus, in this system, attachment of a single copy of these specific NLSs to the N- or C-termini of SpCas9 is unable to achieve adequate nuclear localization. In this example, the C-terminal NLS is the C-terminal NLS of nucleoplasmin (KRPAATKKAGQAKKKK), and the C-terminal NLS is the C-terminal NLS of SV40 large T-antigen (PKKKRKV). Of the versions of SpCas9 tested, only 2xNLS-SpCas9 exhibited nuclear localization ( Figure 2B ).

[0315] The tracrRNA from the CRISPR seat of Streptococcus pyogenes SF370 has two transcription start sites, resulting in two transcripts, 89-nucleotides (nt) and 171nt, which are then processed into the same 75nt mature tracrRNA. Select shorter 89nt tracrRNA for expression in mammalian cells (the expression construct shown in Figure 6, wherein functionality is determined by the results of the Surveyor assay as shown in Figure 6B). The transcription start site is marked as +1, and the transcription terminator and the sequence detected by Northern blot are also indicated. The expression of the processed tracrRNA is also confirmed by Northern blot. Figure 7 C shows the results of Northern blot analysis of the total RNA extracted from 293FT cells, and these cells are transfected with the U6 expression construct carrying long or short tracrRNA and SpCas9 and DR-EMX1 (1)-DR. The left and right figures are respectively from 293FT cells transfected with or without SpRNA enzyme III. U6 indicates a loading control blotted with a probe targeting human U6 snRNA. Transfection of the short tracrRNA expression construct produces abundant levels of the processed form of tracrRNA (approximately 75 bp). Very low amounts of long tracrRNA were detected on Northern blots.

[0316] To facilitate accurate transcription initiation, the RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA ( Figure 2C ). Similarly, a U6 promoter-based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DR, also encompassed by the term “tracr pairing sequence”; Figure 2C The initiator spacer was designed to target the human EMX1 locus ( Figure 2C ) in the 33-base pair (bp) target site (a 30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence that satisfies the NGG recognition motif of Cas9), a locus for a key gene in the development of the cerebral cortex.

[0317] In order to test whether heterologous expression of CRISPR system (SpCas9, SpRNase III, tracrRNA and pre-crRNA) in mammalian cells can achieve targeted cleavage of mammalian chromosomes, HEK 293FT cells were transfected with a combination of CRISPR components. Since DSBs in mammalian nuclei are partially paired by the non-homologous end joining (NHEJ) pathway that leads to the formation of indels, the potential cleavage activity at the target EMX1 seat was detected using Surveyor assay (see, e.g., Guschin et al., 2010, Methods Mol Biol 649: 247). Co-transfection of all four CRISPR components can induce up to 5.0% cleavage in the protospacer (see Figure 2D ). Co-transfection of all CRISPR components (minus SpRNase III) also induced up to 4.7% indels in the protospacer, suggesting the possible presence of endogenous mammalian RNases that can assist in crRNA maturation, such as the related Dicer and Drosha enzymes. Removal of any of the remaining three components abolished the genomic cleavage activity of the CRISPR system ( Figure 2D ). Sanger sequencing of amplicons containing target loci with demonstrated cleavage activity: 5 mutant alleles were found in 43 sequenced clones (11.6%). Similar experiments using a variety of guide sequences produced indel percentages up to 29% (see Figures 4-8, 10, and 11). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells.

[0318] In order to optimize the cutting efficiency, the applicant also tested whether the different subtypes of tracrRNA affect the cutting efficiency and found that in the example system, only the short (89-bp) transcript form can mediate the cutting of the human EMX1 genomic seat. Figure 9 provides another Northern blot analysis of crRNA processing in mammalian cells. Figure 9A shows a schematic diagram showing an expression vector of a single spacer flanked by two directional repeats (DR-EMX1 (1)-DR). The 30bp spacer and directional repeat sequence of the prototype spacer 1 of the human EMX1 seat are shown in the sequence below Figure 9A. The straight line indicates that its reverse complementary series is used to generate the region of the Northern blot probe for EMX1 (1) crRNA detection. Figure 9B shows the Northern blot analysis of the total RNA extracted from 293FT cells, which are transfected with the U6 expression construct carrying DR-EMX1 (1)-DR. The left and right figures are respectively from 293FT cells transfected with or without SpRNA enzyme III. DR-EMX1 (1) -DR is processed into mature crRNA only in the presence of SpCas9 and short tracrRNA and is independent of the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA is about 33bp and shorter than the 39-42bp mature crRNA from Streptococcus pyogenes. These results demonstrate that the CRISPR system can be transplanted into eukaryotic cells and reprogrammed to promote the cutting of endogenous mammalian target polynucleotides.

