Modified CPF1 guide RNA

Abstract

The present invention provides nucleic acids comprising Cpf1 crRNA, a processing sequence 5' of Cpf1 crRNA, and an extension sequence 5' of the processing sequence. The present invention also provides compositions comprising nucleic acids, vectors, and optionally Cpf1. In addition, the present invention provides methods for genetically modifying eukaryotic target cells, comprising contacting eukaryotic target cells with nucleic acids or compositions to genetically modify target nucleic acids in the cells.

Description

[0001] Incorporation by reference of electronically submitted materials

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 567,123, filed on October 2, 2017; U.S. Provisional Patent Application No. 62 / 617,138, filed on January 12, 2018; and U.S. Provisional Application No. 62 / 697,327, filed on July 12, 2018, the entire disclosures of which are incorporated herein by reference. Background Art

[0004] RNA-guided endonucleases have proven to be effective tools for genome engineering in multiple cell types and microorganisms. RNA-guided endonucleases generate site-specific double-stranded DNA breaks or single-stranded DNA breaks in target nucleic acids. When the cleavage of the target nucleic acid occurs intracellularly, the breaks in the nucleic acid can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0005] RNA-guided endonucleases and their gene editing components (e.g., guide RNAs) delivered directly into cells both in vitro and in vivo have great potential as a therapeutic strategy for treating genetic diseases. However, currently, it is challenging to deliver these components directly into cells with reasonable efficiency.

[0006] Therefore, there is a need to identify new compositions and related methods to improve cellular delivery and other properties of RNA-guided endonucleases that will enhance genome engineering. The present invention provides such compositions and related methods. Summary of the invention

[0007] The present invention is a nucleic acid comprising a Cpf1 crRNA with an extension sequence. In one aspect, the nucleic acid comprises a Cpf1 crRNA and an extension sequence at the 5' end of the Cpf1 crRNA, wherein the extension sequence comprises less than about 60 nucleotides. In another aspect, the nucleic acid comprises a Cpf1 crRNA, a processing sequence 5' of the Cpf1 crRNA, and an extension sequence 5' of the processing sequence. Also provided is a composition comprising a nucleic acid, a vector, and optionally a Cpf1 protein or a vector encoding the protein.

[0008] The present invention also provides a method for genetically modifying a eukaryotic target cell. The method involves contacting the eukaryotic target cell with a nucleic acid comprising a Cpf1 crRNA as described herein.

[0009] These and other aspects of the invention are described in more detail in the following sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A is a graph comparing the delivery of unmodified crRNA complexed with Cpf1 using cationic liposomes (lipofectamine) or electroporation (nucleofection).

[0011] Figure 1B and 1C is a graph comparing cationic liposome-mediated delivery and NHEJ generation of Cpf1-crRNA complexes containing unmodified crRNA (41 nucleotides in length) and extended crRNA.

[0012] Figure 1D Schematic diagram of the structure of 41 nucleotide (nt) unmodified crRNA and extended crRNA. Arrows represent Cpf1 cleavage sites.

[0013] Figure 1E It shows about Figure 1D Figure 2. Plot of NHEJ efficiency of the crRNA constructs shown in Figure 2.

[0014] Figure 1F is a diagram depicting cellular delivery of Cpf1 RNPs with different lengths using cationic liposomes and crRNAs labeled with fluorescent dyes.

[0015] Figure 2 A graph showing gene editing efficiency of GFP knockdown according to 5′ extended crRNA delivered to GFP-HEK cells via electroporation (right) and a schematic diagram of crRNA (left) are provided.

[0016] Figure 3A is a schematic diagram of the in vivo study in AI9 mice.

[0017] Figure 3B is a schematic diagram of the gastrocnemius muscle injection site and imaging segment.

[0018] Figure 4A is a graph of HDR frequencies of crRNAs with different extensions delivered with donor DNA using electroporation.

[0019] Figure 4B is a graph of the percentage of GFP- cells with crRNAs of varying extensions delivered along with donor DNA, illustrating the efficiency of NHEJ using electroporation.

[0020] Figure 4C is a graph of the percentage of BFP-cells with crRNA of different extensions delivered together with donor DNA, showing the NHEJ efficiency using electroporation.

