Crispr-based compositions and methods of use

Abstract

The present invention relates to compositions for modification in CRISPR systems and methods of use thereof. In particular, length modified and chemically modified forms of crRNA and tracrRNA are described for use as reconstructed guide RNA for interaction with Cas9 of the CRIPSR system. The resulting length modified and chemically modified forms of crRNA and tracrRNA can be economically produced, and can be tailored to have unique properties related to their biochemical and biological activity in the CRIPSR Cas9 endonuclease system context.

Description

[0001] This application is a divisional application of a patent application filed on December 18, 2015, with application number 202110095191.4 and titled “CRISPR-based compositions and methods of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 USC 119 to U.S. Provisional Patent Applications Serial Nos. 62 / 093,588 and 62 / 239,546, filed on December 18, 2014 and October 9, 2015 and entitled “CRISPR-BASED COMPOSITIONS AND METHODS OF USE,” the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy was created on December 18, 2015, is named IDT01-008-US_ST25.txt, and is 177,163 bytes in size. Technical Field

[0006] The present invention relates to compositions for modification in CRISPR systems, and methods of using the same. Background Art

[0007] Site-specific DNA cleavage using clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins (CRISPR-Cas systems) shows great potential for many biological applications. CRISPR is used for genome editing; genome-scale specific targeting of endogenous genes with transcriptional repressors (CRISPRi) and activators (CRISPRa); and other applications of RNA-guided DNA targeting using Cas enzymes.

[0008] CRISPR-Cas systems are derived from bacteria and archaea to provide adaptive immunity against viruses and plasmids. There are three types of CRISPR-Cas systems that may be potentially suitable for research and therapeutic agents, but type II CRISPR systems have ideal features, using a single CRISPR-associated (Cas) nuclease (particularly Cas9) and a complex of an appropriate guide RNA (a 2-part RNA system similar to the natural complex in bacteria containing a CRISPR activating RNA: trans-activating crRNA (crRNA: tracrRNA) pair or an artificial chimeric single guide RNA (sgRNA)) to mediate double-stranded cleavage of the target DNA. In mammalian systems, these RNAs have been introduced by transfecting a DNA cassette containing an RNA Pol III promoter (e.g., U6 or H1) driving RNA transcription, a viral vector, and a single-stranded RNA after in vitro transcription (see Xu, T. et al., Appl Environ Microbiol, 2014. 80(5): p. 1544-52).

[0009] In the CRISPR-Cas9 system, using, for example, a system present in Streptococcus pyogenes (Streptococcus pyogenes) as an example (S.py. or Spy), the natural crRNA length is about 42bp, containing a 5' region of about 20 bases complementary to the target sequence (also referred to as the protospacer sequence) and a 3' region corresponding to the complementary region of the tracrRNA sequence of about 22 bases in length. The natural tracrRNA length is about 85-90 bases, with a 5' region containing a region complementary to crRNA and about 10 base regions upstream of 5'. The remaining 3' region of tracrRNA includes a secondary structure (referred to herein as "tracrRNA 3' tail").

[0010] Jinek et al. extensively studied the parts of crRNA and tracrRNA required for the normal operation of the CRISPR-Cas9 system (Science, 2012.337(6096): p. 816-21). They designed truncated crRNA:tracrRNA fragments that can still function in CRISPR-Cas9, where crRNA is the wild-type 42 nucleotides and tracrRNA is truncated to 75 nucleotides. They also developed an embodiment in which crRNA and tracrRNA are connected using a linker loop to form a single guide RNA (sgRNA), which varies between 99-123 nucleotides in different embodiments. The configuration of the natural 2-part crRNA:tracrRNA complex is shown in Figure 1and a 99-nucleotide implementation scheme of an artificial sgRNA single guide is shown in Figure 2 middle.

[0011] At least two teams have elucidated the crystal structure of Streptococcus pyogenes Cas9 (SpyCas9). In Jinek, M. et al., the structure did not show the nuclease complexed with the guide RNA or target DNA. They performed molecular simulation experiments to reveal the predicted interactions between the protein in complex form and RNA and DNA (Science, 2014.343, p. 1215, DOI: 10.1126 / science / 1247997).

[0012] In Nishimasu, H. et al., the crystal structure of SpyCas9 was shown at 2.5 angstrom resolution as a complex with sgRNA and its target DNA (Cell, 2014.156(5): 935-49, incorporated herein in its entirety). The crystal structure identified two lobes of the Cas9 enzyme: the recognition lobe (REC) and the nuclease lobe (NUC). The sgRNA: target DNA heteroduplex (negatively charged) is located in the positively charged groove between the two lobes. The REC lobe, which shows no structural similarity to known proteins and is therefore likely to be a Cas9-specific functional domain, interacts with parts of the crRNA and tracrRNA that are complementary to each other.

[0013] Another team, Briner et al. (Mol Cell, 2014.56(2):333-9, incorporated herein in its entirety) identified and characterized six conserved components within the natural crRNA:tracrRNA duplex and sgRNA.

[0014] The CRISPR-Cas9 system is used for genome engineering as follows: a portion of crRNA hybridizes with a target sequence, a portion of tracrRNA hybridizes with a portion of crRNA, and the Cas9 nuclease binds to the entire construct and guides cutting. Cas9 contains two domains homologous to endonucleases HNH and RuvC, wherein the HNH domain cuts the DNA strand complementary to crRNA, and the RuvC-like domain cuts the non-complementary strand. This results in double-strand breaks in genomic DNA. When repaired by non-homologous end joining (NHEJ), the break is usually offset by 1 or more bases, resulting in the destruction of the native DNA sequence, and in many cases, if the event occurs in the coding exon of a protein-coding gene, a frameshift mutation is caused. The break can also be repaired by homology-dependent recombination (HDR), which allows new genetic material to be inserted into the cleavage site generated by Cas9 cutting via experimental manipulation.

[0015] Some current methods for delivering guide RNA into mammalian cells include transfection of double-stranded DNA (dsDNA) containing an RNA Pol III promoter for endogenous transcription, viral delivery, transfection of RNA as an in vitro transcription (IVT) product, or microinjection of IVT products. Each of these methods has disadvantages. Unmodified exogenous RNA introduced into mammalian cells is known to initiate an innate immune response through recognition by Toll-like receptors (TLRs), RIG-I, OASI, and other receptors that recognize pathogen-associated molecular patterns (PAMPs). However, in most published studies, RNA was delivered to cells by in vitro transcribed (IVT) by T7 RNA polymerase. This type of RNA payload has been shown to be a triggering factor for the innate immune response. The above alternative delivery methods also each have their own disadvantages. For example, dsDNA cassettes can lead to integration, guide RNA transcription driven endogenously by RNA Pol II promoters can persist constitutively, and the amount of RNA transcribed is uncontrollable.

[0016] RNA is rapidly degraded by nucleases present in serum and cells. Unmodified CRISPR RNA triggers (crRNA, tracrRNA and sgRNA) prepared by IVT method or chemical synthesis are rapidly degraded during or after delivery to mammalian cells. If RNA is chemically modified to obtain nuclease resistance, greater activity will be achieved. The most effective degradation activity present in serum and cells is 3'-nuclease (Eder et al., Antisense Research and Development, 1: 141-151, 1991). Therefore, "capping" of synthetic oligonucleotides generally improves nuclease stability. Chemical modification of single-stranded antisense oligonucleotides (ASOs) and double-stranded small interfering RNAs (siRNAs) has been well studied, and there are successful methods in practice today (for reviews, see: Kurreck, Eur. J. Biochem., 270: 1628-1644, 2003; Behlke, Oligonucleotides, 18: 305-320, 2008; Lennox et al., Gene Therapy, 18: 1111-1120, 2011). Therefore, it is necessary to develop chemical modification strategies for use with the RNA components of CRISPR / Cas. Although the basic toolbox of available chemical modifications is well known to those skilled in the art, the effects of site-specific modifications on the interaction of RNA substances and effector proteins are not easily predicted, and effective modification patterns must usually be determined empirically. In some cases, the sequence of the RNA may affect the effectiveness of the modification pattern, and the modification pattern for different sequence backgrounds needs to be adjusted, making the practical application of these methods more challenging.

[0017] Therefore, it is necessary to modify the guide RNA to reduce its toxicity to cells and extend its lifespan and functionality in mammalian cells, while still performing its intended purpose in the CRISPR-Cas system. The methods and compositions of the invention described herein provide RNA and modified RNA oligonucleotides for use in the CRISPR-Cas system. These and other advantages of the invention and additional inventive features will be apparent from the description of the invention provided herein. Summary of the invention

[0018] The present invention relates to a modified composition for use in a CRISPR system and a method of using the same. The composition includes modified internucleotide bonds and 2'-O-alkyl and 2'-O-fluorine modified RNA oligonucleotides to serve as guide strands (crRNA:tracrRNA or sgRNA) of a CRISPR-Cas system. The composition also includes terminal modifications, such as reverse dT (inverted-dT) bases or other non-nucleotide modifiers (e.g., propylene glycol groups (C3 spacers), naphthyl-azo modifiers, or other modifiers as well known in the art) that prevent exonuclease attack.

[0019] In a first aspect, an isolated tracrRNA is provided, comprising a length-modified form of SEQ ID NO.: 18. The isolated tracrRNA exhibits activity in a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) endonuclease system.

[0020] In a second aspect, an isolated crRNA is provided, comprising a length-modified form of formula (I):

[0021] 5'-X—Z-3'(I),

[0022] Wherein X represents the sequence of the target-specific protospacer sequence domain, and Z represents the sequence of the universal tracrRNA binding domain, wherein the protospacer sequence domain comprises about 20 target-specific nucleotides, and the tracrRNA binding domain comprises about 20 nucleotides. The isolated crRNA shows activity in a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

[0023] In a third aspect, an isolated tracrRNA is provided, comprising a chemically modified form of one of SEQ ID NOs.: 2, 18, 30-33, and 36-39. The isolated tracrRNA exhibits activity in a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

[0024] In a fourth aspect, an isolated crRNA is provided, comprising a chemically modified form of formula (I):

[0025] 5'-X—Z-3'(I),

[0026] Wherein X represents a sequence comprising a target-specific protospacer sequence domain, and Z represents a sequence comprising a universal tracrRNA binding domain, wherein the protospacer sequence domain comprises about 17 nucleotides to about 20 nucleotides, and the tracrRNA binding domain comprises about 12 nucleotides to about 19 nucleotides. The isolated crRNA exhibits activity in a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The wild-type (WT) native 2-part crRNA:tracrRNA complex with 42 bases of unmodified crRNA (SEQ ID No.: 46) and 89 bases of unmodified tracrRNA (SEQ ID No.: 18) is shown. Lowercase letters represent RNA.

[0028] Figure 2 A 99-base artificial single guide RNA (SEQ ID NO: 428) (sgRNA) is shown in which the crRNA and tracrRNA elements are fused into a single sequence by adding a new hairpin loop. Lowercase letters represent RNA.

[0029] Figure 3 An alignment of the full-length and truncated tracrRNA species studied in Example 2 is shown. The sequences are RNA and are shown 5'-3'. The alignment is based on the 89 base WT tracrRNA sequence above (SEQ ID No.: 18). Internal spaces represent internal truncated / deleted sites. Capital letters represent RNA.

[0030] Figure 4 An alignment of the full-length and truncated crRNA and tracrRNA species studied in Example 3 is shown. The alignment is based on the 42 base WT crRNA (SEQ ID No.: 46) and 89 base WT tracrRNA (SEQ ID No.: 18) sequences above their respective groups. The 20 base 5' domain in the crRNA is sequence specific and targets human HPRT1. The 3' domain is underlined and binds to the region at the 5' end of the tracrRNA. The 5' domain in the tracrRNA is underlined and binds to the 3' end of the crRNA. Capital letters represent RNA.

[0031] Figure 5The diagram shows a truncated 2-part crRNA:tracrRNA complex with 36 bases of crRNA (SEQ ID No.: 48) and 67 bases of tracrRNA (SEQ ID No.: 2). Lowercase letters represent RNA.

[0032] Figure 6 Schematic diagram of the structure of one embodiment of an optimized truncated and chemically modified tracrRNA (SEQ ID No.: 134). The length is 67 bases. RNA is in lowercase and 2'OMe RNA is in uppercase. Phosphorothioate (PS) internucleotide bonds are indicated by "*". Residues that cause significant loss of function when converted from RNA to 2'OMe RNA are identified by large arrows, and residues that cause moderate loss of function when converted from RNA to 2'OMe RNA are identified by small arrows.

[0033] Figure 7 The schematic diagram of shows the structure of one embodiment of an optimized truncated and chemically modified crRNA (SEQ ID No.: 239). The length is 36 bases. RNA is lowercase and 2'OMe RNA is uppercase. The bonds between phosphorothioate (PS) nucleotides are indicated by "*". Residues that cause significant loss of function when converted from RNA to 2'OMe RNA are identified by large arrows, and residues that cause moderate loss of function when converted from RNA to 2'OMe RNA are identified by small arrows. The 5' terminal 20 bases of the protospacer sequence target-specific guide domain are indicated, which in this case is a sequence specific to the human HPRT1 gene. The 3' terminal 16 bases of the tracrRNA binding domain are indicated.

[0034] Figure 8 The schematic diagram of shows the structure of one embodiment of the optimized truncated / modified crRNA:tracrRNA complex used in Example 8. The crRNA is at the top, with the 20 bases of the 5'-protospacer sequence domain underlined, which in this case is specific to the target human HPRT1 site 38285; the 3' end is the 16 base tracrRNA binding domain. The tracrRNA is aligned below. RNA is lowercase, 2'OMe RNA is uppercase, and "*" indicates a phosphorothioate internucleotide bond modification. The figure shows a complex formed by crRNA SEQ ID No.: 178 and tracrRNA SEQ ID No.: 100.

[0035] Fig. 9The schematic diagram of shows the structure of one embodiment of a highly modified optimized truncated / modified crRNA:tracrRNA complex. The crRNA is located above, with the 20 bases of the 5'-protospacer sequence domain underlined, which in this case is specific to the target human HPRT1 site 38285; the 3' end is the 16 base tracrRNA binding domain. The tracrRNA is aligned below. RNA is lowercase, 2'OMe RNA is uppercase, and "*" indicates a phosphorothioate internucleotide bond modification. The figure shows a complex formed by crRNA SEQ ID No.: 446 and tracrRNA SEQ ID No.: 134.

[0036] Fig.10 The schematic diagram shows the crRNA modification pattern used in Example 10. The oligonucleotide sequences (SEQ ID No.: 429-439, respectively, in order of appearance) are shown 5'-3'. Lowercase = RNA; underlined = 2'-O-methyl RNA; C3 = C3 spacer (propylene glycol modifier); * = phosphorothioate internucleotide bond; ZEN = naphthyl-azo modifier. The 5'-target-specific protospacer sequence domain is indicated. The bases are indicated by "N" in this domain because the sequence of each target site is different, but the modification pattern used remains constant. The 3' universal tracrRNA binding domain is indicated. The modification patterns are numbered for reference in Table 10 and Table 10. Fig.10 Reference between.

[0037] Fig.11 10 is a plot of the data in Table 10, which shows functional gene editing observed in mammalian cells using T7E1 assay using crRNA prepared with 11 different modification patterns tested at 12 different sites of the human HPRT1 gene. All crRNA variants are paired with optimized modified tracrRNA (SEQ ID No.: 100).

[0038] Fig.12 The schematic diagram of shows the structure of one embodiment of an optimized truncated / modified crRNA:tracrRNA complex that is highly modified using crRNA modification pattern 6 that is universal and applicable to any sequence background. The crRNA (SEQ ID No.: 440) is located above, with the 20 bases of the 5'-protospacer sequence domain underlined (N-base); the 3' end is the 16 base tracrRNA binding domain. The tracrRNA (SEQ ID No.: 134) is aligned below. RNA is lowercase, 2'OMe RNA is uppercase, and "*" indicates phosphorothioate internucleotide bond modification.

[0039] Fig.13 Shown is a graph of RT-qPCR data from HEK-Cas9 cells transfected with different CRISPR gRNAs, showing the relative expression levels of IFIT1 and IFITM1, 2 genes involved in the interferon signaling pathway. DETAILED DESCRIPTION OF THE INVENTION

[0041] Aspects of the invention relate to compositions for modification in CRISPR systems and methods of use thereof.

[0042] As used herein, the term "oligonucleotide" refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) and any other type of polynucleotide, which is an N-glycoside of a purine or pyrimidine base (a single nucleotide is also referred to as a "base" or "residue"). There is no expected difference in length between the terms "nucleic acid", "oligonucleotide" and "polynucleotide", and these terms can be used interchangeably. These terms refer only to the main structure of the molecule. Therefore, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. For use in the present invention, oligonucleotides may also include nucleotide analogs in which bases, sugars or phosphate backbones are modified, as well as non-purine or non-pyrimidine nucleotide analogs. Oligonucleotides may include ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof.

[0043] The compositions of the present invention include any modification that may reduce activation of the innate immune system. The modification may be exchanged or substituted at conventional phosphodiester bonds, ribose or nucleobases of RNA. Such compositions may include, for example, modified nucleotides, such as 2'-O-methyl modified RNA.