[0319] FIG2 shows the bacterial CRISPR system described in this Example. Figure 2A A schematic diagram showing the CRISPR locus 1 from Streptococcus pyogenes SF370 and the proposed mechanism of CRISPR-mediated DNA cleavage by this system is shown. Mature crRNA processed from the direct repeat-spacer array guides Cas9 to the following genomic targets, which consist of complementary protospacers and protospacer adjacent motifs (PAMs). After target-spacer base pairing, Cas9 mediates double-strand breaks in the target DNA. Figure 2B The engineering of Streptococcus pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) with a nuclear localization signal (NLS) that enables import into mammalian cell nuclei is shown. Figure 2CShown is the mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter and tracrRNA and pre-crRNA arrays (DR-spacer-DR) driven by the RNA Pol3 promoter U6, which promotes precise transcription initiation and termination. The prototype spacer from the human EMX1 locus with a satisfactory PAM sequence is used as a spacer in the pre-crRNA array. Figure 2D Surveyor nuclease assays for minor insertions and deletions mediated by SpCas9 are shown. SpCas9 is expressed with and without SpRNA enzyme III, tracrRNA, and a pre-crRNA array carrying an EMX1-target spacer. Figure 2E shows a schematic representation of base pairing between the target seat and the EMX1-targeting crRNA and an exemplary chromatogram showing a small deletion adjacent to the SpCas9 cleavage site. Figure 2F shows the alleles of mutations identified from sequencing analysis of 43 clonal amplicons, which show a variety of small insertions and deletions. Dashes indicate missing bases, and non-aligned or mismatched bases indicate insertions or mutations. Scale bar = 10 μm.

[0320] To further simplify the three-component system, a chimeric crRNA-tracrRNA hybrid design was adapted in which the mature crRNA (including a guide sequence) was fused to a partial tracrRNA via a stem-loop to mimic the natural crRNA:tracrRNA duplex ( FIG. 3A ).

[0321] The guide sequence can be inserted between the BbsI sites using annealed oligonucleotides. The prototype spacers on the sense strand and antisense strand are indicated above and below the DNA sequence, respectively. The human PVALB and mouse Th seats reached a modification rate of 6.3% and 0.75%, respectively, proving the broad applicability of the CRISPR system in modifying different seats across a variety of organisms. Although for each seat, only one-third of the spacers were detected for cutting using a chimeric construct, when the co-expressed pre-crRNA arrangement was used, all target sequences were cut, with an effective indel generation of 27% (Figures 4 and 5).

[0322] Fig. 5 provides the other diagram of SpCas9 that can be reprogrammed to target multiple genomic loci in mammalian cells.Fig. 5A provides the schematic diagram of people EMX1 seat, shows the position of five prototype spacers, indicated by underlined sequence.Fig. 5B provides the schematic diagram of pre-crRNA / trcrRNA complex, shows the hybridization (top) between the same repeat region of pre-crRNA and tracrRNA, and the schematic diagram of chimeric RNA design, including a 20bp guide sequence and the tracr pairing and tracr sequence composed of part same repeat and the tracrRNA sequence (bottom) hybridized with hairpin structure.The result of the Surveyor determination of the efficiency of the cutting mediated by Cas9 at five prototype spacers in people EMX1 seat is shown in Fig. 5C.Use pre-crRNA / tracrRNA complex (crRNA) or chimeric RNA (chiRNA) of processing to target each prototype spacer.

[0323] Since the secondary structure of RNA can be decisive for intermolecular interactions, the assumed secondary structures of all guide sequences used in the genome targeting experiment (Fig. 3B) were compared using a structure prediction algorithm based on minimum free energy and Boltzmann weighted structure ensembles (see, e.g., Gruber et al., 2008, Nucleic Acids Research, 36: W70). The analysis revealed that in most cases, effective guide sequences in the context of chimeric crRNAs are substantially free of secondary structural motifs, while invalid guide sequences are more likely to form internal secondary structures that can prevent base pairing with target prototype spacer DNA. Therefore, it is possible that when using chimeric crRNAs, the variability of the spacer secondary structure may affect the efficiency of CRISPR-mediated interference.

[0324] Fig. 3 illustrates an example expression vector. Fig. 3A provides a schematic diagram of a bicistronic vector for driving the expression of a synthetic crRNA-tracrRNA chimera (chimeric RNA) together with SpCas9. The chimeric guide RNA includes a 20-bp guide sequence corresponding to the prototype spacer in the target site of the genome. Fig. 3B provides a guide sequence showing the targeting of human EMX1, PVALB and mouse Th loci, together with a schematic diagram of its predicted secondary structure. It is shown that the modification efficiency at each target site is lower than the RNA secondary structure diagram (EMX1, n = 216 amplicon sequencing reads; PVALB, n = 224 reads; Th, n = 265 reads). The folding algorithm produces an output in which each base is colored according to its assumed possibility of the predicted secondary structure, as indicated by the rainbow scale (rainbow scale) reproduced in gray scale in Fig. 3B. An additional vector design for SpCas9 is shown in FIG3A , including a single expression vector incorporating a U6 promoter linked to an insertion site for a guide oligonucleotide and a Cbh promoter linked to the SpCas9 coding sequence.