[0021] Figure 4D is a graph of the percentage of GFP- cells with extended crRNA delivered using electroporation with and without single-stranded DNA (ssDNA) that has no homology to the target sequence.

[0022] Figure 4E is a graph showing gene editing as a percentage of GFP- cells using electroporated crRNA extended with 100nt RNA and 9nt RNA having no homology to the target sequence.

[0023] Figure 4F is a graph of the percentage of GFP- cells using electroporated crRNA with and without a 4nt extension and further modified with chemical moieties.

[0024] Figure 5A and 5B A schematic diagram for conjugating crRNA and donor DNA is provided.

[0025] Figure 5C is an image of a gel electrophoresis separation showing the release of donor DNA and crRNA from the conjugated crRNA / DNA molecules after reduction with thiol.

[0026] Figure 6 is a diagram demonstrating that Cpf1 conjugated to HD-RNA induces NHEJ in GFP-HEK cells after transfection with PAsp(DET), a cationic polymer.

[0027] Figure 7 is a graph demonstrating that Cpf1 conjugated to HD-RNA induces HDR in GFP-HEK cells after transfection with PAsp(DET), a cationic polymer.

[0028] Figure 8 The sequence of the Cpf1 protein is provided.

[0029] Fig. 9 Examples of Cpf1 processing sequences are provided.

[0030] Fig. 10A A schematic diagram of crRNA conjugated to donor DNA is shown.

[0031] Fig. 10B The sequences used in crRNA conjugated to donor DNA are shown.

[0032] Fig. 10C is a graph of the percentage of RFP+ cells in primary Ai9 myoblasts after treatment with various crRNAs and Cpf1 using electroporation.

[0033] Fig. 10D is a graph of the percentage of RFP cells in primary Ai9 myoblasts transfected with 100 nt DNA or RNA.

[0034] Fig. 10E is a graph of NHEJ efficiency in HepG2 cells transfected with Cpf1 RNP with or without 9 nt extension of crRNA targeting Serpina1 gene using electroporation.

[0035] Fig.11A RNA structures that can be used in crRNA extension are shown.

[0036] Fig. 11B Shown are trinucleotide repeats that can be used to provide various RNA structures.

[0037] Fig. 11C Shown is the intersection of hybridized extension sequences of crRNA in the kissing loop, which can be used to form crRNA multimers.

[0038] Fig.11D The intersection of hybridized stretch sequences of crRNA is shown to form a trimer (panel (i)) or an octamer (panel (ii)).

[0039] Fig.12 The editing efficiency (% BPF-) of various Cpf1 crRNAs in HEK293T cells expressing BFP is shown. MS is 2'-OMe 3'-phosphorothioate modification on the first three nucleotides starting from the 5' end, +9du is 2'-deoxy modification on the 9th nucleotide starting from the 5' end, and +9S is phosphorothioate modification on the first 9 nucleotides starting from the 5' end. 7 days after electroporation, BFP knockout efficiency was measured by flow cytometry. Mean ± SE, n = 3. By Student's t test, all extended crRNAs showed statistically significant differences from unmodified crRNA, with p values ​​less than 0.05.

[0040] Fig.13A is a diagram of unmodified Cas9 sgRNA and Cpf1 crRNA; and

[0041] Fig. 13B and 13C is a graph showing the relative activities of Cas9 sgRNA and Cpf1 crRNA according to GFP knockdown.

[0042] Fig.14 Schematic diagram of an extended crRNA modified with biotin and avidin and linked to a targeting molecule comprising biotin.

[0043] Fig.15A Schematic diagram of chemical modification of crRNA with extension.

[0044] Fig. 15B It is a graph quantifying the amount of remaining crRNA after incubation in serum.

[0045] Fig. 15C is a graph of the percentage of GFP- cells after delivery of crRNA with 9 nt extension and chemical modification using cationic liposomes.

[0046] Fig.15D is a graph comparing the percentage of GFP- cells after delivery of extended crRNA with chemical modifications and unmodified extended crRNA together with Cpf1 using electroporation.