[0044] More generally, the term "modified nucleotide" refers to a nucleotide having one or more modifications to a nucleoside, a nucleobase, a pentose ring or a phosphate group. For example, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate and cytidine monophosphate, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. Modifications include naturally occurring modifications produced by modifications of enzymes (e.g., methyltransferases) that modify nucleotides. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Modifications also include base analogs and universal bases. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, such as 2'-O-alkyl (including 2'-O-methyl), 2'-fluoro, 2'-methoxyethoxy, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, bicyclic nucleic acids, 4'-CH2—O-2'-bridge, 4'-(CH2)2—O-2'-bridge, 2'-LNA and 2'-O-(N-methylcarbamate) or those containing base analogs. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No.: 5,672,695 and Matulic-Adamic et al., U.S. Pat. No.: 6,248,878.

[0045] The use of 2'-O-methyl has been documented in the siRNA literature (see Behlke, MA, Oligonucleotides, 2008. 18(4): 305-19) and in mRNA delivery (see Sahin, U. et al., Nat Rev Drug Discov, 2014. 13(10): 759-80). Sahin et al. describe modifications of mRNA therapeutics that extend beyond 2'-OMe modification and "non-immunogenic" mRNA.

[0046] The term "ribonucleotide" encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes in the sugar moiety, changes in the base moiety and / or changes in the linkages between ribonucleotides in the oligonucleotide.

[0047] The term "Cas9 protein" encompasses wild-type and mutant forms of Cas9 that have biochemical and biological activity when combined with an appropriate guide RNA (e.g., sgRNA or a dual crRNA:tracrRNA composition) to form an active CRISPR-Cas endonuclease system. This includes orthologs and Cas9 variants with different amino acid sequences from Streptococcus pyogenes Cas9 used as an example in the present invention.

[0048] The term "length modification" as a term for modified RNA refers to a shortened or truncated form of a reference RNA that lacks a nucleotide sequence, or an extended form of a reference RNA that includes an additional nucleotide sequence.

[0049] The term "chemical modification" as a term for modified RNA refers to a form of the reference RNA containing chemically modified nucleotides or non-nucleotide chemical groups covalently linked to the RNA. Chemically modified RNA as described herein generally refers to synthetic RNA prepared using an oligonucleotide synthesis procedure, wherein the modified nucleotides are introduced during the synthesis of the RNA oligonucleotide. However, chemically modified RNA also includes synthetic RNA oligonucleotides modified with suitable modifiers after synthesis.

[0050] Applicants have discovered novel crRNA and tracrRNA oligonucleotide compositions showing stable activity in clustered regularly spaced short palindromic repeats (CRISPR)-CRISPR-related (Cas) (CRISPR-Cas) endonuclease systems. The oligonucleotide composition includes a length-modified form of crRNA and tracrRNA, and a chemically modified form of crRNA and tracrRNA. The length-modified form of crRNA and tracrRNA enables the use of conventionally available cost-effective and effective oligonucleotide synthesis schemes to prepare the active forms of these RNAs. The chemically modified form of crRNA and tracrRNA provides an activating agent with adjustable (such as improved stability in cells and in vivo backgrounds) certain specific properties. The length-modified form of crRNA and tracrRNA may also include modification, so that a wide variety of compositions with activity in the background of the CRISPR-Cas endonuclease system can be obtained. These oligonucleotide compositions and their properties in the CRISPR-Cas endonuclease system are described below.

[0051] Length modification of crRNA and tracrRNA

[0052] Figure 1A diagram of a wild-type S. pyogenes crRNA:tracrRNA complex is depicted, wherein an exemplary isolated crRNA (SEQ ID No.: 46) is paired with an isolated tracrRNA (SEQ ID No.: 8). In a first aspect, an isolated tracrRNA comprising a length-modified form of SEQ ID NO.: 18 is provided. The isolated tracrRNA exhibits activity in a CRISPR-Cas endonuclease system. In one aspect, the isolated tracrRNA comprises a length-modified form of SEQ ID NO.: 18 nucleotides having missing sequence information. In some embodiments, the length-modified form of SEQ ID NO.: 18 comprises a shortened or truncated form of SEQ ID NO.: 18, wherein SEQ ID NO.: 18 can be shortened by 1 to 20 nucleotides at the 5' end and by 1-10 nucleotides at the 3' end. This shortened or truncated form of SEQ ID NO.: 18 retains activity when paired with a competent crRNA in a CRISPR-Cas endonuclease system. If the 5' end of the tracrRNA is shortened and extended into the sequence paired with the 3' end of the crRNA, chemical modifications that enhance the binding affinity in these domains can be used to obtain improved activity. If the 3' end of the crRNA is shortened and extended into the sequence paired with the 5' end of the tracrRNA, chemical modifications that enhance the binding affinity in these domains can be used to obtain improved activity. Preferred examples of length-modified forms of SEQ ID NO.: 18 with shortened or truncated forms include SEQ ID NO: 2, 30-33, and 36-39. Highly preferred examples of length-modified forms of SEQ ID NO.: 18 with shortened or truncated forms include SEQ ID NO: 2. For each of the above-mentioned exemplary length-modified forms of SEQ ID NO.: 18 with shortened or truncated forms, SEQ ID NO.: 2, 30-33, and 36-69 can be composed of chemically unmodified nucleotides.

[0053] In a second aspect, an isolated crRNA comprising a length-modified form of formula (I) is provided:

[0054] 5'-X—Z-3'(I),

[0055] wherein X represents a sequence including a target-specific protospacer domain, and Z represents a sequence including a tracrRNA binding domain.

[0056] The target-specific protospacer sequence domain (X domain of formula (I)) typically includes about 20 nucleotides complementary to the DNA region targeted by the CRISPR-Cas nuclease system. In most CRISPR nuclease systems, the tracrRNA binding domain (Z domain of formula (I)) typically includes about 20 nucleotides (in the native S.py. form, the domain is 22 nucleotides). The isolated crRNA shows activity in the CRISPR-Cas nuclease system.

[0057] In one aspect, the crRNA of separation includes a length-modified form of formula (I) with missing sequence information. In some embodiments, the length-modified form of formula (I) includes a shortened or truncated form of formula (I), wherein formula (I) can shorten 1-8 nucleotides at the 3' end of the Z domain. The length-modified form of formula (I) can be shortened at the 5 ends of the X domain to accommodate a target-specific protospacer sequence domain with 17, 18, 19 or 20 nucleotides. A very preferred example of this length-modified form of formula (I) includes a target-specific protospacer sequence domain with 19 or 20 nucleotides. An exemplary length-modified form of formula (I) with a shortened or truncated form having a target-specific protospacer sequence (X domain) of 17-20 nucleotides in length and / or 1-8 nucleotides missing at the 3' end of the Z domain can be composed of chemically unmodified nucleotides.

[0058] This shortened or truncated form of formula (I) retains activity when paired with a qualified tracrRNA in a CRISPR-Cas endonuclease system. Preferred embodiments of isolated crRNAs of formula (I) having length-modified forms of formula (I) may include chemically unmodified nucleotides and chemically modified nucleotides.

[0059] Chemically modified forms of crRNA and tracrRNA

[0060] In a third aspect, there is provided an isolated tracrRNA comprising a chemically modified nucleotide or a non-nucleotide chemical modifier. The isolated tracrRNA shows activity in the CRISPR-Cas endonuclease system. In one aspect, the isolated tracrRNA includes a modified chemically modified nucleotide having a modified bond selected from a ribose modification, a terminal modification group, and a modified bond between nucleotides. Exemplary ribose modifications include 2'O-alkyl (e.g., 2'OMe), 2'F, bicyclic nucleic acids, and locked nucleic acids (LNA). Exemplary terminal modification groups include propylene glycol (C3) spacers and naphthyl-azo modifiers (N, N-diethyl-4-(4-nitronaphthalene-1-ylazo)-aniline or "ZEN") and reverse dT residues. Exemplary modified bonds between nucleotides include phosphorothioate modifications. In one aspect, the isolated tracrRNA with a chemically modified form includes SEQ ID NO.: 46 and a length-modified form thereof, such as a shortened or truncated form of SEQ ID NO.: 46. Preferred shortened or truncated forms of SEQ ID NO.: 46 with chemically modified nucleotides include SEQ ID NOs: 2, 30-33, and 36-39 with chemically modified nucleotides. Other examples of isolated tracrRNAs with chemically modified nucleotides that have stable activity in the CRISPR-Cas endonuclease system are presented in the Examples.

[0061] In a fourth aspect, there is provided a crRNA comprising a separation of chemically modified nucleotides. The crRNA of the separation shows activity in the CRISPR-Cas nuclease system. In one aspect, the crRNA of separation includes a modified chemically modified nucleotide having a key selected from ribose modification, a terminal modification group and a modified internucleotide. Exemplary ribose modifications include 2'O-alkyl (e.g., 2'OMe), 2'F, bicyclic nucleic acids, and locked nucleic acids (LNA). Exemplary terminal modification groups include propylene glycol (C3) spacers and naphthyl-azo modifiers (N, N-diethyl-4-(4-nitronaphthalene-1-ylazo)-aniline or "ZEN") and reverse dT residues. The modified key between exemplary nucleotides includes phosphorothioate modification. In one aspect, the crRNA of separation with a chemically modified form includes the crRNA of formula (I) and its length modified form. The preferred shortened or truncated form of the crRNA with formula (I) of chemically modified nucleotides includes SEQ ID NO.: 429-439. Highly preferred examples of isolated crRNAs with chemically modified nucleotides include SEQ ID NOs.: 434 and 435. These specific isolated crRNA species represent "universal" crRNAs with chemically modified nucleotides that exhibit high activity when combined with qualified tracrRNAs in the CRISPR-Cas endonuclease system. Other examples of isolated crRNAs with chemically modified nucleotides that have stable activity in the CRISPR-Cas endonuclease system are presented in the Examples.

[0062] The above-mentioned isolated, length-modified and chemically modified crRNA and tracrRNA preferably include chemical modifications (e.g., 2'OMe, 2'F, bicyclic nucleic acids, locked nucleic acids, etc.) and end-capping modifications (e.g., ZEN, C3 spacer, reverse dT) at the 2'-OH group. The use of these two types of general modifications provides isolated, length-modified and chemically modified crRNA and tracrRNA with biochemical stability and immunological tolerance in a biological context.

[0063] The above-mentioned isolated, length-modified and chemically modified crRNA and tracrRNA can be mixed in different combinations to form an active crRNA:tracrRNA as a guide RNA for Cas9. For example, the isolated length-modified tracrRNA can be combined with the isolated chemically modified crRNA to form an active crRNA:tracrRNA as a guide RNA for Cas9. The embodiment provides an illustration of different combinations of isolated, length-modified and chemically modified crRNA and tracrRNA to produce an active crRNA:tracrRNA as a guide RNA for Cas9.

[0064] The extent to which specific chemically modified nucleotides need to be included in one (or both) of the separated, length-modified and chemically modified crRNA and tracrRNA depends on the application of the active crRNA obtained:tracrRNA as the guide RNA of Cas9. In certain biochemical assays of the CRISPR-Cas endonuclease system, particularly in the case where nucleases can be minimized or not present, it may not be necessary to extensively chemically modify the crRNA and tracrRNA to achieve the stable activity of the guide RNA of the Cas9 of the obtained CRISPR-Cas endonuclease system. This is attributed to the fact that when nucleases can be minimized or nucleases do not exist, chemically modified nucleotides that confer nuclease resistance are unnecessary. In certain biological (in vivo) contexts, a mixture including crRNA and tracrRNA is delivered to cells in a carrier (such as liposome nanoparticles), and the crRNA and tracrRNA separated by length modification and chemical modification may require less chemically modified nucleotides than a mixture of crRNA and tracrRNA that is directly delivered to the bloodstream or injected into an organ system as a separated "naked" RNA mixture. The degree of chemical modification present in chemically modified crRNA and tracrRNA can determine the half-life of the relevant RNA molecule in vivo (i.e., in a relevant biological context, such as in the bloodstream or within a cell). Therefore, the modification profile of chemically modified crRNA and tracrRNA can be used to fine-tune the biochemistry and biological activity of the resulting crRNA:tracrRNA duplex as a guide RNA for Cas9 in the CRISPR-Cas endonuclease system.

[0065] Although the prior art focuses on the structure when Cas9 interacts with sgRNA, the disclosed design pattern described herein covers the above-mentioned crRNA:tracrRNA binary RNA system. Single-stranded guide RNA provides several benefits, such as simplicity of therapeutic design. However, standard solid phase phosphoramidite RNA synthesis shows that the yield of oligonucleotides decreases with increasing length, and this problem becomes more obvious when the length exceeds 60-70 bases. This hinders the stable and cost-effective synthesis of some tracrRNA and chimeric sgRNA, especially at a larger scale required for some commercial or therapeutic applications. Therefore, the present invention not only covers the embodiment of sgRNA as the guide RNA of Cas9, but also covers the embodiment of the guide RNA of Cas9 with alternative binary crRNA:tracrRNA. However, when combined with Cas9 in the CRISPR-Cas nuclease system, it is possible to engineer a separate guide RNA with stable activity by connecting or synthesizing appropriate crRNA and tracrRNA into an artificial single-molecule sgRNA based on the separation, length modification and chemical modification of crRNA and tracrRNA provided herein. Long individual guides of this type can be obtained by direct synthesis or by postsynthetic chemical conjugation of shorter chains.

[0066] The design of length-modified and chemically modified tracrRNA compositions solves potential synthesis problems associated with tracrRNA oligonucleotides of length>80 nucleotides. The coupling efficiency of 2'-OMe-modified RNA monomers (effectively containing protective groups on 2'-OH) is greater than RNA monomer coupling. Incorporation of 2'-OMe-modified RNA provides some advantages. First, it allows longer oligonucleotides to be synthesized as complete 2'-OMe or RNA / 2'-OMe mixed oligonucleotides. Secondly, the methods and compositions of the present invention lead to synthesis and transfection of crRNA:tracrRNA that can escape immune system detection. It is well known that exogenous unmodified RNA triggers innate immune responses in mammalian cells and whole animals. Oligonucleotides modified using 2'OMe can confer RNA stability to nucleases (the third advantage) and reduce cell death and toxicity associated with immunogenic triggering factors. These advantages are not unique to the 2'-OMe modification itself, as other disclosed modified nucleotides with different chemical moieties (eg, 2'F, other 2'O-alkyls, LNA, and other bicyclic nucleotides) may provide similar benefits and advantages in conferring nuclease resistance.

[0067] In another embodiment, the portion of the tracrRNA that is complementary to the crRNA contains at least one modified nucleotide, and in a further embodiment, the portion of the tracrRNA that is complementary to the crRNA comprises more than 10% modified residues, and in a further embodiment, the portion of the tracrRNA that is not complementary to the crRNA comprises more than 50% modified residues, and in a further embodiment, the portion of the tracrRNA that is not complementary to the crRNA comprises more than 90% modified residues.

[0068] In another embodiment, the crRNA portion is unmodified and the tracrRNA portion comprises at least one modified nucleotide. In another embodiment, the crRNA portion is unmodified and the tracrRNA portion comprises more than 10% modified bases.

[0069] In another embodiment, the isolated crRNA of formula (I) is designed to have empirically determined modifications. Figure 7 and Fig.10 As depicted in FIG, the 12 nucleotides at the 3' end of the Z domain (tracrRNA binding domain) and the 10-12 nucleotides at the 5' end of the X domain (within the protospacer sequence domain) represent universal nucleotides suitable for replacement with chemically modified nucleotides, wherein the resulting RNA maintains stable activity in the CRISPR-Cas nuclease system. Other nucleotides within the 5' end of the Z domain (tracrRNA binding domain) are not tolerant to replacement with chemically modified nucleotides ( Figure 7). However, the ability of other sites in the crRNA of the separated formula (I) to accept chemically modified nucleotides and retain activity in the CRISPR-Cas nuclease system is largely determined empirically. The tracrRNA binding domain (Z domain) of crRNA is constant (i.e., the sequence does not change with the target site), so regardless of the target site, the modification pattern described herein is universal for all crRNAs and can be widely used. The pre-inter-region sequence (X domain) of crRNA changes with the target, and the tolerance of some base positions in the domain to chemical modification changes with the sequence background, and if the maximum chemical modification of the site is required, it can benefit from empirical optimization. However, some residues in the target-specific pre-inter-region sequence (X) domain can be modified without considering the sequence background. The 10-12 residues at the 5' end of the domain can be replaced by 2'-modified residues, and it is expected that the full activity of the modified crRNA will be maintained. According to the sequence background, the remaining 8-10 bases at the 3' end of the pre-inter-region sequence (X) domain may tolerate modification or may not tolerate modification. A sequence context in which 17 of the 20 bases of the protospacer (X) domain can be modified while maintaining full activity is shown in Figure 7 The sites where modifications impair activity are indicated.

[0070] The applications of Cas9-based tools are numerous and varied. They include (but are not limited to): plant gene editing, yeast gene editing, rapid generation of knockout / knockin animal lines, generation of animal models of disease states, correction of disease states, insertion of reporter genes, and genome-wide functional screening.

[0071] The utility of the present invention is further expanded by including mutant forms of Cas enzymes, such as the D10A and H840A double mutants of Cas9, as fusion proteins with transcriptional activators (CRISPRa) and repressors (CRISPRi) (see Xu, T. et al., Appl Environ Microbiol, 2014. 80(5): p. 1544-52). Cas9-sgRNA complexes can also be used to target single-stranded mRNA (see O'Connell, MR et al., Nature, 516: 263, 2014). In the same manner as for targeting dsDNA, crRNA:tracrRNA can be used with PAMmer DNA oligonucleotides to guide Cas9 cleavage to target mRNA, or it can be used in the mRNA capture assay described by O'Connell.