[0325] In order to test whether the spacer comprising a secondary structure can work in the prokaryotic cell of CRISPR natural operation, the transformation interference (Fig. 3C) of the plasmid with the prototype spacer was tested in the E. coli strain of heterologous expression of Streptococcus pyogenes SF370 CRISPR locus 1. The CRISPR seat was cloned into a low-copy E. coli expression vector, and the crRNA array was replaced by a single spacer flanked by a pair of DR (pCRISPR). The E. coli strains comprising different pCRISPR plasmids were transformed with an excitation plasmid comprising the corresponding prototype spacer and PAM sequence (Fig. 3C). In bacterial assays, all spacers promoted effective CRISPR interference (Fig. 3C). These results show that there may be other factors affecting the CRISPR activity efficiency in mammalian cells.

[0326] In order to study the specificity of CRISPR-mediated cutting, a series of EMX1-targeted chimeric crRNAs with single-point mutations were used to analyze the effects of single nucleotide mutations in the guide sequence on protospacer cutting in mammalian genomes (Fig. 4A). Fig. 4B shows the results of the Surveyor nuclease assay comparing the cutting efficiency of Cas9 when paired with chimeric RNAs of different mutations. Single base mismatches up to 12-bp of the 5' of PAM substantially eliminate the genome cutting performed by SpCas9, while the spacer with mutations further away from the upstream position retains activity against the original protospacer target (Fig. 4B). In addition to PAM, SpCas9 has single base specificity within the last 12-bp of the spacer. In addition, CRISPR can mediate genome cutting as effectively as a pair of TALE nucleases (TALENs) targeting the same EMX1 protospacer. Fig. 4C provides a schematic diagram showing the design of TALEN targeting EMX1 and Figure 4D Surveyor gel comparing the efficiency of TALEN and Cas9 is shown (n=3).

[0327] A set of components for achieving CRISPR-mediated gene editing in mammalian cells has been established through the error-prone NHEJ mechanism, testing the ability of CRISPR to stimulate homologous recombination (HR), a high-fidelity gene repair pathway used to make precise edits in the genome. Wild-type SpCas9 is able to mediate site-specific DSBs that can be repaired by both NHEJ and HR. In addition, an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase (SpCas9n; shown in FIG5A ) (see, e.g., Sapranausaks et al., 2011, Nucleic Acids Research, 39:9275; Gasunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579), such that the nicked genomic DNA undergoes high-fidelity homology-directed repair (HDR). The Surveyor assay confirmed that SpCas9n does not produce indels at the EMX1 protospacer target. As shown in FIG5B , co-expression of EMX1-targeted chimeric crRNA with SpCas9 produces indels in the target site, while co-expression with SpCas9n does not produce indels (n=3). In addition, the sequencing of 327 amplicons did not detect any indel induced by SpCas9n. The same seat was selected to test the HR mediated by CRISPR by co-transfection of HEK 293FT cells with chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n and for introducing a pair of restriction sites (HindIII and NheI) near the prototype spacer. Fig. 5C provides a schematic diagram of the HR strategy, including the relative position and primer annealing sequence (arrow) of the recombination point. SpCas9 and SpCas9n indeed catalyze the integration of the HR template into the EMX1 seat. PCR amplification target region, followed by restriction digestion with HindIII reveals the cleavage product corresponding to the expected fragment size (arrows in the restriction fragment length polymorphism analysis shown in Fig. 5D), wherein SpCas9 and SpCas9n mediate similar levels of HR efficiency. The applicant further confirmed HR (Fig. 5E) using the Sanger sequencing of genomic amplicons. These results demonstrate the practicality of CRISPR in promoting the insertion of targeted genes in mammalian genomes. Given that 14-bp (12-bp from the spacer and 2-bp from the PAM) are specifically targeted to wild-type SpCas9, the accessibility of the nickase can significantly reduce the possibility of off-target modifications because single-strand breaks are not substrates for the error-prone NHEJ pathway.

[0328] Construction of expression constructs that mimic the natural architecture of the CRISPR locus with arrayed spacers ( Figure 2A ) to test the possibility of multiplex sequence targeting. Using a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers, efficient cleavage was detected at both loci ( Figure 5F , showing both the schematic design of the crRNA array and a Surveyor blot showing efficient modification of cleavage). Targeted deletion of larger genomic regions was also detected by coexisting DSBs using spacers for two targets within EMX1 separated by 119 bp, and 1.6% deletion efficiency was detected (3 out of 182 amplicons; Figure 5G ). This demonstrates that the CRISPR system can mediate multiplex editing in a single genome.