[0047] Fig.16 is a graph comparing cationic polymer-mediated delivery and NHEJ generation of Cpf1-crRNA complexes containing unmodified crRNA (41 nt), 9 base pair extended crRNA (50 nt total), or 59 base pair extended crRNA (100 nt total). DETAILED DESCRIPTION

[0048] The present invention provides a modified guide nucleic acid for Cpf1, referred to as "crRNA", which has enhanced properties compared to conventional crRNA molecules. As used herein, crRNA refers to a nucleic acid sequence (e.g., RNA) that binds to the RNA-guided endonuclease Cpf1 and targets the RNA-guided endonuclease to a specific position in the target nucleic acid to be cut by Cpf1. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system, which is related to type V adaptive immunity. Cpf1 does not require tracrRNA molecules like other CRISPR enzymes, and only requires a single crRNA molecule to work. Cpf1 prefers the "TTN" PAM motif, which is located 5' upstream of its target. In addition, the cleavage site of Cpf1 is staggered by about 3-5 bases, which produces a "sticky end" (Kim et al., 2016. "Genome-wide analysis reveals specificities of Cpf1endonucleases in human cells", published online on June 6, 2016). These sticky ends with 3-5 bp overhangs are thought to facilitate NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends.

[0049] Those skilled in the art will appreciate that the Cpf1 crRNA can be from any species or derived or isolated from any synthetic or naturally occurring variant or ortholog from any source. That is, the Cpf1 crRNA can have the required elements (e.g., sequence or structure) of a crRNA that recognizes (binds to) any Cpf1 polypeptide or ortholog from any bacterial species, or a synthetic variant thereof. Examples of Cpf1 crRNA sequences are described in Fig. 9 ; thus, for example, examples of Cpf1 crRNAs include those comprising any one of SEQ ID NOs: 21-39). An example of a Cpf1 crRNA sequence of a synthetic variant Cpf1 is a crRNA corresponding to a MAD7 Cpf1 ortholog by Inscripta, Inc. (CO, USA). Another example of a Cpf1 variant is a Cpf1 modified to reduce or eliminate RNase activity, for example, by introducing a modification (e.g., an H800A mutation or an H→A mutation at a corresponding position) of H800A, K809A, K860A, F864A, and R790A of Acidaminococcus Cpf1 (AsCpf1), or a corresponding position of a different Cpf1 ortholog.

[0050] Typically, crRNA includes a targeting domain and a stem-loop domain located at 5' of the targeting domain. The overall length of crRNA is not particularly limited, as long as it can guide Cpf1 to a specific position in the target nucleic acid. The stem-loop domain is generally about 19-22 nucleotides (nt) in length, and the targeting / guiding domain is about 14-25nt (e.g., at least about 14nt, 15nt, 16nt, 17nt or 18nt) anywhere. In some embodiments, the overall length of Cpf1 crRNA can have a length of 20 to 100nt, 20nt to 90nt, 20nt to 80nt, 20nt to 70nt, 20nt to 60nt, 20nt to 55nt, 20nt to 50nt, 20nt to 45nt, 20nt to 40nt, 20nt to 35nt, 20nt to 30nt or 20nt to 25nt.

[0051] One aspect of the disclosure provides a nucleic acid comprising a Cpf1 crRNA, an extension sequence 5' to the crRNA, and optionally a processing sequence, which may be located between the crRNA and the extension sequence, within the extension sequence, or 5' to the extension sequence.

[0052] Extension sequence

[0053] The nucleic acid of the present invention comprises an extension sequence located at 5' of crRNA. The extension sequence may comprise any combination of nucleic acids (i.e., any sequence). In one embodiment, the extension sequence increases the overall negative charge density of the nucleic acid molecule and improves the delivery of nucleic acids including crRNA.

[0054] In some embodiments, the extension sequence can be cut once in the cell. It is not desirable to be bound by any particular theory or mechanism of action, and it is believed that Cpf1 can cut the extension sequence. However, in some applications, it is desirable to use a construct in which the extension sequence is not cut from Cpf1 crRNA. Therefore, in some embodiments, the extension sequence cannot be cut by Cpf1crRNA. For example, the extension sequence or some of its parts or regions may include one or more modified internucleotide bonds (modified "main chain"), which are resistant to cutting by Cpf1 crRNA (e.g., nuclease resistance). Examples of modified internucleotide bonds include, but are not limited to: phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, bridged nucleic acid (BNA) or phosphotriester modified bonds and combinations thereof. The extension sequence or some of its parts may also include synthetic nucleotides, such as xenonucleic acid (XNA) resistant to nucleases. XNA is a nucleic acid in which the furanose ring of DNA or RNA is replaced with a five- or six-membered modified ribose molecule, such as 1,5-anhydrohexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), and 2'4'-C-(N-methylaminomethylene) bridged nucleic acid (BNA), 2'-O,4'-C-methylene-β-D-ribonucleic acid or locked nucleic acid (LNA), ANA (arabinose nucleic acid), 2'-fluoro-arabinose nucleic acid (FANA), and α-L-threofuranosyl nucleic acid (TNA). In addition, any combination thereof may also be used.