[0072] By utilizing methods for delivering synthetic RNA oligonucleotides for CRISPR / Cas9 applications, it is possible to 1) use mass spectrometry to determine discrete RNA sequences, 2) selectively insert 2'-OMe modified RNA into well-tolerated positions to confer stability and avoid immunogenicity, but still retain functional efficacy, 3) specifically control the amount of RNA introduced into the cell to achieve a controlled transient effect, and 4) eliminate concerns about the introduced dsDNA being endogenously transcribed into RNA but also being a substrate in a homology-directed repair pathway or in a non-homologous end-joining pathway, thereby leading to integration events. These integration events may lead to long-term, undesirable expression of crRNA or tracrRNA elements. In addition, integration may damage other genes in a random and unpredictable manner, thereby changing the genetic material of the cell in an undesirable and potentially harmful manner. Therefore, the present invention is intended to be a means of introducing transient expression of elements of the CRISPR pathway into cells, the introduction of which is transient and does not leave persistent signs or change the genome outside of any variation expected to be guided by the crRNA guide.

[0073] CRISPR-Cas endonuclease system

[0074] Qualified CRISPR-Cas endonuclease system includes a ribonucleoprotein (RNP) complex formed by a separated Cas9 protein and a separated guide RNA selected from a binary crRNA:tracrRNA combination and one of a chimeric sgRNA. In some embodiments, the length modification and / or chemical modification form of the separated crRNA and tracrRNA is combined with purified Cas9 protein (e.g., SEQ ID NO.: 407-410), separated mRNA encoding Cas9 protein (e.g., SEQ ID NO.: 413) or a gene encoding Cas9 protein (e.g., SEQ ID NO.: 411 and 412) in an expression vector. In some assays, the length modification and / or chemical modification form of the separated crRNA and tracrRNA can be introduced into a cell line stably expressing Cas9 protein from an endogenous expression cassette encoding a Cas9 gene. In other experiments, a mixture of the length modification and / or chemical modification form of crRNA and tracrRNA and Cas9 mRNA or Cas9 protein combination can be introduced into a cell.

[0075] Example 1

[0076] This example demonstrates that chemically modified and truncated guide RNAs function in an in vitro Cas9 DNA cleavage assay.

[0077] CrRNA and tracrRNA oligonucleotides were synthesized with various chemical modifications and truncations relative to the indicated WT sequences (Table 1).

[0078] Table 1: In vitro biochemical studies of HPRT1 target DNA cleavage by Cas9 endonuclease with various crRNA and tracrRNA pairs.

[0079]

[0080]

[0081]

[0082]

[0083] Oligonucleotide sequences are shown as 5'-3'. Lowercase = RNA, underline = 2'-O-methyl RNA, italics = 2'-fluoro RNA. The length (base) of the RNA oligonucleotide is indicated. The relative cleavage efficiency of the recombinant Cas9 with each crRNA:tracrRNA pair visualized by agarose gel electrophoresis is indicated for the DNA target, where "+++" indicates complete cleavage, "++" and "+" indicate intermediate levels of cleavage, and "-" indicates no cleavage.

[0084] The crRNA contains a 19-base pre-spacer sequence guide sequence that matches a site in the human HPRT1 gene adjacent to a suitable "NGG" PAM site. A 938 base pair region from the human HPRT1 gene was cloned into a pCR-Blunt vector (Life Technologies). The plasmid was linearized by digestion with the restriction endonuclease XmaI (New England BioLabs) prior to use in Cas9 cleavage assays. The sequence of the HPRT1 target fragment is shown below. The target PAM site is indicated in bold, and the pre-spacer sequence guide sequence binding site is underlined.

[0085] HPRT1 target sequence. SEQ ID No.: 27.

[0086] GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGATTGTATT

[0087] TCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTCTGTACCCATTAATC

[0088] CTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGGTGCAGGTCTCAGAACTGTCC

[0089] TTCAGGGTTCCAGGTGATCAACCAAGTGCCTTGTTCTGTAGTGTCAACTCATTGCTGCCCCTTCCT

[0090] AGTAATCCCCATAATTTAGCTCTCCATTTCATAGTCTTTCCTTGGGTGTGTTAAAAGTGACCAT

[0091] GGTACACTCAGCACGGATGAAATGAAACAGTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATG

[0092] CCCTGTAGTCTCTCTGTATGTTATATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGA

[0093] CCC CACGAAGTGTTGGATATAA GCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAA

[0094] CCATCTTTAACCTAAAAGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGTAGA

[0095] GAGGCACATTTGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGATGAATTATGA

[0096] TTCTTTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGACTATA

[0097] ATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTTTTTCTCACTCATTTTTCAAAACAC

[0098] GCATAAAAATTTAGGAAAGAGAATTGTTTTCTCCTTCCAGCACCTCATAATTTGAACAGACTG

[0099] ATGGTTCCCATTAGTCACATAAAGCTGTAGTCTAGTACAGACGTCCTTAGAACTGGAACCTGG

[0100] CCAGGCTAGGGTGACACTTCTTGTTGGCTGAAATAGTTGAACAGCTT

[0101] The ability of crRNA and tracrRNA pairs to directly degrade linearized plasmid DNA containing cloned fragments of the human HPRT1 gene was tested by recombinant Spy Cas9 (New England BioLabs). CrRNA:tracrRNA was annealed in a duplex buffer (30mM HEPES pH 7.5, 100mM potassium acetate) at a concentration of 150nM. Spy Cas9 (15nM) was pre-incubated with crRNA:tracrRNA at a 1:1 molar ratio at 37°C for 10 minutes. Subsequently, 3nM of the linearized target plasmid was added and incubated at 37°C for 1 hour. The reaction products were separated on a 1% agarose gel at 125V for 1 hour. According to the manufacturer's protocol, the bands were visualized by post-staining with GelRed (Biotium). The gel was imaged on a UV transilluminator, and the results are summarized in Table 1 above.

[0102] The natural wild-type (WT) CRISPR RNA has a 19-20 base protospacer sequence domain (guide, which binds to the target nucleic acid) at the 5' end and a 22 base domain at the 3' end, which binds to tracrRNA. Therefore, the length of WT crRNA is 41-42 bases. The length of WT tracrRNA is 89 bases. We observed that the WT type crRNA:tracrRNA pair supports complete cutting of the target DNA (cr / tracrRNA pair 2D). We also observed that a truncated form of the reagent with a 35-base crRNA (19 base protospacer sequence and 16 base tracrRNA binding domain) paired with a 67-base tracrRNA supports complete cutting of the target RNA (cr / tracrRNA pair 1A). The crRNAtracrRNA binding region is truncated by 6 bases at the 3' end (SEQ ID No.: 1). The tracrRNA is truncated at both ends (SEQID No.: 2). Paired combinations of short crRNA and long tracrRNA showed cleavage, as did long crRNA and short tracrRNA (for 2A). These findings are important because they allow the use of shorter RNA components to guide Cas9 target recognition and cleavage. Compared to longer RNA oligonucleotides, shorter RNA oligonucleotides are cheaper and less difficult to chemically synthesize, require less purification and provide higher yields.

[0103] Some elements of natural crRNA and tracrRNA are missing ( Figure 1) to prepare functional sgRNA ( Figure 2 ). However, the amount of double-stranded nucleic acid that binds crRNA to tracrRNA in sgRNA is limited to 11 base pairs, which is usually too short for duplex formation under biological salt conditions. Due to the unimolecular hairpin structure, the complex is stable in the form of sgRNA, but the sequence is unstable when divided into 2 RNAs. Therefore, it is unclear what length of double-stranded domain is needed to prepare a minimal but functional 2-molecule (2-part) CRISPR complex, or whether the complex will be used to guide Cas9 for target cleavage. This example shows that for a 2-part crRNA:tracrRNA complex with as few as 15 bases, the paired bases allow the function of the 2-part crRNA:tracrRNA complex, which effectively guides the activity of the Cas9 nuclease against a target complementary to the crRNA protospacer sequence domain (SEQ ID No.: 1 and 2).

[0104] Complete chemical modification of crRNA with 2'OMe RNA was not tolerated (pairs 3A and 5A). In addition, complete 2'OMe modification of the 22-base tracrRNA binding domain of crRNA did not support target cleavage (pairs 4A and 6A), and complete 2'OMe modification of the protospacer sequence guide domain did not support cleavage (pair 7A). Complete chemical modification of tracrRNA with 2'OMe RNA was also not tolerated (pairs 1B, 1C and 2B, 2C).

[0105] Importantly, some highly 2'OMe modified forms of both CRISPR RNA species supported cleavage. Pair 1K showed high cleavage activity for tracrRNA with 29 2'OMe residues at the 3' end (SEQ ID No.: 11). Pair 1L showed high cleavage activity for 9 2'OMe residues at the 5' end and 29 2'OMe residues at the 3' end (SEQ ID No.: 13). Thus, 38 of the 67 RNA residues in the short form of tracrRNA can be converted to 2'OMe RNA (57%) in an in vitro cleavage assay without loss of activity.

[0106] Pair 14A shows that 11 bases at the 3' end of crRNA (50% of the 22-base tracrRNA binding domain) can be modified with 2'OMe RNA and support target cleavage (SEQ ID No.: 14). When paired with modified tracrRNA (pair 14L, Seq ID No.: 13 and 14), the modified crRNA remains fully active. Modification of the 11 bases at the 5' end of crRNA (in the guide protospacer sequence domain, bases 2-12) supports target cleavage (pair 15A), and this modification is also functional when paired with modified tracrRNA (pair 15L, SEQ ID No.: 13 and 15). Even when paired with modified tracrRNA (SEQ ID No.: 13 and 16 for 16L), 2'OMe modifications at the 5' and 3' ends of the crRNA can be combined (SEQ ID No.: 16) such that 22 of the 35 residues are modified (63%) and still support cleavage (for 16A).

[0107] The above-mentioned crRNA:tracrRNA pairs all use 2'OMe RNA as a modifier. Additional studies have shown that 2'F modification is also tolerated by Cas9 and is able to cut target DNA. 9A uses crRNA with 2'F modification on all pyrimidine bases (SEQ ID No.: 23), and the design supports complete target cutting. Complete 2'F modification of crRNA also supports complete target cutting (for 10A, SEQ ID No.: 24). The combined use of 2'OMe and 2'F modifications may allow complete modification of crRNA and tracrRNA. The research in this embodiment utilizes in vitro biochemical analysis. In the context of mammalian gene editing, performance may change, where the sequence must work in the nuclear environment.

[0108] Example 2

[0109] This example demonstrates that truncated tracrRNA functions in mammalian cells to guide genome editing by the Spy Cas9 nuclease.

[0110] Functional Cas9 nuclease and RNA trigger (single sgRNA or dual crRNA:tracrRNA pair) must be present in the mammalian cell nucleus for CRISPR genome editing. Transfection of large plasmid vectors expressing Cas9 is ineffective and increases the variability of experimental results. In order to accurately evaluate the effects of changes in the length and chemical composition of crRNA and tracrRNA in mammalian cells in the absence of other variables, a cell line stably expressing Spy Cas9 was constructed.

[0111] A HEK293 cell line with constitutive expression of SpyCas9 (human codon optimization) with stable vector integration and selection under G418 was developed as described below. Human optimized Spy Cas9 was connected to pcDNA3.1 expression vector (Life Technologies) and transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies). Transfected cells were grown for 2 days using neomycin and then placed under selective pressure. After 7 days, cells were inoculated into single colonies using limiting dilution techniques. The Cas9 activity of monoclonal colonies was screened, and clones with the highest expression levels were used for future studies. Single copy integration events of Cas9 were determined using droplet digital PCR (ddPCR). Western blotting using anti-Cas9 antibodies showed low but constant expression of Cas9 protein. This cell line is hereinafter referred to as "HEK-Cas9".

[0112] HEK-Cas9 cell lines were used in subsequent studies. In a reverse transfection mode, the anti-HPRT1 crRNA:tracrRNA complex was mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into HEK-Cas9 cells. Transfection was performed in a 96-well plate format with 40,000 cells per well. RNA was introduced at a final concentration of 30 nM in 0.75 μl of lipid reagent. The cells were incubated at 37 ° C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the target HPRT region (HPRT forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID No.: 28); HPRT reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID No.: 29)). The PCR products were melted and reannealed in NEB buffer 2 (New England BioLabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England BioLabs) at 37°C for 1 hour. The digestion products were visualized on a fragment analyzer (Advanced Analytics). The percentage of cleavage of the targeted DNA was calculated as: average molar concentration of cleavage products / (average molar concentration of cleavage products + molar concentration of uncut bands) × 100.

[0113] TracrRNA (Table 2) was synthesized with deletions at the 5' end, the 3' end, internally, or a combination thereof. The tracrRNA was complexed with an unmodified truncated anti-HPRT1 crRNA SEQ ID No.: 1 (Table 1) having a 19-base protospacer sequence domain targeting HPRT1 at the 5' end and a 16-base tracrRNA binding domain at the 3' end. The paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and treated as described above. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurements of the products using a fragment analyzer. A description of the wild-type Saccharomyces cerevisiae crRNA:tracrRNA complex is provided in Figure 1 (which pairs crRNA SEQ ID No.: 46 with tracrRNA SEQ ID No.: 18). The relative positions of the deletions in the tracrRNA tested in this example are Figure 3 The sequences are shown in sequence alignment format.

[0114] Table 2: Effect of length truncation in tracrRNA on gene editing efficiency of Cas9 endonuclease in mammalian cells.

[0115]

[0116]

[0117] Oligonucleotide sequences are shown as 5'-3'. Lower case = RNA. The length of the RNA oligonucleotide is indicated (in bases). The number of RNA residues removed at the 5' end, 3' end and internal (int) in the truncation study is indicated. The relative functional activity of each substance is indicated by the % cleavage in the T7EI heteroduplex assay.

[0118] This example shows that for gene editing in mammalian cells, tracrRNA can tolerate significant deletions at the 5' and 3' ends and retain full functionality. Deleting 18 bases from the 5' end is well tolerated. Deleting 20 bases from the 5' end results in reduced activity, which may be due to the low binding affinity of crRNA. If Tm enhancement modification is used to stabilize the short duplex forming region, this shortened length or shorter length may be functional. Deleting up to 10 bases from the 3' end is well tolerated. Additional deletions result in loss of activity. Internal deletions that destroy the interval between hairpin elements or hairpin elements are not functional.

[0119] In summary, this embodiment shows that tracrRNA is truncated from 89 bases of wild type (WT, SEQ ID No.: 18) to 67 bases (SEQ ID No.: 2) or truncated to 62 bases (SEQ ID No.: 38) or truncated to 61 bases (SEQ ID No.: 39) to maintain high functional activity. Using this shortened tracrRNA will be cheaper and easier to manufacture by chemical methods compared to WT 89 bases RNA. Some truncated substances (SEQ ID No.: 2, SEQ ID No.: 38 and SEQ ID No.: 39) show functional activity increased than 89 alkaline WT tracrRNA. Therefore, the shortened tracrRNA of the present invention, in addition to being lower in cost and easier to manufacture by chemical methods, also shows improved activity.

[0120] Example 3

[0121] Examples 1 and 2 show that crRNA:tracrRNA complexes that are 42 and 89 bases shorter than WT, respectively, can show higher functional activity in mammalian gene editing. This example shows further optimization of the length of these RNA species.

[0122] A series of crRNAs and tracrRNAs were synthesized (Table 3) with different lengths as shown. Truncations were made at the 3' end of the crRNA, the 5' end of the tracrRNA, and / or the 3' end of the tracrRNA. The crRNA and tracrRNA were paired as shown in Table 3. The crRNAs all used a 20-base protospacer sequence domain targeting HPRT1 at the 5' end and a variable length 3' end (tracrRNA binding domain). The alignment of the crRNA and tracrRNA sequences studied in this example is Figure 4 to illustrate the truncation positions relative to each functional domain.

[0123] Paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and processed as described in Example 2. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurement of the product using a fragment analyzer. The results are shown in Table 3. The relative positions of the deletions are shown in sequence alignment format. Figure 4 middle.

[0124] Table 3: Effect of length truncation in crRNA and tracrRNA on gene editing efficiency of Cas9 endonuclease in mammalian cells.

[0125]

[0126]

[0127] Oligonucleotide sequences are shown as 5'-3'. Lowercase = RNA. The length of the RNA oligonucleotide is indicated (in bases). The relative functional activity of each crRNA:tracrRNA pair is indicated by the % cleavage in the T7EI heteroduplex assay.

[0128] All compounds studied directed CRISPR / Cas editing at the HPRT1 locus in HEK-Cas9 cells. The efficiency varied widely between 6% and 57%. The most effective crRNA+tracrRNA combination was a 36-base crRNA (SEQ ID No.: 48) with a 67-mer tracrRNA (SEQ ID No.: 2). A schematic diagram of the truncated, optimized crRNA:tracrRNA complex is shown in Figure 5 In this case, the tracrRNA binding domain of crRNA is truncated from the WT 22 base sequence (3' end) to 16 bases. This hybridizes with the crRNA binding domain at the 5' end of tracrRNA. The tracrRNA is truncated by 18 bases at the 5' end and 4 bases at the 3' end, producing an active 67-base product. For this pair, a blunt end is formed when the 3' end of crRNA hybridizes with the 5' end of tracrRNA. Other forms also show high activity, including a 42-base (WT) crRNA (SEQ ID No.: 46) paired with a 70-base tracrRNA (SEQ ID No.: 51).