[0329] Example 2: CRISPR system modifications and alternatives

[0330] The ability to program sequence-specific DNA cleavage using RNA defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system could be further improved to increase the efficiency and versatility of CRISPR targeting. Optimized Cas9 activity may rely on free Mg in a concentration lower than that present in the mammalian cell nucleus. 2+ High levels of free Mg 2+ The availability of (see, e.g., Jinek et al., 2012, Science, 337:816), and the preference for the NGG motif just downstream of the protospacer limits the ability to target an average of every 12-bp in the human genome. Some of these constraints can be overcome by exploring the diversity of CRISPR loci across microbial metagenomes (see, e.g., Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci can be transplanted into a mammalian cell environment by methods similar to those described in Example 1. It was shown that the modification efficiency at each target site was lower than these RNA secondary structures. The algorithm that generated the structure colors each base according to the probability of the secondary structure predicted by its hypothesis. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively. Statistical analysis of the cleavage activity across biological replicates at these two protospacer sites is also provided in Figure 7.

[0331] Example 3: Sample target sequence selection algorithm

[0332] A software program is designed to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on the desired guide sequence length and CRISPR motif sequence (PAM) for a given CRISPR enzyme. For example, one can identify candidate CRISPR target sequences on both strands of an input DNA sequence by searching for 5'-N on both the input sequence and the reverse complement of the input sequence. x -NGG-3' to identify the target site of Cas9 from S. pyogenes with the PAM sequence NGG. Similarly, the target site of Cas9 of S. pyogenes CRISPR1 with the PAM sequence NNAGAAW can be identified by searching for 5'-Nx-NNAGAAW-3' on both the input sequence and the reverse complement of the input sequence. Similarly, the target site of Cas9 with the PAM sequence NNAGAAW can be identified by searching for 5'-Nx-NNAGAAW-3' on both the input sequence and the reverse complement of the input sequence. x -NGGNG-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR3 with the PAM sequence NGGNG. N can be fixed by the program or specified by the user. x The value "x" in , such as 20.

[0333] Since multiple occurrences in the genome of the DNA target site can lead to non-specific genome editing, after identifying all potential sites, the program filters out the sequence based on the number of times it appears in the relevant reference genome. For those CRISPR enzymes whose sequence specificity is determined by the 'seed' sequence (such as 11-12bp 5' from the PAM sequence, including the PAM sequence itself), the filtering step can be based on the seed sequence. Therefore, in order to avoid editing at other genomic loci, the results are filtered based on the number of times the seed: PAM sequence appears in the relevant genome. The user can be allowed to select the length of the seed sequence. For the purpose of passing the filter, the user can also be allowed to specify the number of times the seed: PAM sequence appears in the genome. Unique sequences are screened by default. The filtering level is changed by changing both the length of the seed sequence and the number of times the sequence appears in the genome. The program can additionally or alternatively provide the sequence of the guide sequence complementary to the one or more target sequences reported by providing the reverse complement of the one or more target sequences identified.

[0334] Further details of methods and algorithms for optimizing sequence screening can be found in US Application Serial No. TBA (Broad Reference No. BI-2012 / 084 44790.11.2022), incorporated herein by reference.

[0335] Example 4: Evaluation of various chimeric crRNA-tracrRNA hybrids

[0336] This example describes the chimeric RNA (chiRNAs) of the tracr sequence with wild-type tracrRNA sequences of different lengths incorporated; Including guide sequence, tracr pairing sequence and tracr sequence in a single transcript) and the results obtained. Figure 18a shows a schematic diagram of a bicistronic expression vector of chimeric RNA and Cas9. Cas9 is driven by the CBh promoter and the chimeric RNA is driven by the U6 promoter. The chimeric guide RNA is composed of a 20bp guide sequence (N) joined to the tracr sequence (from the first "U" of the lower chain to the end of the transcript), which is truncated at each point as indicated. The guide and tracr sequence are separated by the tracr-pairing sequence GUUUUAGAGCUA, followed by the loop sequence GAAA. The results of the SURVEYOR assay of the indel mediated by Cas9 at the human EMX1 and PVALB seats are shown in Figures 18b and 10c, respectively. The arrow indicates the expected SURVEYOR fragment. ChiRNA is indicated by its "+n" mark, and crRNA refers to the hybrid RNA of guiding and tracr sequence being expressed as separated transcripts. The quantitative results of these results carried out in triplicate are shown in the histogram of Figures 11a and 11b, corresponding to Figures 10b and 10c ("ND" indicates that indel is not detected). Prototype spacer ID and its corresponding genomic target, prototype spacer sequence, PAM sequence and chain position are provided in Table D. The guide sequence is designed to be complementary to the entire prototype spacer sequence (in the case of the separated transcript in the hybrid system), or only partially underlined (in the case of chimeric RNA).