[0055] The length of the extended sequence is not particularly limited, as long as the extended sequence increases the overall negative charge density. For example, the extended sequence can have at least about 2 nucleotides (nt) up to about 1000nt (for example, at least about 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,300,400,500,600,700,800 or 900, and up to about 1000nt) length. In one aspect, the extended sequence length is no more than about 100 nucleotides, for example, a length is no more than about 80 nucleotides, a length is no more than about 60 nucleotides or a length is no more than about 40 nucleotides (for example, a length is no more than about 30 nucleotides or a length is no more than about 20 nucleotides). Any one of the aforementioned lower limit and the upper limit about length can be expressed as a scope. Shorter sequences (e.g., no more than about 15 nucleotides, or no more than about 10 nucleotides) may also be used. In some embodiments, the extended sequence comprises at least about 2 nucleotides, such as at least about 4 nucleotides, at least about 6 nucleotides, or even at least about 9 nucleotides. Any of the foregoing may be expressed as a range. Thus, for example, the extension sequence can be about 2-60 nucleotides (e.g., about 2-40 nucleotides, about 2-30 nucleotides, about 2-20 nucleotides, about 2-15 nucleotides, or about 2-10 nucleotides), about 4-60 nucleotides (e.g., about 4-40 nucleotides, about 4-30 nucleotides, about 4-20 nucleotides, about 4-15 nucleotides, or about 4-10 nucleotides); about 6-60 nucleotides (e.g., about 6-40 nucleotides, about 6-30 nucleotides, about 6-20 nucleotides, about 6-15 nucleotides, or about 6-10 nucleotides); or about 9-60 nucleotides (e.g., about 9-40 nucleotides, about 9-30 nucleotides, about 9-20 nucleotides, about 9-15 nucleotides, or about 9-10 nucleotides).

[0056] In some embodiments, the extension sequence has no function other than conferring a greater overall negative charge density to the nucleic acid construct. In this embodiment, for example, the extension sequence is a random or non-coding sequence. In some cases, for example, when a processing sequence is used, the sequence can be degraded when the processing sequence is cut and released from the nucleic acid construct.

[0057] In other embodiments, the extension sequence has a function of being separated and isolated from the nucleic acid construct by giving a larger overall negative charge density. The extension sequence may have any additional function. For example, the extension sequence may provide a hybridization site for another nucleic acid, such as a donor nucleic acid. In addition, in some embodiments, the extension sequence may be an aptamer and / or promote cell binding. However, it is sometimes undesirable to recruit proteins other than the RNA-guided endonuclease and the combination of the guide RNA. In addition, the aptamer sequence generally has a complex folding pattern, which may be large and not compact. Therefore, in other embodiments, the extension sequence is not an aptamer sequence.

[0058] In some embodiments, the extension sequence may include a sequence encoding a protein whose expression is desired in the target cell to be edited. For example, the extension sequence may include a sequence encoding an RNA-guided endonuclease, such as an RNA-guided endonuclease paired with (i.e., recognized and guided by) the crRNA used in the nucleic acid construct. The extension sequence may include, for example, a sequence of an mRNA of an RNA-guided endonuclease.

[0059] In some embodiments, the extension part is self-folded (self-hybridized) to provide a structured extension part. There is no restriction on the type of structure provided. The extension part can have a random coil structure; however, in some embodiments, the structure that the extension part has is more compact than the random coil structure of the same number of nucleotides, which provides a larger negative charge density. By increasing the overall length of the extension part, the negative charge of the molecule increases. When a more compact structure is used, the overall negative charge density of the molecule is further increased. Tightness or charge density can be determined according to the mobility in gel electrophoresis. More specifically, if gel electrophoresis is performed for two nucleic acids with the same number of nucleotides running together on the same gel, the nucleic acid with higher mobility (moving the farthest in the gel) is considered to have a more compact structure.