[0129] The length of the shortest crRNA tested is 34 bases (SEQ ID No.: 49), and generally shows lower activity compared to longer variants. The shorter duplex domain formed between the variant and tracrRNA has a reduced binding affinity (Tm) compared to 36 base crRNA variants, and the 34 base complexes are less stable at 37 ° C. Chemical modifications such as 2'OMe RNA, 2'F RNA or LNA residues that increase binding affinity (Tm) will increase the stability of the short duplex domain, and will result in improved activity, thereby allowing the use of very short crRNAs of this design. Extensive use of Tm enhancement modifications will allow the use of shorter tracrRNA binding domains in crRNA, such as 13 bases, or 12 bases, or 11 bases, or 10 bases, or 9 bases, or 8 bases or shorter, depending on the type and quantity of the modified residues used.

[0130] Example 4

[0131] Examples 1, 2 and 3 show that crRNA:tracrRNA complexes shorter than the WT length of 42 and 89 bases, respectively, can show higher functional activity in mammalian gene editing. In these examples, all truncations were performed in the universal domain of RNA. This example tests the effect of truncation on the target-specific pre-spacer sequence domain of the guide crRNA.

[0132] A series of crRNAs were synthesized (Table 4) with protospacer sequence domains of 20, 19, 18 or 17 bases in length as shown. The 5' end of the crRNA was truncated using a 16-mer universal tracrRNA binding sequence at the 3' end. The crRNA was paired with an unmodified 67-mer tracrRNA (SEQ ID No.: 2). The crRNAs targeted 4 different sites in the same exon of the human HPRT1 gene.

[0133] Paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and processed as described in Example 2. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurement of the product using a fragment analyzer. The results are shown in Table 4.

[0134] Table 4: Effect of length truncation in the 5'-protospacer sequence domain of crRNA on the gene editing efficiency of Cas9 endonuclease in mammalian cells.

[0135]

[0136]

[0137] Oligonucleotide sequences are shown 5'-3'. Lower case = RNA. Target specific protospacer sequence domains are underlined and length (bases) indicated. The relative functional activity of each substance is indicated by % cleavage in the T7EI heteroduplex assay.

[0138] Of the four sites studied, one (site 38087) showed high activity for all four crRNAs, with no change in activity as the protospacer sequence domain shortened. Site 38285 showed similar efficacy for 20- and 19-base protospacer sequence crRNAs (SEQ ID No.: 48 and 1), slightly reduced for the 18-base form (SEQ ID No.: 54), and greatly reduced for the 17-base form (SEQ ID No.: 55). Site 38094 showed similar efficacy for 20- and 19-base protospacer sequence crRNAs (SEQ ID No.: 64 and 65), moderately reduced for the 18-base form (SEQ ID No.: 66), and inactive for the 17-base form (SEQ ID No.: 67). Position 38358 showed good activity for the 20-base form (SEQ ID No.: 60), lower activity for the 19-base form (SEQ ID No.: 61), even lower activity for the 18-base form (SEQ ID No.: 62), and no activity for the 17-base form (SEQ ID No.: 63).

[0139] Compared with the wild-type 20 base domain, the use of a shortened 17 base pre-spacer sequence guide domain can reduce the occurrence of undesirable off-target events (Fu et al., Nature: Biotechnology, 32: 279, 2014). We observed that the on-target efficacy changed in a sequence context-specific manner, and the 20 base and 19 base pre-spacer sequence guide domains were generally effective, but the activity began to decrease when the 18 base pre-spacer sequence domain was used, and the activity was significantly reduced when the 17 base pre-spacer sequence domain was used. Therefore, in order to maintain the desired on-target efficiency, 20 and 19 base target-specific pre-spacer sequence guide domains are used in this article. The significant truncation of the pre-spacer sequence guide domain reduces the on-target cleavage of the DNA target by the Cas9 endonuclease in many cases. The use of chemical modifications that enhance Tm (increasing the binding affinity of the crRNA protospacer target-specific domain to the target DNA sequence) can allow the use of shorter sequences, such that a 17-base protospacer guide domain can show similar on-target efficacy as an unmodified 20-base protospacer guide domain.

[0140] Example 5

[0141] This example shows that truncated crRNA:tracrRNA complexes show improved gene editing activity at multiple sites. Previous examples studied the efficacy of truncated RNA at a single site in the human HRPT1 gene as a trigger for CRISPR gene editing in mammalian cells. There may be site / sequence-specific effects. This example shows that the truncated species of the present invention have improved performance at 12 sites in the exons of the human HPRT1 gene.

[0142] A series of crRNAs (Table 5) were synthesized with a 20-base protospacer sequence domain specific for 12 sites in the human HPRT1 gene and a 16-mer universal tracrRNA binding sequence at the 3' end. The crRNAs were paired with unmodified 67-mer tracrRNA (SEQ ID No.: 2). The same 12 sites were studied using WT length crRNA:tracrRNA complexes, where the crRNA contained a 20-base protospacer sequence guide domain paired with a 22-mer universal tracrRNA binding sequence at the 3' end, which was complexed with a WT 89-mer tracrRNA (SEQ ID No.: 18).

[0143] Paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and treated as described in Example 2. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurement of the product using a fragment analyzer. The results are shown in Table 5.

[0144] Table 5: Effect of length truncation in crRNA and tracrRNA on gene editing efficiency of Cas9 endonuclease in mammalian cells.

[0145]

[0146]

[0147] Oligonucleotide sequences are shown as 5'-3'. Lowercase = RNA. The length of the RNA oligonucleotide is indicated (in bases). The relative functional activity of each crRNA:tracrRNA pair is indicated by the % cleavage in the T7EI heteroduplex assay.

[0148] The relative efficiency of CRISPR-mediated gene editing in HEK-Cas9 cells varies with sequence background. However, in all cases, shorter optimized RNA guides (36-mer crRNA and 67-mer tracrRNA) show higher efficiency compared to WT RNA (42-mer crRNA and 89-mer tracrRNA). Using the shortened optimized guide RNA of the present invention to improve the Cas9 cutting of targeted DNA relative to WT RNA, thereby increasing the gene editing rate.

[0149] Example 6

[0150] Example 1 describes chemical modification patterns that work with Cas9 in an in vitro biochemical cleavage assay of target DNA. This example demonstrates that chemically modified tracrRNA works in mammalian cells to guide genome editing by Spy Cas9 nuclease. The optimal modification pattern differs between in vitro and in vivo use.

[0151] A series of tracrRNAs (Table 6) were synthesized with various chemical modifications, including: ribose-modified 2'OMe RNA and LNA; terminal modification groups propanediol spacer and naphthyl-azo modifier (N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-aniline, or "ZEN"); and selected internucleotide bonds with thiophosphate modifications. For the structure of naphthyl-azo modifiers and the use of naphthyl-azo modifiers and propanediol modifiers as end groups to prevent exonuclease attack, see: Lennox et al., Molecular Therapy Nucleic Acids, 2:e117 2013. The tracrRNAs listed in Table 6 were complexed with an unmodified truncated anti-HPRT1 crRNA SEQ ID No.: 1 (Table 1) having a 19-base protospacer sequence domain targeting HPRT1 at the 5' end and a 16-base tracrRNA binding domain at the 3' end. Paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and treated as described above. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurement of the products using a fragment analyzer.

[0152] Table 6: Optimization of tracrRNA oligonucleotide modification patterns in mammalian cells.

[0153]

[0154]

[0155]

[0156] Oligonucleotide sequences are shown 5'-3'. Uppercase = DNA; Lowercase = RNA; Underline = 2'-O-methyl RNA; Italics = 2'-fluoro RNA; +a, +c, +t, +g = LNA; C3 = C3 spacer (propylene glycol modifier); * = phosphorothioate internucleotide linkage; ZEN = naphthyl-azo modifier; Inv-dT = inverted dT. The relative functional activity of each substance is indicated by the % cleavage in the T7EI heteroduplex assay.

[0157] Due to the presence of exonucleases and endonucleases that degrade unmodified oligonucleotides, modification is usually necessary for synthetic nucleic acids to fully function in the intracellular environment. A wide variety of modifications that confer nuclease resistance to oligonucleotides have been described. The precise combination and order of modifications used that fully function for a given application can vary with the nature of the protein interactions required for the sequence background and biological function. A large number of previous studies have been conducted on the chemical modification of antisense oligonucleotides (which interact with RNase H1) and siRNA (which interacts with DICER, AGO2 and other proteins). It is expected that chemical modification will improve the function of CRISPR crRNA:tracrRNA complexes. However, it is impossible to predict what kind of modification and / or modification pattern will be compatible with the functional compound of synthetic RNA and Cas9. The present invention defines the minimum, medium and large amounts of chemical modification patterns of tracrRNA, which maintain a high level of function to guide Cas9-mediated gene editing in mammalian cells.

[0158] The results in Table 6 show that a large number of modifications are tolerated throughout the 5' and 3' terminal domains of tracrRNA. Modifications of the internal domains of tracrRNA showed reduced activity, which may be due to structural changes in the folded RNA and / or blocking of protein contact points by 2'-OH of key RNA residues through 2'OMe modification. For example, compound SEQ ID No.: 100 has 39 / 67 residues (58%) modified with 2'OMe RNA and remains fully active compared to the unmodified sequence. SEQ ID No.: 134 has 46 / 67 residues (69%) modified with 2'OMe RNA and remains nearly fully active compared to the unmodified sequence ( Figure 6). SEQ ID No.: 134 is a truncated 67-mer variant of tracrRNA. Using SEQ ID No.: 134 as a model, modification of 11 consecutive residues in the 5' domain with 2'OMe RNA was tolerated without loss of activity. Modification of 35 consecutive residues in the 3' domain with 2'OMe RNA was tolerated without loss of activity. It is noteworthy that the two hairpin structures present in the 3' domain are essential for function, as the absence of either of these features results in a loss of activity (Examples 2, Figure 3 ), but both domains can be fully modified with 2'OMe RNA without compromising function. Note that SEQ ID No.: 134 and 100 have phosphorothioate (PS) modified internucleotide bonds at both the 5' and 3' ends, which provide additional protection from exonuclease attack.

[0159] Specific residues were identified that, when modified, resulted in a substantial reduction or complete loss of activity. Using the 67 base tracrRNA (e.g., SEQ ID No.: 134) as a reference, starting from the 5' end of the sequence, substitution of the native RNA with 2'OMe RNA at residues U12, A15, G26, U27, G30, U31, and U32 resulted in a substantial loss of activity ( Figure 6 ). In addition, specific residues were identified that resulted in less but significantly reduced activity when modified. Using the 67 base tracrRNA (e.g., SEQ ID No.: 134) as a reference, starting from the 5' end of the sequence, replacing the native RNA with 2'OMe RNA at residues U13, U18, C23, U24, and C28 resulted in reduced activity ( Figure 6 ). This study was performed using 2'OMe RNA. Other modifications such as 2'F, LNA, DNA, etc. at these positions may be better tolerated. The central 21 residue domain of the unmodified RNA in SEQ ID No.: 134 was fully modified (SEQ ID No.: 141) or partially modified (SEQ ID No.: 142 and 143) with 2'-F RNA. These variants are not functional. The central 21 residue domain of the unmodified RNA in SEQ ID No.: 134 was fully modified (SEQ ID No.: 138) or partially modified (SEQ ID No.: 139 and 140) with DNA. These variants are not functional. Modification of isolated residues in this domain may work, but continuous large blocks of modifications in this domain reduce the activity of tracrRNA.

[0160] To further investigate which individual residues can be modified using 2'OMe RNA within the central domain of tracrRNA, a single base modification 2'OMe RNA 'walk' (SEQ ID No.: 144-162) was performed. In this series, modifications of residues A14, A19, A20, G21, G22, A25, and C29 showed no loss of activity and are candidates for modification.

[0161] Antisense oligonucleotides are usually made using complete PS modification, where each internucleotide bond is phosphorothioate modified. This large level of modification is possible because the protein effector molecule RNase H1 (which mediates ASO-guided mRNA degradation) tolerates PS modification in ASO when a functional substrate / enzyme complex is formed. On the other hand, siRNA cannot tolerate complete PS modification; a large amount of PS modification destroys the effective interaction with the effector protein AGO2 (which mediates siRNA-guided mRNA degradation). A large amount of PS modification of tracrRNA in the internal RNA loop destroys the functional interaction with Cas9 (Seq ID No.: 133; 29 PS modifications). Limited PS end modification can be performed without loss of activity (SEQ ID No.: 98 and 101; 2-3 PP bonds at each end). Less PS modification can be tolerated in the central domain. In particular, RNAse cleavage mapping (where incubation of tracrRNA in a series of serum or cell extract dilutions is used to find the most sensitive sites to RNAse attack) can be used to identify critical sites, where only one or a few PS modifications of bonds can stabilize RNA without destroying function.

[0162] There are applications where PS modification contributes to chemical toxicity. In such cases, other methods need to be used to block exonuclease attack. Options include terminal modifiers such as reversed dT or abasic groups such as dSpacer, C3 spacer (propylene glycol), ZEN (naphthyl-azo modifier), etc. The placement of such terminal modification groups can eliminate the need for a bond between the terminal PS nucleotides.

[0163] Example 7

[0164] Example 1 describes chemical modification patterns that work with Cas9 in an in vitro biochemical cleavage assay of target DNA. This example demonstrates that chemically modified crRNA works in mammalian cells to guide Spy Cas9 nuclease for genome editing. The optimal modification pattern differs between in vitro and in vivo use.

[0165] A series of crRNAs (Table 7) were synthesized with a variety of chemical modifications, including: ribose-modified 2'OMe RNA, 2'F, and LNA; terminal modification groups propanediol spacers and naphthyl-azo modifiers (N,N-diethyl-4-(4-nitronaphthalene-1-ylazo)-aniline or "ZEN"), and reverse dT residues; and selected internucleotide bonds with thiophosphate modifications. For the structure of naphthyl-azo modifiers and the use of naphthyl-azo modifiers and propanediol modifiers as end groups to prevent exonuclease attack, see: Lennox et al., Molecular Therapy Nucleic Acids, 2:e117 2013. The crRNA has a 19-base protospacer sequence domain targeting HPRT1 at the 5' end (SEQ ID No.: 1, 9, 10, 14-16, 22-24, 163-173) or a 20-base protospacer sequence domain targeting the same site (SEQ ID No.: 48, 174-237) and a 16-base tracrRNA binding domain at the 3' end. The crRNAs listed in Table 7 were compounded with unmodified truncated (67 bases) tracrRNA SEQ ID No.: 2 (Table 1) or chemically modified truncated (67 bases) tracrRNA SEQ ID No.: 100 (Table 6). Using two tracrRNAs can determine whether the function of the chemically modified crRNA will change when paired with the modified tracrRNA. The paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and treated as described above. Relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with quantitative measurement of the products using a fragment analyzer.

[0166] Table 7: Optimization of crRNA oligonucleotide modification patterns in mammalian cells.

[0167]

[0168]

[0169]

[0170] Oligonucleotide sequences are shown from 5' to 3'. Uppercase = DNA; Lowercase = RNA; Underline = 2'-O-methyl RNA; Italics = 2'-fluoro RNA; +a, +c, +t, +g = LNA; C3 = C3 spacer (propylene glycol modifier); * = phosphorothioate internucleotide bond; ZEN = naphthyl-azo modifier; InvT = inverted dT. The relative functional activity of each substance is indicated by the % cleavage in the T7EI heteroduplex assay when the indicated crRNA is paired with the indicated tracrRNA. ND = not determined.

[0171] Due to the presence of exonucleases and endonucleases that degrade unmodified oligonucleotides, synthetic nucleic acids generally require some chemical modification to fully function in the intracellular environment. A wide variety of modifications that confer nuclease resistance to oligonucleotides have been described. The exact combination and order of modifications used that fully function for a given application can vary with the nature of the protein interactions required for the sequence context and biological function. A large number of previous studies have been conducted on chemical modifications of antisense oligonucleotides (which interact with RNase H1) and siRNA (which interact with DICER, AGO2 and other proteins). Chemical modifications are expected to improve the function of CRISPR crRNA:tracrRNA complexes. However, it is impossible to predict what kind of modification and / or modification pattern will be compatible with the combination of RNA and Cas9 in a functional manner. The present invention defines the minimum, medium and large chemical modification patterns of crRNA for guiding Cas9-mediated gene editing to maintain high levels of function in mammalian cells. The study in Example 7 was conducted by targeting a single site in the human HPRT1 gene. It should be noted that the modification pattern of the 20 base 5' terminal protospacer sequence guide domain of the well-performing crRNA may vary with the sequence context. However, when targeting different sites, when the sequence of the adjacent protospacer sequence domain changes, it is likely to affect the modification pattern of the well-behaved 3' end tracrRNA binding domain as defined herein, so the 3' domain modification pattern shown here will be "universal".

[0172] The results in Table 7 show that a large number of modifications are tolerated throughout the 5' and 3' ends of crRNA. Modifications at certain selected positions within the internal domain of crRNA result in reduced activity or complete blockage of activity, which may be due to structural changes in folded RNA and / or modification by 2'OMe so that protein contact points are blocked by 2'-OH of key RNA residues. For example, compound SEQID No.: 204 has 21 / 36 residues (58%) modified with 2'OMe RNA and remains fully active compared to the unmodified sequence. Compound SEQ ID No.: 239 has 30 / 36 residues (83%) modified with 2'OMe RNA and remains fully active compared to the unmodified sequence. Both compounds also have 3 phosphorothioate (PS)-modified internucleotide bonds at the 5' and 3' ends, which provide additional protection for exonuclease attacks. In contrast, SEQ ID No.: 165 has only 4 / 36 residues (11%) modified with 2'OMe RNA, but completely loses activity.