[0337] Table D:

[0338]

[0339]

[0340] Cell culture and transfection

[0341] Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2mM GlutaMAX (Life Technologies), 100U / mL penicillin and 100μg / mL streptomycin at 37°C with 5% CO2 incubation. 293FT cells were seeded at a density of 150,000 cells / well into 24-well plates (Corning) 24 hours before transfection. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer's recommended protocol. A total of 500ng of plasmid was used for each well of the 24-well plate.

[0342] SURVEYOR assay for genome modification

[0343] As described above, 293FT cells were transfected with plasmid DNA. After transfection, cells were incubated at 37 ° C for 72 hours before genomic DNA extraction. Following the manufacturer's protocol, genomic DNA was extracted using QuickExtract DNA extraction solution (Epicentre). In brief, pelleted cells were resuspended in QuickExtract solution and incubated at 65 ° C for 15 minutes and at 98 ° C for 10 minutes. For each gene, PCR amplification (primers are listed in Table E) was performed in the genomic region flanking the CRISPR target site, and the manufacturer's protocol was followed to purify the product using QiaQuick Spin columns (Qiagen). A total of 400 ng of purified PCR products were mixed with 2 μl 10X Taq DNA polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 μl and subjected to a reannealing process to allow heteroduplex formation: 95°C for 10 min, decreasing from 95°C to 85°C at –2°C / s, decreasing from 85°C to 25°C at –0.25°C / s, and holding at 25°C for 1 minute. After reannealing, the products were treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) following the manufacturer's recommended protocol and analyzed on 4-20% Novex TBE polyacrylamide gels (Life Technologies). The gel was stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged with the GelDoc gel imaging system (Bio-rad). Quantification was performed based on relative band intensity.

[0344] Table E:

[0345]

[0346] In silico identification of unique CRISPR target sites

[0347] To identify unique target sites for the Streptococcus pyogenes SF370 Cas9 (SpCas9) enzyme in the human, mouse, rat, zebrafish, fruit fly, and Caenorhabditis elegans genomes, we developed a software package to scan both strands of a DNA sequence and identify all possible SpCas9 target sites. For this example, each SpCas9 target site was operationally defined as a 20 bp sequence followed by an NGG protospacer adjacent motif (PAM) sequence, and we identified all chromosomes that satisfied this 5'-N 20 -NGG-3'. To prevent non-specific genome editing, after identifying all potential sites, all target sites are filtered based on the number of times the sequence appears in the relevant reference genome. In order to exploit the sequence specificity of Cas9 activity conferred by a 'seed' sequence (which can be, for example, a sequence of approximately 11-12bp starting from 5' of the PAM sequence), the 5'-NNNNNNNNNN-NGG-3' sequence is selected to be unique in the relevant genome. All genome sequences were downloaded from the UCSC genome browser (human genome hg19, mouse genome mm9, rat genome rn5, zebrafish genome danRer7, fruit fly genome dm4, and Caenorhabditis elegans genome ce10). Comprehensive search results can be obtained using the UCSC genome browser information. Exemplary visualization of some target sites in the human genome is provided at Figure 22 middle.

[0348] Initially, three sites in the EMX1 seat in the targeting human HEK 293FT cells. The genome modification efficiency of each chiRNA is assessed using SURVEYOR nuclease assay, which detects the mutation caused by DNA double-strand break (DSB) and the subsequent repair thereof by non-homologous end joining (NHEJ) DNA damage repair pathway. The construct designated as chiRNA (+n) indicates that the straight + nth nucleotide of wild-type tracrRNA is included in the chimeric RNA construct, wherein 48,54,67 and 85 values ​​are used for n. Chimeric RNA all includes the DNA cutting of the wild-type tracrRNA (chiRNA (+67) and chiRNA (+85)) mediation of long fragments at all three EMX1 target sites, wherein chiRNA (+85) especially shows the DNA cutting (Figure 10 b and 10 a) of the crRNA / tracrRNA hybrid significantly higher level than the corresponding crRNA expressing guide sequence and tracr sequence in the form of separated transcripts. ChiRNAs were also used to target two sites in the PVALB locus that did not produce detectable cleavage using a hybrid system (guide and tracr sequences expressed as separate transcripts). chiRNA (+67) and chiRNA (+85) were able to mediate significant cleavage at both PVALB protospacers (Figures 10c and 10b).