[0060] In another embodiment, the extended sequence comprises at least one semi-stable hairpin structure, a stable hairpin structure, a pseudoknot structure, a G-quadruplex structure, a bulge loop structure, an inner loop structure, a branched loop structure, or a combination thereof. These types of nucleotide structures are known in the art and are described in detail in the literature. Fig.11ASchematically illustrated in. It should be understood that the illustration is only for the purpose of illustrating the general structure, and is not expected to be the detailed illustration of the actual molecular structure. Those skilled in the art recognize that the hairpin structure, for example, can have a non-complementary region of intersperse, which produces a "protrusion" or other changes in the structure, and other depicted structures can include similar changes. The structure of a given nucleotide sequence can be determined using an available algorithm (for example, by "The mfold Web Server" operated by the Rensselaer Polytechnic Institute and The RNA Institute, College of Arts and Sciences, State University of New York in Albany; also referring to M.Zuker, DH Mathews & D.H. Turner. Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide In RNA Biochemistry and Biotechnology, 11-43, J. Barciszewski and BFC Clark, editors, NATO ASI Series, Kluwer Academic Publishers, Dordrecht, NL, (1999)).

[0061] The provided structural types can use repetitive trinucleotide motifs (e.g., Fig. 11B) is controlled. The repetitive trinucleotide motif is a motif of three nucleotides that are repeated at least twice (e.g., repeated twice or more times, three times or more times, four times or more times, five times or more times, six times or more times, seven times or more times, eight times or more times, or ten times or more times) in the sequence. Therefore, the extension sequence can include a repetitive trinucleotide motif. In one embodiment, the extension sequence includes a repetitive trinucleotide motif of CAA, UUG, AAG, CUU, CCU, CCA, UAA or a combination thereof, which provides a random coil sequence. In another embodiment, the extension sequence includes a repetitive trinucleotide motif of CAU, CUA, UUA, AUG, UAG or a combination thereof, which provides a semi-stable hairpin structure. In another embodiment, the extension sequence includes a repetitive CNG trinucleotide motif (e.g., CGG, CAG, CUG, CCG), a repetitive trinucleotide motif of CGA or CGU or a combination thereof, which provides a stable hairpin structure. In another embodiment, the extended sequence comprises a repetitive trinucleotide motif of AGG, UGG or a combination thereof, which provides a quadruplex (or G-quadruplex) structure. In yet another embodiment, the extended sequence comprises a combination of the aforementioned trinucleotide motifs and a combination of different structures produced therefrom. For example, the extended sequence can have a region comprising a random coil structure, a region comprising a semi-stable hairpin, a region comprising a stable hairpin and / or a region comprising a quadruplex. Therefore, each region can comprise a repetitive trinucleotide motif associated with the structure shown. Non-limiting examples of structures are presented in the following table:

[0062] Table 1. Representative RNA extension sequences (35 nucleotides) and their corresponding structures.

[0063]

[0064] Extension sequence can also be used to produce crRNA polymers; Therefore, in another embodiment, there is provided a crRNA polymer, which comprises two or more crRNA molecules (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or even 8 or more crRNA molecules), wherein each crRNA comprises an extension sequence as described herein, and the crRNA molecules of the polymer are connected by its extension sequence, for example, via base pairing or hybridization. Therefore, in one embodiment, each crRNA of the polymer comprises an extension sequence, and the extension sequence comprises a region fully complementary to the extension region of another crRNA of the polymer, to promote hybridization. The complementary region can have any suitable length (e.g., 4nt or more, 6nt or more, 8nt or more, 10nt or more, 15nt or more, etc.) that promotes interaction. The crRNA polymer can be used for example to deliver multiple crRNAs simultaneously, for example, when multiple crRNAs are needed for a specific treatment strategy. An example of such use is exon skipping, in which a DNA fragment is cut by two crRNAs to restore a functional reading frame (e.g., Ousterout DG et al. (2015), Multiplex CRISPR / Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne Muscular Dystrophy. Nat Commun. 6: 6244). Exon skipping requires two crRNAs, each of which targets a different site in the nucleus for targeting (one at the 5' site and the other at the 3' site). Ideally, the ratio of the two crRNAs should be 1: 1; however, it is difficult to maintain this ratio. By pairing the crRNAs in the polymer (e.g., each containing a different targeting sequence) via an appropriate extension structure, delivery at a desired ratio can be facilitated.