[0173] Large sequences are tolerant to 2'OMe modifications at the 5' and 3' ends of crRNA, but modifications of certain residues in the central part of the molecule lead to inactivation. In order to further study which individual residues can be modified using 2'OMeRNA in the central domain of crRNA, single base modification 2'OMe RNA 'walking' (SEQ ID No.: 272-286) was performed. Specific residues (positions within crRNA) that cause a large reduction in activity or complete loss were identified. Using crRNA SEQ ID No.: 239 of 36 genes as a model and numbering from the 5' end of the sequence, the natural RNA that replaces residues U15 and U16 with 2'OMe RNA results in a significant loss of activity, and the natural RNA that replaces residue U19 results in a moderate loss of activity ( Figure 7 These three sites are located within the target-specific protospacer sequence guide domain and therefore vary in sequence with the target (residues 15, 16, and 19, Figure 7 ). In certain sequence contexts, these sites may tolerate modification. In the universal tracrRNA binding domain (residues 21-36), replacing the native RNA of residues U22, U23, and U24 with 2'OMe RNA resulted in a large loss of activity. Given that this domain does not change with sequence context, the modification tolerance of these sites may not change with target sequence changes. The sequence-specific effects of modifications in the 20-base target-specific protospacer sequence guide domain are studied in more detail in Example 10.

[0174] Antisense oligonucleotides with complete PS modification are usually prepared, in which the bond between each nucleotide is phosphorothioate modified. This large amount of modification level is possible because the protein effector molecule RNase H1 tolerates PS modification in ASO when forming a functional substrate / enzyme complex. On the other hand, siRNA cannot tolerate complete PS modification; a large amount of PS modification destroys the productive interaction with the effector protein AGO2. Limited PS end modification of crRNA can be performed without loss of activity (SEQ ID No.: 177, 178, 239, etc., with 3 PS bonds at each end). End modification is required because it increases additional protection against exonuclease attack. PS modification of selected internal sites can also be tolerated and may provide additional protection for endonuclease attack. Using SEQ ID No.: 264 as the base modification pattern, PS modifications were made to internal bonds in the tracrRNA binding domain (SEQ ID No.: 265), in the 3' end (seed region) of the protospacer sequence guide domain (SEQ ID No.: 266), or in both regions (SEQ ID No.: 267). Increasing levels of PS modification resulted in reduced functional activity, with SEQ ID No.: 267 having approximately 50% of the activity of the less modified SEQ ID No.: 264 variant. 21 of the 35 internucleotide bonds in SEQ ID No.: 267 were modified and would be stable to nuclease exposure. In situations where exposure to a high nuclease environment is desired (e.g., direct IV administration for research or therapeutic indications), this highly modified variant may actually show higher activity than the less modified variant (which would degrade more quickly).

[0175] There are experimental settings in which PS modifications promote chemical toxicity. In this case, other methods need to be used to block exonuclease attacks. The crRNA can have a C3 isolation group (propylene glycol modifier) ​​or ZEN (naphthyl-azo modifier) ​​placed on either or both of the 5' end and the 3' end to block exonuclease attacks, thereby avoiding the need for PS modification. This strategy can be used to eliminate PS end-capping modifications (see SEQ ID No.: 179-186). This strategy can be used to reduce the PS content of more highly modified crRNA variants. SEQ ID No.: 271 has an internal protospacer sequence domain and a PS-modified racrRNA binding domain with the same pattern as SEQ ID No.: 267, but only 15 PS internucleotide bonds (instead of 21) are used, and improved activity is shown. Therefore, the combination of non-base end-capping and internal PS modification can be used to improve nuclease stability while maintaining high activity.

[0176] Example 8

[0177] The following examples demonstrate the improved efficacy of the modified CRISPR crRNA and tracrRNA of the present invention. Example 2-7 uses crRNA:tracrRNA complexes to be transfected into human HEK-Cas9 cells at a concentration of 30nM. Experimental tests have previously shown that this dose represents the upper limit of the dose-response curve, so that the use of higher doses of RNA cannot improve gene editing efficiency, but the use of lower doses results in lower gene editing efficiency. These measurements are performed using unmodified RNA. This example re-studies the dose response of the new optimized chemically modified RNA of the present invention compared to unmodified RNA, and demonstrates that chemical modification (ie, nuclease stabilization) results in more effective compounds that can be used at lower doses.

[0178] Example 5 shows that the truncated guide RNA of the present invention outperforms WT RNA at 12 sites in the human HPRT1 gene. Four of these sites (38087, 38231, 38133, and 38285) were selected for comparison of unmodified RNA with modified RNA in this example. Unmodified crRNA was paired with unmodified tracrRNA (SEQ ID No.: 2) at a 1: 1 molar ratio. Unmodified crRNA was paired with modified tracrRNA (SEQ ID No.: 100) at a 1: 1 molar ratio. Modified crRNA was paired with modified tracrRNA (SEQ ID No.: 100) at a 1: 1 molar ratio. The sequences are shown in Table 8. RNA was transfected into HEK-Cas9 cells at 30nM, 10nM, and 3nM concentrations as described above. Cells were incubated at 37°C for 48 hours before DNA was processed and investigated for evidence of gene editing activity comparing the cleavage rate at the HPRT1 locus in the T7EI mismatch endonuclease assay with quantitative measurement of the products using a fragment analyzer as previously described. The results are shown in Table 8.

[0179] Table 8: Increased efficacy of modified versus unmodified crRNA:tracrRNA complexes in directing Cas9-mediated gene editing in mammalian cells.

[0180]

[0181]

[0182] Oligonucleotide sequences are shown 5'-3'. Lowercase = RNA; Underlined = 2'-O-methyl RNA; * = phosphorothioate internucleotide bonds. Unmodified crRNA = Un-cr. Unmodified tracrRNA = Un-tr. Modified crRNA = Mod-cr. Modified tracrRNA = Mod-tr. For each dose studied, the relative functional activity of each species is indicated by the % cleavage in the T7EI heteroduplex assay.

[0183] In general, when RNA is transfected with high doses (wherein RNA is present in excess), the modification of crRNA and tracrRNA has little effect on gene editing efficiency. At lower doses, improved reagents show improved efficacy, and in some cases show significantly improved efficacy. The degree of improvement varies with the site. Very effective site 38087 shows high-efficiency gene editing of all crRNA / tracrRNA variants tested at 30nM and 10nM dosages, but at 3nM, modified tracrRNA (with any crRNA) is used to show improved activity. Low-efficiency sites, such as 38231, also show improved gene editing efficiency even at the highest dose (30nM) tested using modified RNA. Single tracrRNA modification shows benefits, but when crRNA and tracrRNA are modified, maximum benefits are achieved. Figure 8 A schematic diagram showing an effective modified crRNA (SEQ ID No.: 178) specific to HPRT1 site 38285 paired with a modified tracrRNA (SEQ ID No.: 100). Fig. 9 A schematic diagram of a more highly modified pair of a highly functional crRNA (SEQ ID No.: 239) also specific for HPRT1 site 38285 paired with a modified tracrRNA (SEQ ID No.: 134) is shown.

[0184] In the present embodiment, crRNA:tracrRNA complex is transfected into HEK-Cas9 cells, wherein Cas9 protein is constitutively expressed. Therefore, the transfected RNA can immediately bind to Cas9 protein, minimizing the risk of nuclease degradation in the cytoplasm. It can be expected that in the case where the transfected RNA must remain active (survive) after being exposed to cell nucleases and simultaneously produce Cas9 protein, the benefits of the chemical modification of crRNA and / or tracrRNA will be greater, such as when using a scheme where Cas9 mRNA or Cas9 expression vector is co-transfected with a targeting RNA so that Cas9 is not yet expressed in cells. The benefits of using highly modified RNA will be the greatest for in vivo applications (such as medical therapeutic agents), wherein RNA may be exposed to two nucleases present in serum (after IV administration) and cell cytoplasmic nucleases.

[0185] Example 9

[0186] Examples 2-8 show that truncated and / or chemically modified CRISPR crRNA and / or tracrRNA trigger the activity of Cas9-mediated genome editing in mammalian cells constitutively expressing Cas9. This example shows that the truncated modified RNA composition of the present invention can bind to the Cas9 protein, and the complex can be transfected into human cells, and in addition, transfection of the ribonucleoprotein (RNP) complex is sufficient to trigger efficient genome editing.

[0187] In this example, a reagent specific for human HPRT1 site 38285 was used. Unmodified crRNA was paired with unmodified tracrRNA at a 1:1 molar ratio. Unmodified crRNA was paired with modified tracrRNA at a 1:1 molar ratio. Modified crRNA was paired with modified tracrRNA at a 1:1 molar ratio. The sequences are shown in Table 9. RNA was transfected into unmodified HEK293 cells as described above, except that an increased amount of RNAiMAX lipid transfection reagent (1.2 μL, an increase from the 0.75 μL used per 100 μL transfection for only 30 nM RNA transfection in HEK-Cas9 cells in a 96-well format) was used, and a 1:1 complex of Cas9 protein (Caribou Biosciences) and crRNA:tracrRNA was used at a concentration of 10 nM. Cells were incubated at 37°C for 48 hours, and then DNA was processed and investigated for evidence of gene editing activity, comparing the cleavage rate at the HPRT1 locus in the T7EI mismatch endonuclease assay with quantitative measurement of the product using a fragment analyzer as previously described. The results are shown in Table 9. Table 9: Increased efficacy of modified vs. unmodified crRNA:tracrRNA complexes to direct Cas9-mediated gene editing in mammalian cells.

[0188]

[0189] Oligonucleotide sequences are shown 5'-3'. Lowercase = RNA; Underline = 2'-O-methyl RNA; * = phosphorothioate internucleotide bonds. Unmodified crRNA = Un-cr. Unmodified tracrRNA = Un-tr. Modified crRNA = Mod-cr. Modified tracrRNA = Mod-tr. For each dose studied, the relative functional activity of each complex is indicated by the % cleavage in the T7EI heteroduplex assay.

[0190] All 3 kinds of CRISPR RNA complexes perform well in the RNP transfection scheme of mammalian genome editing. In the RNP scheme, unmodified crRNA+ unmodified tracrRNA pairs (SEQ ID No.: 48 and 2) and unmodified crRNA+ modified tracrRNA pairs (SEQ ID No.: 48 and 100) are 2.5 times better than the performance in the HEK-Cas9 scheme at 10nM dosage, which is consistent with the less modified RNA that suffers degradation between the transfection of Cas9 protein and the final compound in the cytoplasm or nucleus. Therefore, unmodified RNA requires a higher dosage, and in some cases, unmodified RNA is likely to be unable to guide any genome editing activity. On the other hand, the modified crRNA+ modified tracrRNA (SEQ ID NO.: 178 and 100) has high efficiency in both schemes.

[0191] The modified, truncated CRISPR RNAs of the present invention work well with the direct Cas9 RNP transfection method.

[0192] Example 10

[0193] The chemical modification optimization studies carried out in Examples 6 and 7 have studied the activity of crRNAs with various modification patterns paired with tracrRNAs with various modification patterns. tracrRNA is universal and uses the same sequence at all target sites. It is expected that the performance of various modification patterns of tracrRNA will be similar between different target sites. However, the sequence of crRNA changes between different target sites. In the optimized form tested in Examples 7 and 8, the 5'-20 bases of crRNA have target specificity (i.e., "protospacer sequence domain") and 3'-16 bases are universal (i.e., "tracrRNA binding domain"). Like tracrRNA, it is expected that the performance of various modification patterns in the universal 16 base 3' domains of crRNA will be similar at all target sites. However, it is possible that the performance of different modification patterns may be affected by the sequence background present in the 5'-20 base target specific domains.

[0194] It is well established that the effective modification patterns of small interfering RNA (siRNA) are affected by the sequence context (Behlke, Oligonucleotides, 18: 305-320, 2008). For siRNA, certain "limited modification" patterns can be applied to all sites, while for "extensive modification", it is impossible to predict which pattern will be functional for a given sequence, and empirical testing is necessary. This example studies the effect of sequence context on crRNA, testing different modification patterns within the 5'-20 base target-specific domain at different sites.

[0195] The modification studies in Examples 6 and 7 used a single crRNA PAM site in the human HPRT1 gene. This study examined 12 sites in the human HPRT1 gene, including previously examined sites, which compared the functional performance of different modification patterns and established a single modification pattern that can be used with good results at all sites. For other studies on these 12 sites, see Example 5.

[0196] A series of crRNAs (Table 10) were synthesized with a 20-base protospacer sequence domain specific for 12 sites in the human HPRT1 gene and a 16-mer universal tracrRNA binding sequence at the 3' end. The crRNAs were prepared using a variety of chemical modifications, including: ribose-modified 2'OMe RNA, terminal modification groups propanediol spacer and naphthyl-azo modifier (N,N-diethyl-4-(4-nitronaphthalene-1-ylazo)-aniline or "ZEN"), reversed dT residues; and selected internucleotide bonds with phosphorothioate modifications. Schematic diagrams of the different modification patterns used are shown in Fig.10 middle.

[0197] The crRNA was paired with a highly modified 67-mer tracrRNA (SEQ ID No.: 100). The paired crRNA:tracrRNA RNA oligonucleotides were transfected into HEK-Cas9 cells and processed as described in Example 2. The relative gene editing activity was assessed by comparing the cleavage rate of the HPRT1 gene obtained using the T7EI mismatch endonuclease cleavage assay with the quantitative measurement of the product using a fragment analyzer. The results are shown in Tables 10 and Fig.11 middle.

[0198] Table 10: Optimization of crRNA oligonucleotide modification patterns at 12 target sites in mammalian cells.

[0199]

[0200]

[0201]

[0202] Oligonucleotide sequences are shown 5'-3'. Lowercase = RNA; Underline = 2'-O-methyl RNA; C3 = C3 spacer (propylene glycol modifier); * = phosphorothioate internucleotide bond; ZEN = naphthyl-azo modifier. The relative functional activity of each substance is indicated by the % cleavage in the T7EI heteroduplex assay when the indicated crRNA is paired with the indicated tracrRNA at each of the 12 sites in human HRPT1.

[0203] Modified crRNA adopts fixed modification mode in 16 bases 3' terminal domains, which is universal and combined with tracrRNA. Different modification modes (i.e., sequence varies with target site) are tested / compared in target-specific 5' terminal domains. The test group includes variants with 0, 3, 4, 6, 8, 10, 12, 13 or 14 continuous 2'OMe RNA residues starting at 5' end and advancing toward 3' end. The modification mode avoids the position (embodiment 7) that has previously been proven to reduce crRNA functional performance. The use of only non-base modifier end groups (C3 spacers or ZEN) (without additional modification) is also tested. When the functional activity of all 12 sites is compared in the investigation, all sites tested show complete activity when the 0-10 RNA residues at the 5' end are replaced by 2'OMe RNA residues. Only one of the 12 sites showed a slight decrease in activity when residue 12 was modified, but 3 / 12 sites showed decreased activity when residue 13 was modified, and 4 / 12 sites showed decreased activity when residue 14 was modified. The terminal modifiers (C3, ZEN) showed full activity at all sites.

[0204] The highest levels of crRNA modifications showing full activity at all sites tested included modification patterns 6 and 7 ( Fig.10 ). This represents that 61% and 67% of the bases in the crRNA are modified with 2'OMe RNA, respectively.

[0205] n * n * n * nnnnnnn nnnnnnnnnnguuu uagagcuau * g * c * u Modification Mode 6

[0206] (SEQ ID NO.:434)

[0207] n * n * n * nnnnnnnnn nnnnnnnnguuu uagagcuau * g * c * u Modification Mode 7

[0208] (SEQ ID NO.:435)

[0209] The data in this example also show that individual sites can be modified at a higher level and retain potency. For example, 8 of the 12 sites studied using modification pattern 8 showed full activity, with 72% of the residues modified. In addition, Example 7 shows that the crRNA targeting site 38285 in HPRT1 (SEQ ID No.: 239) is fully active, and 30 / 36 residues are modified (83%, with only 6 unmodified RNA residues remaining). Base modification patterns such as modification pattern 6 or modification pattern 7 can be used as a starting point for research to empirically determine the extent to which a specific sequence can be modified before loss of activity. Fig.12 A schematic diagram showing the pairing of modification pattern 6crRNA with highly modified tracrRNA (SEQ ID No.: 134) is shown.

[0210] Embodiment 11

[0211] The examples herein use the Cas9 endonuclease from Streptococcus pyogenes. The natural amino acid sequence of S.py.Cas9 (SpyCas9) is shown below (SEQ ID No.: 407).

[0212] The native Cas9 DNA sequence is codon optimized for expression in E. coli and has elements added for mammalian nuclear localization (nuclease localization signal) and to aid protein purification (His tag). The final amino acid sequence of the recombinant protein is shown (SEQ ID No.: 408). The DNA sequence used to express the recombinant protein in E. coli is shown (SEQ ID No.: 409).

[0213] The native Cas9 DNA sequence is codon-optimized for expression in human cells and has elements added for antibody recognition (V5 epitope) and mammalian nuclear localization (nuclease localization signal, NLS). The final amino acid sequence is shown (SEQ ID No.: 410), and the DNA sequence is as follows (SEQ ID No 411).