[0349] For all five targets in EMX1 and PVALB seats, the genome modification efficiency that increases consistently with the increased tracr sequence length is observed. Without wishing to be bound by any theory, the secondary structure formed by the 3' end of tracrRNA can play a role in enhancing the CRISPR complex formation rate. Figure 21 provides a diagram of the predicted secondary structure of each of the chimeric RNAs used in this example. Using the minimum free energy and partition function algorithm, RNA folding (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAfold.cgi) is used to predict the secondary structure. The false color of each base (reproduced in grayscale) indicates the possibility of pairing. Because chiRNA with a longer tracr sequence can cut targets that are not cut by natural CRISPR crRNA / tracrRNA hybrids, it is possible that chimeric RNA can be more effectively loaded onto Cas9 than its natural hybrid counterpart. To facilitate the application of Cas9 in site-specific genome editing in eukaryotic cells and organisms, all predicted unique target sites of the Streptococcus pyogenes Cas9 were identified in silico in the human, mouse, rat, zebrafish, Caenorhabditis elegans, and Drosophila genomes. Chimeric RNAs can be designed against Cas9 enzymes from other microorganisms to expand the target space of CRISPR RNA-programmable nucleases.

[0350] Figures 11 and 21 show schematic diagrams of exemplary bicistronic expression vectors for expressing chimeric RNAs including up to +85 nucleotides of a wild-type tracr RNA sequence and SpCas9 with a nuclear localization sequence. SpCas9 is expressed from a CBh promoter and terminated by a bGH polyA signal (bGH pA). The extended sequence immediately following the schematic diagram corresponds to the region surrounding the guide sequence insertion site and includes the 3'-portion of the U6 promoter (first shielded region), the BbsI cleavage site (arrow), partial direct repeats (tracr pairing sequence GTTTTAGAGCTA, underlined), the loop sequence GAAA, and the +85 tracr sequence (underlined sequence after the loop sequence) from 5' to 3'. An exemplary guide sequence insert is shown after the guide sequence insertion site, with the nucleotides of the guide sequence represented by "N" for the selected target.

[0351] The sequences described in the above examples are as follows (polynucleotide sequences are 5' to 3'):

[0352] U6-short tracrRNA (Streptococcus pyogenes SF370):

[0353] (Bold = tracrRNA sequence; Underline = Termination sequence) U6-long tracrRNA (Streptococcus pyogenes SF370):

[0354] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTGATTTCTCCTTGATTATTTGTTATAAAAGTTATAAAAATAATCTTGTTGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0355] U6-DR-BbsI backbone - DR (Streptococcus pyogenes SF370):

[0356] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTTCGAGAAGACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC

[0357] U6 - chimeric RNA - BbsI backbone (Streptococcus pyogenes SF370)

[0358] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

[0359] NLS - SpCas9 - EGFP:

[0360]

[0361] SpCas9-EGFP-NLS:

[0362]

[0363] NLS-SpCas9-EGFP-NLS:

[0364]

[0365] NLS-SpCas9-NLS:

[0366]

[0367] NLS-mCherry-SpRNA enzyme 3:

[0368] MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV

[0369] SpRNA enzyme 3-mCherry-NLS:

[0370] MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK

[0371] NLS-SpCas9n-NLS (The D10A nickase mutation is in lowercase letters):

[0372]

[0373] hEMX1-HR Template-HindII-NheI:

[0374]

[0375] NLS-StCsn1-NLS:

[0376]

[0377] U6-St_tracrRNA(7-97):

[0378] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAAT GGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA

[0379] U6-DR-Spacer-DR (Streptococcus pyogenes SF370)

[0380] (lowercase underlined = direct repeat; N = guide sequence; bold = terminator)

[0381] Chimeric RNA, containing +48 tracr RNA (Streptococcus pyogenes SF370)

[0382] (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0383] Chimeric RNA, containing +54 tracr RNA (Streptococcus pyogenes SF370)

[0384] (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0385] Chimeric RNA, containing +67 tracr RNA (Streptococcus pyogenes SF370)

[0386] (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0387] Chimeric RNA, containing +85 tracr RNA (Streptococcus pyogenes SF370)

[0388] (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0389] CBh-NLS-SpCas9-NLS

[0390] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccacc ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCC CCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCG GCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAA CACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACC CGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCA ACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCA CGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTG AGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGT TCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCA ACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCA GCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCC CCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGA GCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGA AGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGG AGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAG ATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGA GCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC ACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGC AGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTT CAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATAC CACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCG TGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGA CAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCA CGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAG CTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGA AGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAA AGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATG TACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGA AGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGA AGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGAC AATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAA CCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCT GATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAA GTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAA AGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAG CGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATT ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATA AGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCA GACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGG GACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGG GCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCC CTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCC TGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAG AGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAG AGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACAC CACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGC CTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTA GCAGCAGGACGCTTTAA (Underline = NLS-hSpCas9-NLS)

[0391] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0392] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0393] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0394] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0395] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0396] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0397] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0398] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0399] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR1 Cas9 (with a PAM of NNAGAAW) (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)

[0400] Example chimeric RNA targeting S. thermophilus LMD-9 CRISPR3 Cas9 (with a PAM of NGGNG) (N = guide sequence; first underlined = tracr mate sequence; second underlined = tracr sequence; bold = terminator)

[0401] A codon-optimized version of Cas9 from the S. thermophilus LMD-9 CRISPR3 locus (with an NLS at both the 5' and 3' ends)

[0402]

[0403] Example 5: Optimization of guide RNA for Streptococcus pyogenes Cas9 (referred to as SpCas9).