[0214] The native S.py Cas9 DNA sequence is codon optimized for expression in human cells and assembled into a T7 RNA polymerase expression cassette (SEQ ID No.: 412). The sequence contains a T7 RNA polymerase promoter, a V5 epitope tag, a nuclear localization signal, a codon-optimized Cas9 sequence, a second nuclear localization signal, and a BGH (bovine growth hormone) gene 3'-UTR element with a polyadenylation signal. The sequence of the mRNA prepared by the expression cassette is shown (SEQ ID No.: 413).

[0215] S.py.Cas9 amino acid sequence (SEQ ID No.: 407).

[0216] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRT

[0217] ARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT

[0218] IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEENPI

[0219] NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK

[0220] DTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL

[0221] KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

[0222] QRTFDNGSIPHQIHLGELHILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE

[0223] ETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR

[0224] KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK

[0225] DKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN

[0226] GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK

[0227] GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV

[0228] ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK

[0229] SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV

[0230] AQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAYNLAVVGTA

[0231] LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE

[0232] TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK

[0233] KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII

[0234] KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ

[0235] HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDT

[0236] TIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0237] S.py Cas9 amino acid sequence expressed from DNA codon optimized for expression in E. coli containing three NLS sequences and a purification His tag (SEQ ID No.: 408).

[0238] MGSSAPKKKRKVGIHGVPAAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK

[0239] NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK

[0240] HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS

[0241] DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL

[0242] GLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT

[0243] KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL

[0244] EKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIETFRI

[0245] PYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL

[0246] YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI

[0247] SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV

[0248] MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV

[0249] SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR

[0250] ERMKRIEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ

[0251] SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGLS

[0252] ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

[0253] REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS

[0254] NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK

[0255] ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

[0256] EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS

[0257] HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE

[0258] NIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAPKKKR

[0259] KVDPKKKRKVAAALEHHHHHH

[0260] S.pyCas9 DNA sequence codon-optimized for expression in E. coli containing three NLS sequences and a purification His tag (SEQ ID No.: 409).

[0261] ATGGGCAGCAGCGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCAT

[0262] GGACAAAAAGTACTCTATTGGCCTGGATATCGGGACCAACAGCGTCGGGTGGGCTGTTATCACC

[0263] GACGAGTATAAAGTACCTTCGAAAAAGTTCAAAGTGCTGGGCAACACCGATCGCCATTCAATC

[0264] AAAAAGAACTTGATTGGTGCGCTGTTGTTTGACTCCGGGGAAACCGCCGAGGCGACTCGCCTT

[0265] AAACGTAACAGCACGTCGCCGGTACACTCGCGGTAAGAATCGCATTGCTATTTGCAGGAAATCT

[0266] TTAGCAACGAGATGGCAAAGTCGATGACTCGTTTTTCCACCGCCTCGAGGAAAGCTTTCTGG

[0267] TGGAGGAAGACAAAAAGCATGAGCGTCACCCGATCTTCGGCAACATTGTCGATGAAAGTAGCGT

[0268] ATCATGAAAAAATACCCAACCATTTACCACTTACGCAAAAAGCTGGTGGACAGCACTGACAAAG

[0269] CTGATTTGCGCCTTATCTATTTAGCCCTGGCACATATGATTAAGTTTCGTGGTCACTTCCTGATCG

[0270] AAGGAGACTTAAATCCCGACAACAGTGATGTTGATAAATTGTTTATTCAGCTTGTCCAAACTTAC

[0271] AATCAACTGTTCGAGAAAACCCGATCAATGCTCCCGGTGTGGATGCAAAAGCCATTTTAAGTG

[0272] CACGCCTTAGCAAGTCCCGTCGCTTAGAAACCTTATCGCGCAGCTGCCCGGCGAGAAAAAGA

[0273] ATGGTTTGTTTGGGAACCTTATTGCCTTGAGCTTAGGCCTCACCCCGAATTTTCAAAGTAATTTC

[0274] GATCTTGCAGAAGACGCCAAATTACAACTGTCGAAGGATACTTATGATGACGATCTCGATAATCT

[0275] GTTAGCGCAGATTGGTGACCAATACGCCGATCTTTTTCTGGCGGCTAAAAATCTGAGCGACGCC

[0276] ATCTTGCTTTCGGATATTCTCCGCGTTAACACCGAAATCACGAAAGCGCCTCTTAGTGCCAGCAT

[0277] GATTAAACGTTATGATGAACACCACCAGGACCTGACCTTACTCAAAGCGTTGGTTCGCCAGCAA

[0278] CTGCCAGAGAAGTACAAAGAAATCTTCTTTGATCAGTCAAAGAATGGTTATGCCGGCTATATTG

[0279] ACGGGGGTGCAAGCCAAGAGGAATTCTACAAATTTATCAAGCCTATTCTGGAGAAAATGGATG

[0280] GCACCGAAGAGTTATTGGTGAAGCTTAACCGTGAAGACCTCCTGCGGAAACAGCGCACATTCG

[0281] ATAATGGTTCGATCCCACACCAAATCCATTTGGGGGAGTTACACGCTATTTTGCGTCGCCAGGA

[0282] AGACTTTTACCCTTTCCTGAAGGATAACCGGGAGAAAATTGAGAAGATCCTTACCTTTCGTATTC

[0283] CGTATTACGTAGGCCCCTTAGCACGGGGTAATAGCCGTTTCGCGTGGATGACACGGAAGTCGGA

[0284] AGAGACGATCACCCCGTGGAACTTCGAAGAGGTAGTCGACAAGGGCGCATCAGCGCAGTCTTT

[0285] TATTGAACGTATGACGAATTTCGATAAAAACTTGCCCAATGAGAAGGTGCTTCCGAAACATTCC

[0286] TTGTTATATGAATATTTTACAGTTTACAACGAGCTGACCAAGGTTAAATACGTGACGGAAGGAAT

[0287] GCGCAAGCCCGCTTTTCTTAGCGGTGAGCAAAAAAAGGCGATCGTCGACCTGTTATTCAAAAC

[0288] GAATCGTAAGGTGACTGTAAAGCAACTCAAAGAAGATTACTTCAAAAAGATTGAGTGCTTCGA

[0289] CAGCGTCGAAATCTCTGGGGTAGAGGATCGGTTTAACGCAAGTTTAGGTACCTACCATGACCTG

[0290] CTTAAAATCATTAAGGATAAAGACTTCTTAGATAATGAAGAGAACGAAGATATTCTCGAGGACA

[0291] TCGTCTTGACGTTAACCTTATTTGAGGATCGTGAAATGATTGAGGAACGCCTCAAAACTTATGC

[0292] CCACCTGTTCGACGATAAGGTGATGAAGCAGCTGAAACGTCGGCGCTACACAGGATGGGGCCG

[0293] CTTGAGTCGCAAACTTATTAACGGAATCCGTGACAAGCAATCCGGCAAAACGATTCTGGATTTC

[0294] TTGAAGTCGGACGGATTTGCTAATCGCAACTTCATGCAGTTGATCCATGATGACTCCCTGACTTT

[0295] TAAAGAGGATATTCAAAAGGCGCAGGTTAGTGGTCAAGGCGACAGCTTACACGAACACATCGC

[0296] AAATTTGGCTGGTTCGCCGGCCATTAAAAAGGGGATCCTCCAGACCGTGAAAGTTGTAGATGA

[0297] GCTTGTTAAGGTCATGGGTCGTCATAAGCCCGAAAACATCGTGATTGAAATGGCGCGGGAGAAT

[0298] CAAACGACCCAGAAAGGACAAAAGAATAGCCGTGAACGGATGAACGGAAGGCAT

[0299] TAAAGAGCTGGGGTTCCAAATCTTGAAGGAAACCCCTGTGGAGAACACTCAGCTCCAAAATGA

[0300] AAAACTTTACCTGTACTATTTGCAGAACGGACGCGATATGTACGTGGACCAAGAGTTGGATATT

[0301] AATCGGCTGAGTGACTACGACGTTGATCATATCGTCCCGCAGAGCTTCCTCCAAAGACGATTCTA

[0302] TTGACAATAAGGTACTGACGCGCTCTGATAAAAAACCGTGGTAAGTCGGACAACGTGCCCTCCG

[0303] AAGAGGTTGTGAAAAAAGATGAAAATTATTGGCGCCAGCTTTTAAACGGAAGCTGATCACAC

[0304] AACGTAAATTCGATAATTTGACCAAGGCTGAACGGGGTGGCCTGAGCGAGTTAGATAAGGCAG

[0305] GATTTTATTAAACGCCAGTTAGTGGAGACTCGTCAAATCACCAAACATGTCGCGCAGATTTTGGA

[0306] CAGCCGGATGAACACCAAGTACGAATGAAAATGACAAACTGATCCGTGAGGTGAAAGTCATTAC

[0307] TCTGAAGTCCAAATTAGTTAGTGATTTCCGGAAGGACTTTCAATTCTACAAAGTCCGTGAAATT

[0308] AATAACTATCATCACGCACATGACGCGTACCTGAATGCAGTGGTTGGGACCGCCCTTATCAAGA

[0309] AATATCCTAAGCTGGAGTCGGAGTTTGTCTATGGCGACTATAAGGTATACGATGTTCGCAAAATG

[0310] ATTGCGAAATCTGAGCAGGAGATCGGTAAGGCAACCGCAAAATTTTTCTTTACTCAAACATTAT

[0311] GAATTTCTTTAAGACAGAAATCACTCTGGCCAACGGGAGATTCGCAAACGTCCGTTGATCGA

[0312] AACAAACGGCGAGACTGGCGAAATTGTTTGGGACAAAGGGCGTGATTTCCGACGGTGCGCA

[0313] AGGTACTGAGCATGCCTCAAGTCAATATTGTTAAGAAAACCGAAGTGCAGACGGGCGGGTTTT

[0314] CCAAGGAAAGCATCTTACCCAAACGTAATTCAGATAAACTTATTGCACGCAAAAAGGACTGGG

[0315] ATCCGAAAAGTATGGAGGCTTCGACAGTCCAACCGTAGCCTACTCTGTTCTCGTTGTAGCGAA

[0316] AGTAGAAAAAGGGTAAATCCAAGAAACTGAAATCTGTCAAGGAGTTGCTTGGAATCACCATTAT

[0317] GGAGCGTAGCTCCTTCGAGAAGAACCCGATTGACTTTCTGGAAGCCAAAGGATATAAAGAGGT

[0318] CAAGAAAGATCTTATCATTAAGCTGCCTAAGTATTCACTCTTCGAGCTGGAAAATGGTCGTAAA

[0319] CGCATGCTCGCTTCTGCCGGCGAGTTGCAGAAGGGCAATGAATTAGCACTTCCATCAAAGTACG

[0320] TTAACTTCCTGTATTTGGCCAGCCATTACGAGAAACTGAAGGGGTCTCCAGAGGACAACGAAC

[0321] AGAAACAATTATTTGTAGAGCAGCACAAGCATTATCTTGATGAAATCATTGAGCAAATTTCCGAA

[0322] TTCAGTAAACGCGTAATCCTGGCCGATGCAAACCTCGACAAGGTGCTGAGCGCTTACAATAAGC

[0323] ATCGCGACAAACCTATCCGTGAGCAGGCTGAAAATATCATTCACCTGTTCACATTAACGAACCT

[0324] GGGCGCTCCGGCCGCTTTTAAATATTTCGACACGACAATCGACCGTAAGCGCTATACCAGTACG

[0325] AAAGAAGTGTTGGATGCGACCCTTATTCACCAGTCAATTACAGGATTATATGAGACCCGTATCGA

[0326] CCTTAGCCAATTAGGTGGGGATGCGGCCCCGAAGAAAAAACGCAAAGTGGATCCGAAGAAAA

[0327] AACGCAAAGTGGCGGCCGCACTCGAGCACCACCACCACCACCACTGA

[0328] S.py Cas9 amino acid sequence expressed from DNA codon-optimized for expression in human cells containing a V5 epitope tag and two NLS sequences (SEQ ID No.: 410).

[0329] MGKPIPNPLLGLDSTAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG

[0330] NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE

[0331] SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLI

[0332] EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL

[0333] FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLS

[0334] DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE

[0335] EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE

[0336] KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE

[0337] KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK

[0338] KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY

[0339] AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF

[0340] KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT

[0341] QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD

[0342] YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

[0343] TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR

[0344] KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

[0345] TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE

[0346] VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL

[0347] GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK

[0348] YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH

[0349] RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG

[0350] GDSRADPKKKRKVEFHHTGLVDPSSVPSLSLNR

[0351] The S.pyCas9 DNA sequence (SEQ ID No.: 411) optimized for expression in human cells and containing a V5 epitope tag and two NLS sequences.

[0352] ATGGGCAAGCCCATCCCTAACCCCCTGTTGGGGCTGGACAGCACCGCTCCCAAAAAGAAAAGG

[0353] AAGGTGGGCATTCACGGCGTGCCTGCGGCCGACAAAAGTACAGCATCGGCCTTGATATCGGC

[0354] ACCAATAGCGTGGGCTGGCCGTTATCACAGACGAACGAATACAAGGTACCAGCAAGAAGTTCAAG

[0355] GTGCTGGGGAATACAGACAGGCACTCTATCAAGAAAAAACCTTATCGGGGCTCTGCTGTTTGACT

[0356] CAGGCGAGACCGCCGAGGCCACCAGGTTGAAGAGGACCGCAAGCGAAGGTACACCCGGAG

[0357] GAAGAACAGGATCTGCTATCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAG

[0358] CTTCTTCCACAGGCTGGAGGAGAGCTTCCTTGTCGAGGAGGATAAAGAAGCACGAACGACACCC

[0359] CATCTTCGGCAACATAGTCGACGAGGTCGCTTATCACGAGAAGTACCCCACCATCTACCACCTG

[0360] CGAAAGAAATTGGTGGATAGCACCGATAAAGCCGACTTGCGACTTATCTACTTGGCTCTGGCGC

[0361] ACATGATTAAGTTCAGGGGCCACTTCCTGATCGAGGGCGACCTTAACCCCGACAACAGTGACG

[0362] TAGACAAATTGTTCATCCAGCTTGTACAGACCTAATAACCAGCTTCGAGGAAAAACCCTATTAA

[0363] CGCCAGCGGGGTGGATGCGAAGGCCATACTTAGCGCCAGGCTGAGCAAAAGCAGGCGCTTGG

[0364] AGAACCTGATAGCCCAGCTGCCCGGTGAAAAGAAGAACGGCCTCTTCGGTAATCTGATTGCCC

[0365] TGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCAGAAGATGCCAAGCTGC

[0366] AGTTGAGTAAGGACACCTATGACGACGACTTGGACAATCTGCTCGCCCAAATCGGCGACCAGT

[0367] ACGCTGACCTGTTCCTCGCCGCCAAGAACCTTTCTGACGCAATCCTGCTTAGCGATATCCTTAG

[0368] GGTGAACACAGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGGTACGACGAGC

[0369] ACCATCAGGACCTGACCCTTCTGAAGGCCCTGGTGAGGCAGCAACTGCCCGAGAAGTACAAG

[0370] GAGATCTTTTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGAGCCAGCCAA

[0371] GAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGATGGCACCGAGGAGCTGCTG

[0372] GTGAAGCTGAACAGGGAAGATTTGCTCCGGAAGCAGAGGACCTTTGACAACGGTAGCATCCCC

[0373] CACCAGATCCACCTGGGCGAGCTGCACGCAATACTGAGGCGACAGGAGGATTTCTACCCCTTC

[0374] CTCAAGGACAATAGGGAGAAAATCGAAAAGATTCTGACCTTCAGGATCCCCTACTACGTGGGC

[0375] CCTCTTGCCAGGGGCAACAGCCGATTCGCTTGGATGACAAGAAAGAGCGAGGAGACCATCACC

[0376] CCCTGGAACTTCGAGGAAGTGGTGGACAAAGGAGCAAGCGCGCAGTCTTTCATCGAACGGAT

[0377] GACCAATTTCGACAAAAACCTGCCTAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTTTACGA

[0378] GTACTTCACCGTGTACAACGAGCTCACCAAGGTGAAATATGTGACCGAGGGCATGCGAAAACC

[0379] CGCTTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGGA

[0380] AGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTTGATAGCGTGG

[0381] AAATAAGCGGCGTGGAGGACAGGTTCAACGCCAGCCTGGGCACCTACCACGACTTGTTGAAGA

[0382] TAATCAAAGACAAGGATTTCCTGGATAATGAGGAGAACGAGGATATACTCGAGGACATCGTGCT

[0383] GACTTTGACCCTGTTTGAGGACCGAGAGATGATTGAAGAAAGGCTCAAAACCTACGCCCACCT

[0384] GTTCGACGACAAAGTGATGAAACAACTGAAGAGACGAAGATACACCGGCTGGGGCAGACTGT

[0385] CCAGGAAGCTCATCAACGGCATTAGGGACAAGCAGAGCGGCAAGACCATCCTGGATTTCCTGA

[0386] AGTCCGACGGCTTCGCCAACCGAAACTTCATGCAGCTGATTCACGATGACAGCTTGACCTTCA

[0387] AGGAGGACATCCAGAAGGCCCAGGTTAGCGGCCAGGCGACTCCCTGCACGAACATATTGCAA

[0388] ACCTGGCAGGCTCCCTGCGATCAAGAAGGGCATACTGCAGACCGTTAAGGTTGTGGACGAAT

[0389] TGGTCAAGGTCATGGGCAGGCCACAAGCCCGAAAACATAGTTTATAGAGATGGCCAGAGAGAACC

[0390] AGACCACCCAAAAGGGCCAGAAGAACAGCCGGGAGCGCATGAAAAGGATCGAGGAGGGTATC

[0391] AAGGAACTCGGAAGCCAGATCCTCAAAGAGCACCCCGTGGAGAATACCCAGCTCCAGAACGA

[0392] GAAGCTGTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTTGACCAGGAGTTGGACAT

[0393] CAACAGGCTTTCAGACTATGACGTGGATCACATAGTGCCCCAGAGCTTTCTTAAAGACGATAGC

[0394] ATCGACAACAAGGTCCTGACCCGCTCCGACAAAACAGGGGCAAAAGCGACAACGTGCCAAG

[0395] CGAAGAGGTGGTTAAAAAGATGAAGAACTACTGGAGGCAACTGCTCAACGCGAAATTGATCAC

[0396] CCAGAGAAAGTTCGATAACCTGACCAAGGCCGAGAGGGGCGGACTCTCCGAACTTGACAAAG

[0397] CGGGCTTCATAAAGAGGCAGCTGGTCGAGACCCGACAGATCACGAAGCACGTGGCCCAAATCC

[0398] TCGACAGCAGAATGAATACCAAGTACGATGAGAATGACAAACTCATCAGGGAAGTGAAAGTGA

[0399] TTACCCTGAAGAGCAAGTTGGTGTCCGACTTTCGCAAAGATTTCCAGTTCTACAAGGTGAGGG

[0400] AGATCAACAACTACCACCATGCCCACGACGCATACCTGAACGCCGTGGTCGGCACCGCCCTGA

[0401] TTAAGAAGTATCCAAAGCTGGAGTCCGAATTTGTCTACGGCGACTACAAAGTTTACGATGTGAG

[0402] GAAGATGATCGCTAAGAGCGAACAGGAGATCGGCAAGGCCACCGCTAAGTATTTCTTCTACAG

[0403] CAACATCATGAACTTTTTCAAGACCGAGATCACACTTGCCAACGGCGAAATCAGGAAGAGGCC

[0404] GCTTATCGAGACCAACGGTGAGACCGGCGAGATCGTGTGGGACAAGGGCAGGGACTTCGCCA

[0405] CCGTGAGGAAAGTCCTGAGCATGCCCCAGGTGAATATTGTGAAAAAAACTGAGGTGCAGACA

[0406] GGCGGCTTTAGCAAGGAATCCATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCCGGAA

[0407] GAAGGACTGGGACCCTAAGAAGTATGGAGGCTTCGACAGCCCCACCGTAGCCTACAGCGTGCT

[0408] GGTGGTCGCGAAGGTAGAGAAGGGGAAGAGCAAGAAACTGAAGAGCGTGAAGGAGCTGCTC

[0409] GGCATAACCATCATGGAGAGGTCCAGCTTTGAGAAGAACCCCATTGACTTTTTGGAAGCCAAG

[0410] GGCTACAAAGAGGTCAAAAAGGACCTGATCATCAAACTCCCCAAGTACTCCCTGTTTGAATTG

[0411] GAGAACGGCAGAAAGAGGATGCTGGCGAGCGCTGGGGAACTGCAAAAGGGCAACGAACTGG

[0412] CGCTGCCCAGCAAGTACGTGAATTTTCTGTACCTGGCGTCCCACTACGAAAAGCTGAAAGGCA

[0413] GCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCATTACCTGGACGAG

[0414] ATAATCGAGCAAATCAGCGAGTTCAGCAAGAGGGTGATTCTGGCCGACGCGAACCTGGATAAG

[0415] GTCCTCAGCGCCTACAACAAGCACCGAGACAAACCCATCAGGGAGCAGGCCGAGAATATCATA

[0416] CACCTGTTCACCCTGACAAATCTGGGCGCACCTGCGGCATTCAAATACTTCGATACCACCATCG

[0417] ACAGGAAAAGGTACACTAGCACTAAGGAGGTGCTGGATGCCACCTTGATCCACCAGTCCATTA

[0418] CCGGCCTGTATGAGACCAGGATCGACCTGAGCCAGCTTGGAGGCGACTCTAGGGCGGACCCAA

[0419] AAAAGAAAAGGAAGGTGGAATTCCACCACACTGGACTAGTGGATCCGAGCTCGGTACCAAGC

[0420] TTAAGTTTAAACCGCTGA

[0421] S.py Cas9 DNA sequence (SEQ ID No.: 412) codon-optimized for expression as a T7 RNA polymerase expression cassette in human cells. The sequence contains a T7 RNA polymerase promoter, a V5 epitope tag, a nuclear localization signal, a codon-optimized Cas9 sequence, a second nuclear localization signal, and a BGH (bovine growth hormone) gene 3'-UTR element with a polyadenylation signal.

[0422] TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGACTCGAG

[0423] CGGCCGCCACCATGGGCAAGCCCATCCCTAACCCCCTGTTGGGGCTGGACAGCACCGCTCCCA

[0424] AAAAGAAAAGGAAGGTGGGCATTCACGGCGTGCCTGCGGCCGACAAAAAGTACAGCATCGGC

[0425] CTTGATATCGGCACCAATAGCGTGGGCTGGGCCGTTATCACAGACGAATACAAGGTACCCAGCA

[0426] AGAAGTTCAAGGTGCTGGGGAATACAGACAGGCACTCTATCAAGAAAAACCTTATCGGGGCTC

[0427] TGCTGTTTGACTCAGGCGAGACCGCCGAGGCCACCAGGTTGAAGAGGACCGCAAGGCGAAGG

[0428] TACACCCGGAGGAAGAACAGGATCTGCTATCTGCAGGAGATCTTCAGCAACGAGATGGCCAAG

[0429] GTGGACGACAGCTTCTTCCACAGGCTGGAGGAGAGCTTCCTTGTCGAGGAGGATAAGAAGCA

[0430] CGAACGACACCCCATCTTCGGCAACATAGTCGACGAGGTCGCTTATCACGAGAAGTACCCCAC

[0431] CATCTACCACCTGCGAAAGAAATTGGTGGATAGCACCGATAAAGCCGACTTGCGACTTATCTAC

[0432] TTGGCTCTGGCGCACATGATTAAGTTCAGGGGCCACTTCCTGATCGAGGGCGACCTTAACCCCG

[0433] ACAACAGTGACGTAGACAAATTGTTCATCCAGCTTGTACAGACCTATAACCAGCTGTTCGAGGA

[0434] AAACCCTATTAACGCCAGCGGGGTGGATGCGAAGGCCATACTTAGCGCCAGGCTGAGCAAAAG

[0435] CAGGCGCTTGGAGAACCTGATAGCCCAGCTGCCCGGTGAAAAGAAGAACGGCCTCTTCGGTAA

[0436] TCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCAGAAGA

[0437] TGCCAAGCTGCAGTTGAGTAAGGACACCTATGACGACGACTTGGACAATCTGCTCGCCCAAAT

[0438] CGGCGACCAGTACGCTGACCTGTTCCTCGCCGCCAAGAACCTTTCTGACGCAATCCTGCTTAGC

[0439] GATATCCTTAGGGTGAACACAGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGG

[0440] TACGACGAGCACCATCAGGACCTGACCCTTCTGAAGGCCCTGGTGAGGCAGCAACTGCCCGAG

[0441] AAGTACAAGGAGATCTTTTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGA

[0442] GCCAGCCAAGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGATGGCACCGAG

[0443] GAGCTGCTGGTGAAGCTGAACAGGGAAGATTTGCTCCGGAAGCAGAGGACCTTTGACAACGG

[0444] TAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCAATACTGAGGCGACAGGAGGATTT

[0445] CTACCCCTTCCTCAAGGACAATAGGGAGAAAATCGAAAAGATTCTGACCTTCAGGATCCCCTAC

[0446] TACGTGGGCCCTCTTGCCAGGGGCAACAGCCGATTCGCTTGGATGACAAGAAAGAGCGAGGA

[0447] GACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAAGGAGCAAGCGCGCAGTCTTTCAT

[0448] CGAACGGATGACCAATTTCGACAAAAACCTGCCTAACGAGAAGGTGCTGCCCAAGCACAGCCT

[0449] GCTTTACGAGTACTTCACCGTGTACAACGAGCTCACCAAGGTGAAATATGTGACCGAGGGCATG

[0450] CGAAAACCCGCTTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGAC

[0451] CAACAGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTTG

[0452] ATAGCGTGGAAATAAGCGGCGTGGAGGACAGGTTCAACGCCAGCCTGGGCACCTACCACGACT

[0453] TGTTGAAGATAATCAAAGACAAGGATTTCCTGGATAATGAGGAGAACGAGGATATACTCGAGGA

[0454] CATCGTGCTGACTTTGACCCTGTTTGAGGACCGAGAGATGATTGAAGAAAGGCTCAAAACCTA

[0455] CGCCCACCTGTTCGACGACAAAGTGATGAAACAACTGAAGAGACGAAGATACACCGGCTGGG

[0456] GCAGACTGTCCAGGAAGCTCATCAACGGCATTAGGGACAAGCAGAGCGGCAAGACCATCCTG

[0457] GATTTCCTGAAGTCCGACGGCTTCGCCAACCGAAACTTCATGCAGCTGATTCACGATGACAGCT

[0458] TGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTTAGCGGCCAGGGCGACTCCCTGCACGAA

[0459] CATATTGCAAACCTGGCAGGCTCCCCTGCGATCAAGAAGGGCATACTGCAGACCGTTAAGGTTG

[0460] TGGACGAATTGGTCAAGGTCATGGGCAGGCACAAGCCCGAAAACATAGTTATAGAGATGGCCA

[0461] GAGAGAACCAGACCACCCAAAAGGGCCAGAAGAACAGCCGGGAGCGCATGAAAAGGATCGA

[0462] GGAGGGTATCAAGGAACTCGGAAGCCAGATCCTCAAAGAGCACCCCGTGGAGAATACCCAGCT

[0463] CCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTTGACCAGGA

[0464] GTTGGACATCAACAGGCTTTCAGACTATGACGTGGATCACATAGTGCCCCAGAGCTTTCTTAAA

[0465] GACGATAGCATCGACAACAAGGTCCTGACCCGCTCCGACAAAAACAGGGGCAAAAGCGACAA

[0466] CGTGCCAAGCGAAGAGGTGGTTAAAAAGATGAAGAACTACTGGAGGCAACTGCTCAACGCGA

[0467] AATTGATCACCCAGAGAAAGTTCGATAACCTGACCAAGGCCGAGAGGGGCGGACTCTCCGAAC

[0468] TTGACAAAGCGGGCTTCATAAAGAGGCAGCTGGTCGAGACCCGACAGATCACGAAGCACGTG

[0469] GCCCAAATCCTCGACAGCAGAATGAATACCAAGTACGATGAGAATGACAAACTCATCAGGGAA

[0470] GTGAAAGTGATTACCCTGAAGAGCAAGTTGGTGTCCGACTTTCGCAAAGATTTCCAGTTCTACA

[0471] AGGTGAGGGAGATCAACAACTACCACCATGCCCACGACGCATACCTGAACGCCGTGGTCGGCA

[0472] CCGCCCTGATTAAGAAGTATCCAAAGCTGGAGTCCGAATTTGTCTACGGCGACTACAAAGTTTA

[0473] CGATGTGAGGAAGATGATCGCTAAGAGCGAACAGGAGATCGGCAAGGCCACCGCTAAGTATTT

[0474] CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATCACACTTGCCAACGGCGAAATCAGG

[0475] AAGAGGCCGCTTATCGAGACCAACGGTGAGACCGGCGAGATCGTGTGGGGACAAGGGCAGGGA

[0476] CTTCGCCACCGTGAGGAAAGTCCTGAGCATGCCCCAGGTGAATATTGTGAAAAAAAACTGAGGT

[0477] GCAGACAGGCGGCTTAGCAAGGAATCCATCCTGCCCAAGAGGAACAGCGACAAGCTGATCG

[0478] CCCGGAAGAAGGACTGGGACCCTAAGAAGTATGGAGGCTTCGACAGCCCCACCGTAGCCTACA

[0479] GCGTGCTGGTGGTCGCGAAGGTAGGAAGGGGAAGAGCAAGAAACTGAAGAGCGTGAAGGA

[0480] GCTGCTCGGCATAACCATCATGGAGAGGTCCAGCTTTGAGAAGAACCCCATTGACTTTTTGGAA

[0481] GCCAAGGGCTACAAAGAGGTCAAAAAGGACCTGATCATCAAACTCCCCAAGTACTCCCTGTTT

[0482] GAATTGGAGAACGGCAGAAAGAGGATGCTGGCGAGCGCTGGGGAACTGCAAAAGGGCAACG

[0483] AACTGGCGCTGCCCAGCAAGTACGTGAATTTTCTGTACCTGGCGTCCCACTACGAAAAGCTGA

[0484] AAGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCATTACCTG

[0485] GACGAGATAATCGAGCAAATCAGCGAGTTCAGCAAGAGGGTGATTCTGGCCGACGCGAACCTG

[0486] GATAAGGTCCTCAGCGCCTACAACAAGCACCGAGACAAACCCATCAGGGAGCAGGCCGAA

[0487] TATCATACACCTGTTCACCCTGACAAATCTGGGCGCACCTGCGGCATTCAAATACTTCGATACCA

[0488] CCATCGACAGGAAAAGGTACACTAGCACTAAGGAGGTGCTGGATGCCACCTTGATCCACCAGT

[0489] CCATTACCGGCCTGTATGAGACCAGGATCGACCTGAGCCAGCTTGGAGGCGACTCTAGGGCGG

[0490] ACCCAAAAAAGAAAAGGAAGGTGGAATTCCACCACACTGGACTAGTGGATCCGAGCTCGGTA

[0491] CCAAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTG

[0492] TTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAA

[0493] AATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC

[0494] AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCT

[0495] ATGGC

[0496] S.py Cas9 mRNA (SEQ ID No.: 413) prepared from the expression cassette (SEQ ID No.: 412). The sequence contains a V5 epitope tag, a nuclear localization signal, a codon-optimized Cas9 sequence, a second nuclear localization signal, and a 3'-UTR element and a poly A tail of the BGH (bovine growth hormone) gene.

[0497] GGGAGACCCAAGCUGGCUAGCGUUUAAACGGGCCCUCUAGACUCGAGCGGCCGCCACCAUG

[0498] GGCAAGCCCAUCCCUAACCCCCUGUUGGGGCUGGACAGCACCGCUCCCAAAAAGAAAGGA

[0499] AGGUGGGCAUUCACGGGCUGCCCUGCGGCCGACAAAAAGUACAGCAUCGGCCUUGAAUUCGG

[0500] CACCAAUAGCGUGGGCUGGGCCGUUAUCACAGACGAAUACAAGGUACCCAGCAAGAAGUUC

[0501] AAGGUGGCGGGAAUACAGACAGGCACUCUAUCAAGAAAAACCUUAUCGGGGCUCUGCUG

[0502] UUUGACUCAGGCGAGACCGCCGAGGCCACCAGGUUGAAGAGGACCGCAAGGCGAAGGUACA

[0503] CCCGGAGGAAGAACAGGAUCUGCUAUCUGCAGGAGAUCUUCAGCAACGAGAUGGCCAAGG

[0504] UGGACGACAGCUUCUUCCACAGGCUGGAGGAGAGCUUCCUUGUCGAGGAGGAUAAGAAGC

[0505] ACGAACGACACCCAUCUUCGGCAACAUAGUCGACGAGGUCGCUUAUCACGAGAAGUACCC

[0506] CACCAUCUACCACCUGCGAAAGAAAUUGGUGGAUAGCACCGAUAAAGCCGACUUGCGACUU

[0507] AUCUACUUGGCUCUGGCGCCAUGAUUAAGUCAGGGGCCACUUCCUGAUCGAGGGCGACC

[0508] UUAACCCCCGACAACAGUGACGUAGACAAAUUGUUCAUCCAGCUUGUACAGACCUAAACCA

[0509] GCUGUUCGAGGAAAACCCUAUAACGCCAGCGGGGUGGAUGCGAAGCCAUACUUAGCGCC

[0510] AGGCUGAGCAAAAGCAGGCGCUUGGAGAACCUGUAUAGCCCAGCUGCCCGGUGAAAAAAG

[0511] AACGGCCUCUUCGGUAAUCUGAUGCCCUGAGCCUGGGCCUGACCCCCAACUUCAAGAGCA

[0512] ACUUCGACCUGGCAGAAGAUGCCAAGCUGCAGUUGAGUAAGGACACCUAUGACGACGACU

[0513] UGGACAAUCUGCUCGCCCAAAUCGGCGACCAGUACGCUGACCUGUUCCUCGCCGCCAAGAA

[0514] CCUUUCUGACGCAAUCCUGCUUAGCGAUAUCCUUAGGGUGAACACAGAGAUACCAAGGCC

[0515] CCCCUGAGCGCCAGCAUGAUCAAGAGGUACGACGAGCACCAUCAGGACCUGACCCUUCUGA

[0516] AGGCCCUGGUGAGGCAGCAACUGCCCGAGAAGUACAAGGAGAUUUUUUCGACCAGAGCA

[0517] AGAACGGCUACGCCGGCUACAUCGACGGCGGAGCCAGCCAAGAGGAGGUUCUACAAGUUCAU

[0518] CAAGCCCAUCCUGGAGAAGGAUGGCACCGAGGAGCUGCUGGUGAAGCUGAACAGGGGA

[0519] AGAUUUGCUCCGGGAAGCAGAGGACCUUUGAACGGUAGCAUCCCCCACCAGAUCCACCUG

[0520] GGCGAGCUGCACGCAAUACUGAGGCGACAGGAGGAUUUCACCCCUUCCUCAAGGACAAUA

[0521] GGGAGAAAUCGAAAAGAUUCUGACCUUCAGGAUCCCCUACUACGUGGCCCUCUUGCCAG

[0522] GGGCAACAGCCGAUUCGCUUGGAAUGACAAGAAAGAGCGAGGAGACCAUCCCCCUGGAAC

[0523] UUCGAGGAAGUGGUGGACAAAGGAGCAAGCGCGCGAGUCUUUCAUCGAACGGAUGACCAAU

[0524] UUCGACAAAAACCUGCCUAACGAGAAGGUGCUGCCCAAGCACAGCCUGCUUUACGAGUACU

[0525] UCACCGUGUAACGAGCUCACCAAGGUGAAAAUUGUGACCGAGGGCAUGCGAAACCCGC

[0526] UUUCCUGAGCGGCGAGCAGAAGAGGCCAUCGUGGACCUGCUGCUGUUCAAGACCAACAGGAA

[0527] GGUGACCGUGAAGCAGCUGAAGGAGACUAUUCAAGAGAUCGAGUGCUUUGAUAGCGU

[0528] GGAAAUAAAGCGGCGUGGAGGACAGGUUCAACGCCAGCCUGGGCACCUACCACGACUUGUUG

[0529] AAGAUAAUCAAGACAAGGAUUUCCUGGAUAAUGAGGAGAACGAGGAUAUACUCGAGGAC

[0530] AUCGUGCUGACUUUGACCCUGUUGAGGACCGAGAGAUGAUUGAAGAAAGGCUCAAAACC

[0531] UACGCCCACCUGUUCGACGACAAAGUGAUGAAACACUGAAGAGACGAAGAUACACGGCU

[0532] GGGGCAGACUGUCCAGGAAGCUCAUCAACGGCAUUAGGGACAAGCAGAGCGCGCAAGACCA

[0533] UCCUGGAUUUCCUGAAGUCCGACGGCUUCGCCAACCGAAACUUCAUGCAGCUGAUUCACGA

[0534] UGACAGCUUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUUAGCGGCCAGGGCGACUCC

[0535] CUGCACGAACAUAUUGCAAACCUGGCAGGCUCCCCUGCGAUCAAGAAGGGCAUACUGCAGA

[0536] CCGUUAAGGUUGUGGACGAAUUGGUCAAGGUCAUGGCAGGCACAAGCCCGAAAACAUAG

[0537] UUAUAGAGAUGGCCAGAGAGAACCAGACCACCCAAAGGGCCAGAAGAACACCCGGGGGC

[0538] GCAUGAAAAGGAUCGAGGAGGGUAUACAAGGAACUCGGAAGCCAGAUCCUCUAAGAGCACC

[0539] CCGUGGAGAAUACCCAGCUCCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCAG

[0540] GGACAUGUACGUGACCAGGAGGUUGGACAUCAACAACGGGCUUUCAGACGUAUGACGUGGAUCA

[0541] CAUAGUGCCCCAGAGCUUUCUUAAAGACGAUAGCAUCGACAACAAGGUCCUGACCCGCUCC

[0542] GACAAAACAGGGGCAAAAGCGACAACGUGCCAAGCGAAGAGGGUGGUUAAAAAAGAUGAAG

[0543] AACUACUGGAGGCAACUGCUCAACGCGAAAUUGAUCACCCAGAAAAGUUCGAUAACCUG

[0544] ACCAAGGCCGAGAGGGGCGGACUCUCCGAACUUGACAAAGCGGGCUUCAUAAAGGGCAGC

[0545] UGGUCGAGACCCGACAGAUCACGAAGCACGUGGGCCCAAAUCCUCGACAGCAGAAUGAAUAC

[0546] CAAGUACGAUGAAAUGACAAACUCAUCAGGGAAGGAAGUGAUAUACCCUGAGAGCAA

[0547] GUUGGUGUCCGACUUUCGCAAAGAUUUCCAGUUCUACAAGGUGAGGGAGAUCAACACUA

[0548] CCACCAUGCCCACGACGCAUACCUGAACGCCGUGGUCGGCACCGCCCUGAUUAAGAAGUAU

[0549] CCAAAGCUGGAGUCCGAAUUGUCUACGGCGACUAAAAAUUUACGAUGUGAGGAAUG

[0550] AUGCCUAAGAGCGAACAGGAGAUCGGCAAGGCCACCGCUAAGUAUUUCUUCACAGCAACA

[0551] UCAUGACUUUUUCAAGACCGAGUACACAUCUUGCCAACGGCGAAAUCAGGAAGGGCCGCU

[0552] UAUCGAGACCAACGGUGAGACCGGCGAGAUCGUGUGGGGACAAGGCAGGGACUUCGCCACC

[0553] GUGAGGAAAGCCUGAGCAUGCCCCAGGUGAAUAUUGUGAAAAAAACUGAGGGUGCAGACA

[0554] GGCGGCUUUAGCAAGGAAUCCAUCCUGCCCAAGAGGAACAGCGACAAGCUGAUGCCCCGGA

[0555] AGAAGGACUGGGACCCUAAGAAGUAUGGAGGCUUCGACAGCCCCACCGUAGCCUACAGCGU

[0556] GCUGGUGGUCGCGAAGGUAGGAAGGGGAAGAGAAACUGAGAGGCGUGAAGGAGCU

[0557] GCUCGGCAUAACCAUCAUGGAGAGGUCCAGCUUUGAGAGAACCCCAUUGACUUUUGGA

[0558] AGCCAAGGGCUACAAAGAGGUCAAAAAGGACCUGAUCAUAACUCCCCAAGUACUCCCUG

[0559] UUGAAUUGGAGAACGGCAGAAAGAGGAUGCUGGCGAGCGUGGGAACUGCAAAAGGGC

[0560] AACGAACUGGCCUGCCCAGCAAGUACGUGAAUUUUCUGUACCUGGCGUCCCACUACGAAA

[0561] AGCUGAAAGGCAGCCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCA

[0562] UUACCUGGACGAGAUAAUCGAGCAAAUCAGCGAGUUCAGCAAGAGGGUGAUUCUGGCCGA

[0563] CGCGAACCUGGAUAAGGUCCUCAGCGCCUACAACAAGCACCGAGACAAACCCAUCAGGGAG

[0564] CAGGCCGAGAAUAUCAUACACCUGUUCACCCUGACAAAUCUGGGCGCACCUGCGGCAUUCA

[0565] AAUACUUCGAUACCACCAUCGACAGGAAAAGGUACACUAGCACUAAGGAGGUGCUGGAUG

[0566] CCACCUUGAUCCACCAGUCCAUUACCGGCCUGUAUGAGACCAGGAUCGACCUGAGCCAGCU

[0567] UGGAGGCGACUCUAGGGCGGACCCAAAAAAGAAAAGGAAGGUGGAAUUCCACCACACUGG

[0568] ACUAGUGGAUCCGAGCUCGGUACCAAGCUUAAGUUUAAACCGCUGAUCAGCCUCGACUGUG

[0569] CCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAG

[0570] GUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUA

[0571] GGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGCAGGCAUGCUGGGGAUGCGGUGGGCUCUAUGGC–POLYA

[0572] Example 12

[0573] The following examples demonstrate that the truncated chemically modified crRNA:tracrRNA complexes of the invention reduce stimulation of the innate immune system in mammalian cells when compared to unmodified IVT sgRNA.

[0574] Mammalian cells have a variety of receptors that are expected to identify and respond to foreign RNA as part of antiviral immunity. This includes receptors such as TLR-3, TLR-7, TLR8, RIG-I, MDA5, OAS, PKR, etc. In general, short or chemically modified RNAs (e.g., 2'OMe RNAs) present in mammalian cells escape detection or are less stimulating than long, unmodified RNAs. This example compares the stimulation levels of two immune response-related genes (IFIT1 and IFITM1) when mammalian HEK293 cells are transfected with truncated unmodified or truncated modified crRNA:tracrRNA complexes of the present invention with commercial IVT sgRNAs (Thermo Fisher Scientific, Waltham, MA).

[0575] CRISPR guide RNA specific for human HPRT1 site 38285 was used. The sequences are shown in Table 11 below. Unmodified crRNA:tracrRNA complexes (SEQ ID No.: 48 and 2), modified crRNA:tracrRNA complexes (SEQ ID No.: 178 and 100), and sgRNA (SEQ ID No.: 414) were transfected into HEK-Cas9 cells at 30 nM concentration as outlined in Example 2 above. RNA was prepared 24 hours after transfection using the SV96 Total RNA Isolation Kit (Promega, Madison, WI). Using random hexamers and oligo dT priming, 150 ng of total RNA was used with SuperScript TM cDNA was synthesized using HER2-II reverse transcriptase (Invitrogen, Carlsbad, CA). Transfection experiments were performed at least three times.

[0576] For each 10 μL reaction, use 10 ng of cDNA and Immolase TM Quantitative real-time PCR was performed using DNA polymerase (Bioline, Randolph, MA), 200 nM primers and 200 nM probe. The cycling conditions used were: 95°C for 10 min, followed by 40 cycles of 2-step PCR at 95°C for 15 sec and 60°C for 1 min. The ABI Prism TMPCR and fluorescence measurements were performed using a 7900 sequence detector (Applied Biosystems Inc., Foster City, CA). All reactions were performed in triplicate using dual-color multiplexing. Expression data were normalized to the mean of two internal control genes. The copy number standard was a linearized cloned amplicon for all assays. Unknowns were extrapolated relative to the standard to establish absolute quantitative measurements. The home internal control normalization assay was HPRT1 (primers and probes SEQ ID No.: 415-417) and SFRS9 (primers and probes SEQ ID No.: 418-420). The immune activation pathway assay was IFITM1 (primers and probes SEQ ID No.: 421-423) and IFIT1 (primers and probes SEQ ID No.: 424-426). The results were normalized using non-transfected cells as a baseline and are shown in Fig.13 middle.

[0577] Table 11. Nucleic acid reagents used in the immune activation experiment in Example 12.

[0578]

[0579] 1 Compound 1 is an oligonucleotide having the formula SEQ ID NO:417-(ZEN)-SEQ ID NO:441.

[0580] 2 Compound II is an oligonucleotide having the formula SEQ ID NO:420-(ZEN)-SEQ ID NO:442.

[0581] 3 Compound III is an oligonucleotide having the formula SEQ ID NO:423-(ZEN)-SEQ ID NO:443.

[0582] 4 Compound IV is an oligonucleotide having the formula SEQ ID NO:426-(ZEN)-SEQ ID NO:444.

[0583] Oligonucleotide sequences are shown 5'-3'. Capital letters = DNA; lowercase = RNA; underlined = 2'-O-methyl RNA; * = phosphorothioate internucleotide linkage; ppp = triphosphate; ZEN = naphthyl-azo modifier, dark quencher; FAM = 6-carboxyfluorescein; HEX = hexachlorofluorescein.

[0584] Treatment with unmodified or chemically modified truncated crRNA:tracrRNA complexes did not result in a detectable increase in IFIT1 or IFITM1 expression above baseline. In contrast, treatment with the longer IVT sgRNA resulted in a 45-fold induction of IFITM1 and a 220-fold induction of IFIT1. Thus, the use of the short crRNA:tracrRNA complexes of the present invention is absent of the significant stimulation of the innate immune system that occurs with sgRNAs.

[0585] Embodiment 13

[0586] The following examples combine the modification patterns identified in Examples 6 and 7, which are particularly effective in demonstrating new highly modified crRNA and tracrRNA compositions that perform with high efficiency in mammalian CRISPR genome editing applications.

[0587] A series of crRNA and tracrRNA (Table 12) were synthesized, which had chemical modifications as shown. The crRNA used a 20-base pre-spacer sequence domain targeting the same site (38285) in the human HPRT1 gene at the 5' end and a 16-base tracrRNA binding domain at the 3' end. Using a 67-nucleotide or 62-nucleotide truncated form of the tracrRNA sequence, tracrRNA with the chemical modifications shown was synthesized. The crRNA and tracrRNA listed in Table 12 were paired as shown and transfected into HEK-Cas9 cells at a concentration of 30 nM and treated as described in the previous example. The relative gene editing activity was evaluated by comparing the cleavage rate in the HPRT1 gene obtained by cleavage with T7EI mismatch endonuclease and the quantitative measurement of the product performed using a fragment analyzer.

[0588] Table 12: Activity of highly modified crRNA:tracrRNA complexes in directing Cas9-mediated gene editing in mammalian cells.

[0589]

[0590] Oligonucleotide sequences are shown 5'-3'. Lowercase = RNA; Underlined = 2'-O-methyl RNA; Lowercase italics = 2'F RNA; * = phosphorothioate internucleotide linkages. For each dose studied, the relative functional activity of each complex is indicated by the % cleavage in the T7EI heteroduplex assay.

[0591] crRNA:tracrRNA pairs #1 and #2 show that highly 2'F RNA modified crRNA (SEQ ID No.: 448, wherein 22 / 36 residues are modified, or 61%) is highly functional when paired with unmodified tracrRNA (SEQ ID No.: 2) or highly 2'OMe modified tracrRNA (SEQ ID No.: 100). crRNA:tracrRNA pairs #3 and #4 show that tracrRNA compositions with moderate (SEQ ID No.: 450, wherein 19 / 67 residues are modified, or 28%) or high (SEQ ID No.: 449, wherein 46 / 67 residues are modified, or 69%) 2'F RNA modification levels are highly functional. Information derived from Example 6 (particularly the 2'OMe 'walk', SEQ ID No.: 144-162) was used to identify specific residues that can be modified within the internal domain of the tracrRNA (see Figure 6 ). crRNA:tracrRNA pair #5 demonstrates that a very highly modified tracrRNA, which in this case is a truncated 62 nucleotide design (SEQ ID No.: 451, 51 / 62 residues modified by 2'OMe RNA, or 82%), is highly potent in triggering CRISPR genome editing in mammalian cells. Thus, the original 89 RNA nucleotide wild-type tracrRNA has been optimized herein to a form with as few as 11 RNA residues (11 / 62) remaining, thereby significantly reducing the risk of RNA-based activation of the mammalian innate immune system and minimizing the nuclease-sensitive RNA content of the tracrRNA.

[0592] All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0593] Unless otherwise specified herein or the context is obviously contradictory, the use of the terms "one" and "said" and similar indicators in the context of describing the present invention (especially in the context of the appended claims) should be interpreted as covering the singular and plural. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" will be interpreted as open terms (that is, meaning "including but not limited to"). Unless otherwise specified herein, the description of the numerical range herein is intended only to be used as a shorthand method for individually referring to each individual numerical value falling within the range, and each individual numerical value is incorporated into this specification as if it is individually narrated herein. Unless otherwise specified herein or the context is obviously contradictory, all methods described herein can be performed in any suitable order. Unless otherwise specified, the use of any and all examples or exemplary language (e.g., "such as") provided herein is only intended to better illustrate the present invention, without limiting the scope of the present invention. The language in this specification should not be interpreted as indicating that any unclaimed element is necessary for the practice of the present invention.

[0594] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, changes to these preferred embodiments may become apparent to those of ordinary skill in the art. The inventor expects that such changes will be appropriately used by those skilled in the art, and the inventor intends to implement the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter described in the appended claims as allowed under applicable law. In addition, unless otherwise specified herein or the context is clearly contradictory, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

Claims

1. An isolated crRNA comprising a chemically modified form of formula (I): 5'-XZ-3'(I), wherein X represents a sequence comprising a target-specific protospacer domain, and Z represents a sequence comprising a tracrRNA binding domain, the protospacer domain comprising 17 nucleotides to 20 nucleotides, the tracrRNA binding domain comprising 12 nucleotides to 19 nucleotides, wherein the isolated crRNA exhibits activity in a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system, The chemically modified form of formula (I) comprises a chemically modified nucleotide having a terminal modification group selected from: a propylene glycol (C3) spacer, N,N-diethyl-4-(4-nitronaphthalene-1-ylazo)-aniline ("ZEN"), and a reversed dT residue.

2. The isolated crRNA according to claim 1, wherein the chemically modified form of formula (I) further comprises chemically modified nucleotides having additional modifications selected from ribose modifications and internucleotide modified bonds.

3. The isolated crRNA according to claim 2, wherein the chemically modified nucleotide has an additional modification consisting of a ribose modification selected from 2'OMe, 2'F, bicyclic nucleic acid and locked nucleic acid (LNA).

4. The isolated crRNA of claim 2, wherein the chemically modified nucleotides have additional modifications consisting of internucleotide modified bonds consisting of phosphorothioate modifications.

5. The isolated crRNA according to claim 2, wherein the chemically modified form of formula (I) is selected from SEQ ID NO.: 163, 179-181, 235-237, 240-243, 256, 257, 268-271 and 385-406.

6. An isolated crRNA selected from SEQ ID NO.: 14, 164, 166-169, 171, 174-178, 186, 187, 189-207, 212-214, 221-224, 226-228, 233-249, 252, 256-259, 264-271, 291-374, 376, 377, 379-384, and 445-448, wherein the isolated crRNA is active in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein endonuclease system.

7. An isolated crRNA selected from SEQ ID NO.: 430-439, wherein the isolated crRNA is active in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein endonuclease system.

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