[0404] Applicants mutate the tracrRNA and direct repeat sequences, or mutate the chimeric guide RNA to enhance the RNA in the cell.

[0405] The optimization is based on the observation that there is an extension of thymine (T) in the tracrRNA and guide RNA, which can lead to early transcription termination by the pol 3 promoter. Therefore, the applicant generated the following optimized sequence. The optimized tracrRNA and the corresponding optimized direct repeat are shown in pairs. Optimized tracrRNA 1 (mutation underlined):

[0406] GGAACCATTCA t AACAGCATAGCAAGTTA t AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT

[0407] Optimized direct repeat 1 (mutations are underlined):

[0408] GTT a TAGAGCTATGCTGTT a TGAATGGTCCCAAAAC

[0409] Optimized tracrRNA 2 (mutations are underlined):

[0410] GGAACCATTCAA t ACAGCATAGCAAGTTAA t ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT

[0411] Optimized direct repeat 2 (mutations are underlined):

[0412] GT a TTAGAGCTATGCTGT a TTGAATGGTCCCAAAAC

[0413] Applicants also optimized the chimeric guide RNA for optimal activity in eukaryotic cells.

[0414] Original guide RNA:

[0415] NNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0416] Optimized chimeric guide RNA sequence 1:

[0417] NNNNNNNNNNNNNNNNNNNNGT A TTAGAGCTAGAAATAGCAAGTTAA T ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0418] Optimized chimeric guide RNA sequence 2:

[0419] NNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGGAAACAAAACAGCATGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0420] Optimized chimeric guide RNA sequence 3:

[0421] NNNNNNNNNNNNNNNNNNNNGT A TTAGAGCTATGCTGT A TTGGAAACAA T ACAGCATAGCAAGTTAA T ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0422] Applicants revealed that the optimized chimeric guide RNA worked better, as shown in Figure 9. The experiment was performed by co-transfecting 293FT cells with Cas9 and a U6-guide RNA DNA cassette to express one of the four RNA forms shown above. The target of the guide RNA is the same target site in the human EMX1 locus: "GTCACCTCCAATGACTAGGG"

[0423] Example 6: Optimization of Streptococcus thermophilus LMD-9 CRISPR1 Cas9 (called St1Cas9).

[0424] The applicant designs Figure 12The guide chimeric RNA shown in .

[0425] The St1Cas9 guide RNA can be subjected to the same type of optimization as performed for the SpCas9 guide RNA by breaking polythymine (T) for extension.

[0426] Example 7: Improvement of Cas9 system for in vivo applications

[0427] The applicant performed a metagenomic search of Cas9 with a small molecular weight. Most Cas9 homologs are quite large. For example, SpCas9 is about 1368aa in length, which is too large for easy packaging into a viral vector for delivery. Some sequences may have been misannotated and therefore the exact frequency of each length is not necessarily accurate. Nevertheless, it provides a vague understanding of the distribution of Cas9 proteins and indicates that there are shorter Cas9 homologs.

[0428] Through computational analysis, the applicant discovered that in the bacterial strain Campylobacter, there are two Cas9 proteins with less than 1000 amino acids. The sequence of a Cas9 from Campylobacter jejuni is presented below. With this length, CjCas9 can be easily packaged into AAV, lentivirus, adenovirus and other viral vectors for robust delivery into primary cells and into animal models.

[0429] >Campylobacter jejuni Cas9 (CjCas9)

[0430] 。

[0431] The putative tracrRNA elements of this CjCas9 are:

[0432] TATAATCTCATAAGAAATTTAAAAAGGGACTAAAATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTAAAATT

[0433] The direct repeat sequences are:

[0434] ATTTTACCATAAAGAAATTTAAAAAGGGACTAAAAC

[0435] The co-folded structure of tracrRNA and direct repeats is provided in FIG6 .

[0436] An example of a chimeric guide RNA for CjCas9 is:

[0437] NNNNNNNNNNNNNNNNNNGUUUUAGUCCCGAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCUAAAACCGCUUUU

[0438] Applicants have also optimized Cas9 guide RNA using in vitro methods. Figure 18 shows data from in vitro optimization of St1Cas9 chimeric guide RNA.

[0439] Although preferred embodiments of the present invention have been shown and described herein, it should be apparent to those of ordinary skill in the art that such embodiments are provided by way of example only. Numerous changes, modifications, and substitutions will now be thought of by those of ordinary skill in the art without departing from the present invention. It should be understood that different alternatives of the embodiments of the present invention described herein may be adopted in practicing the present invention. It is intended that the scope of the present invention be defined in accordance with the following claims, and methods and structures within the scope of these claims and their equivalents are also included within the scope of the present invention.

[0440] Example 8: Sa sgRNA optimization

[0441] The applicant designed five sgRNA variants of SaCas9 for the optimized truncated structure with the highest cutting efficiency. In addition, the natural direct repeat: tracr duplex system was tested next to the sgRNA. The guide with a specified length was co-transfected with SaCas9 and tested for its activity in HEK 293FT cells. A total of 100ng sgRNA U6-PCR amplicon (or 50ng of direct repeat and 50ng of tracrRNA) and 400ng of SaCas9 plasmid were co-transfected into 200,000 Hepa1-6 mouse hepatocytes, and DNA was harvested 72 hours after transfection for SURVEYOR analysis. The results are shown in Figure 23.

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[0487] 45. Gibson, DG et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature: Methods 6, 343-345 (2009).

[0488] 46. ​​Tangri S et al., Rationally engineered therapeutic proteins with reduced immunogenicity, J Immunol. 2005 May 15;174(6):3187-96.

[0489] ****

[0490] Although preferred embodiments of the present invention have been shown and described herein, it should be apparent to those of ordinary skill in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those of ordinary skill in the art without departing from the present invention. It should be understood that different alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention.

Claims

1. A composition comprising a CRISPR-Cas system chimeric RNA (chiRNA), wherein the chiRNA comprises from 5 to 3': (a) a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a eukaryotic cell nucleus, (b) a tracr mate sequence that is capable of hybridizing to the tracr sequence, and (c) a tracr sequence having at least 30 nucleotides in length, The chiRNA is capable of forming a CRISPR-Cas complex with the Cas9 protein and guiding the CRISPR-Cas complex to sequence-specifically bind to a target sequence adjacent to the PAM in eukaryotic cells.

2. A Cas9 enzyme derived from Streptococcus pyogenes or Streptococcus thermophilus.

3. A kit comprising a Cas9 enzyme.

4. A fusion protein comprising RNAse III, mCherry and a nuclear localization signal (NLS).

5. A composition comprising one or more PCR primers selected from the group consisting of AAAACCACCCTTCTCTCTGGC, GGAGATTGGAGACACGGAGAG, CTGGAAAGCCAATGCCTGAC, and GGCAGCAAACTCCTTGTCCT.

6. The composition of claim 1, wherein the tracr sequence is at least 40 nucleotides in length.

7. The composition of claim 1, wherein the tracr sequence is at least 50 nucleotides in length.

8. The composition of claim 1, wherein the guide sequence is 15-25 nucleotides in length.

9. The composition of claim 1, wherein the guide sequence is at least 20 nucleotides in length.

10. The composition of claim 1, wherein the chiRNA comprises an RNA sequence selected from the group consisting of: (a)NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCCGGUGCUUU; (b)NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG; (c)NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA.

11. The composition of claim 1, wherein the chiRNA is encoded by a DNA sequence selected from the group consisting of: (a)NNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG GCACCGAGTCGGTGCTTTTTT; (b)NNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG TTTTTTT; (c)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCATTTTTTTT.

12. The composition of claim 1, wherein the chiRNA comprises a methylated nucleotide or a nucleotide analog.

13. The composition of claim 1, wherein the chiRNA comprises a chemical modification selected from the group consisting of 2'-O-methyl analogs, 2'-deoxy analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine, phosphorothioate backbones, or combinations thereof.

14. The composition according to any one of claims 1 and 6-13, further comprising a Cas9 protein or a polynucleotide encoding a Cas9 protein.

15. The composition of claim 14, wherein Cas9 is fused to one or more NLSs independently selected from PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, POPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

16. The composition of claim 14, wherein the Cas9 protein is Streptococcus pyogenes Cas9.

17. The composition of claim 14, wherein the Cas9 protein is Streptococcus thermophilus Cas9.

18. Use of the composition according to any one of claims 1 and 6-17 in the manufacture of a medicament for treating a genetic disease or disorder.

19. Use of the composition according to any one of claims 1 and 6-17 for modifying eukaryotic cells in vitro or in vitro.

20. A eukaryotic cell comprising the composition of any one of claims 1 and 6-17.

21. A non-human organism comprising the eukaryotic cell of claim 20, wherein the non-human organism is not an animal or plant species.

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