[0065] In one embodiment, two or more crRNAs with structured stretches participate in RNA "kissing" interactions (aka loop-loop interactions), which occur when an unpaired nucleotide in one structured stretch sequence (e.g., a hairpin loop) base pairs with an unpaired nucleotide in another structure (e.g., another hairpin loop) on a second crRNA. Examples of this type of interaction are described in Fig. 11C The formation of kissing loops or other structures polymerizes two or more crRNA molecules. This strategy can be used to connect several crRNA molecules.

[0066] Hybridization of complementary sequences in the extension on each crRNA can also be used to promote polymerization. For example, a supramolecular crRNA structure can be constructed via an extension region with self-assembly ability. For example, a trimer can be formed by three RNA molecules with appropriately placed hybridization regions (e.g., Fig.11D , panel (i); Shu D, Shu Y, Haque F, Abdelmawla S, & Guo P (2011) Thermodynamically stable RNA three-way junctions as platform for constructing multi-functional nanoparticles for delivery of therapeutics. Nat Nanotechnol. 6 (10): 658-667.)). Similarly, RNA octamers can be generated by assembling sixteen RNA molecules ( Fig.11D , inset (ii); Yu J, Liu Z, Jiang W, Wang G, & Mao C (2014) De novo design of anRNAtile that self-assembles into a homo-octameric nanoprism. Nat Commun. 6: 5724)).

[0067] Any of the aforementioned types of extensions can be used with or without a processing sequence. In some embodiments, a nucleic acid can include multiple processing sequences and extension sequences. For example, a nucleic acid can further include a second processing sequence 5' of a first extension sequence and a second extension sequence 5' of a second processing sequence. The second processing and extension sequence can be the same as the first processing and extension sequence (e.g., repeated), or either or both of the second processing sequence and the second extension sequence can be different from the first processing sequence and / or extension sequence. Nucleic acids are not particularly limited to any number of processing and extension sequences, and can have 2, 3, 4, 5, etc. processing and / or extension sequences.

[0068] The 5' end of the nucleic acid construct (i.e., the processing sequence or extension sequence at the 5' end, where applicable) can be further modified as desired. For example, the 5' end can be modified with a functional group (e.g., a functional group that participates in a bioorthogonal or "click" chemistry). For example, the 5' end of the nucleic acid can be chemically modified with an azide, a tetrazine, an alkyne, a strained olefin, or a strained alkyne. Such modifications can use appropriately paired functional groups to facilitate attachment of a desired chemical moiety or molecule to the construct.

[0069] The 5' end of the nucleic acid can be optionally modified to include a biofunctional molecule via the above-mentioned bioorthogonal or "click" chemistry. The biofunctional molecule can be any molecule that enhances the delivery or activity of an RNA-guided endonuclease, or provides some other desired function, such as targeting a nucleic acid to a specific destination (e.g., targeting a portion of a specific protein, cell receptor, tissue, etc.), or facilitating tracking of the construct (e.g., a detectable marker, such as a fluorescent marker, a radioactive marker, etc.). Examples of biofunctional molecules include, for example, endolytic enzyme polymers, donor DNA molecules, amino sugars (e.g., N-acetylgalactosamine (GalNAc) or tri-GalNAc), guide and / or tracer RNA (e.g., a single guide RNA), and other peptides, nucleic acids, and targeting ligands (e.g., antibodies, ligands, cell receptors, aptamers, galactose, sugars, small molecules). In one embodiment, the crRNA comprises a biotin or avidin (or streptavidin) molecule conjugated to a crRNA extension, allowing the modified crRNA to bind to another molecule (e.g., a targeting molecule or peptide) conjugated to avidin / streptavidin or biotin, as appropriate (see, e.g., Fig.14 ). In another embodiment, the crRNA extension portion can be covalently linked to an amino sugar in any suitable manner, for example, via a linker. As used herein, "amino sugar" is a sugar molecule in which the hydroxyl group has been replaced by an amine group (e.g., galactosamine) and / or a nitrogen that is part of a complex functional group (e.g., N-acetylgalactosamine (GalNAc); tri-N-acetylgalactosamine (triantennary N-acetylgalactosamine)). Amino sugars can be modified to contain an optional spacer. Examples of amino sugars include N-acetylgalactosamine (GalNAc), trivalent GalNAc, or triantennary N-acetylgalactosamine. An example of an amino sugar group includes the following:

[0070] Wherein the joint can be any commonly known in the art, and each joint can be identical or different from each other.Usually, joint is saturated or unsaturated aliphatic or heteroaliphatic chain.Aliphatic or heteroaliphatic chain usually comprises 1-30 member (for example 1-30 carbon, nitrogen and / or oxygen atom), and can be replaced by one or more functional groups (for example one or more ketones, ethers, esters, amides, alcohols, amines, ureas, thioureas, sulfoxides, sulfones, sulfonamides and / or disulfide groups).In some cases, use shorter aliphatic or heteroaliphatic chain (for example, about 1-15 member in chain, about 1-10 member, about 1-5 member, about 3-15 member, about 3-10 member, about 5-15 member or about 5-10 member). In other cases, longer aliphatic or heteroaliphatic chains are used (e.g., about 5-30 members, about 5-25 members, about 5-20 members, about 10-30 members, about 10-25 members, about 10-20 members, about 15-30 members, about 15-25 members, or about 15-20 members in the chain). Examples of spacers include substituted and unsubstituted alkyls, alkenyls, and polyethylene glycols (e.g., PEG 1-10 or PEG 1-5), or combinations thereof. More specific examples are provided for illustration as follows:

[0071]

[0072] Prior to conjugation to the linker, the amino sugar may comprise a functional group (e.g., an azide, a tetrazine, an alkyne, a strained olefin, or a strained alkyne) that allows conjugation to an appropriately paired functional group attached to the crRNA extension (e.g., at the 5' end). Thus, for example, prior to conjugation to the extended crRNA, the amino sugar may comprise:

[0073]

[0074] Among them A 2 Contains azides, tetrazines, alkynes, strained alkenes or strained alkynes, as described herein. More specific examples are as follows:

[0075]

[0076] Among them A 2 Containing an azide, tetrazine, alkyne, strained alkene or strained alkyne, as described herein, for example:

[0077]

[0078] Processing sequence

[0079] In some embodiments, crRNA includes a processing sequence. The processing sequence is a nucleic acid sequence that is self-cut in vitro or in vivo by Cpf1 without the need for a guide / targeting sequence. It is not desirable to be bound by any particular theory or mechanism of action, and it is believed that when present, the processing sequence is cut after entering the cell, and the crRNA is released from any extension sequence. The processing sequence can be located between the crRNA and the extension sequence. In this configuration, after the processing sequence is cut, the crRNA is released from the extension sequence of the nucleic acid construct provided herein.

[0080] The processing sequence can also be located within the extension sequence, at the 5' of the extension sequence, or can serve as an extension sequence. In addition, multiple processing sequences can be used. For example, the second processing sequence can serve as an extension sequence alone or together with another nucleotide sequence. However, the extension sequence is generally different from the processing sequence (if present). In addition, in one embodiment, the extension sequence does not include a processing sequence and / or any other complete (complete) crRNA sequence.

[0081] In some embodiments, the processing sequence is located at the tight 5' of the crRNA (i.e., directly attached to the crRNA sequence). In other embodiments, the spacer sequence may be present between the crRNA and the processing sequence. The spacer sequence may have any length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt), provided that it does not prevent Cpf1 cleavage of the processing sequence or the function of the crRNA released after cleavage.

[0082] In one embodiment, the processing sequence comprises a fragment of a direct repeat sequence of a Cpf1 array. The Cpf1 array (sometimes also referred to as pre-crRNA) is a naturally occurring array that comprises a direct repeat sequence and a spacer sequence between each direct repeat. The direct repeat portion of the array includes two parts: a crRNA sequence portion and a processing portion. Within a given direct repeat, the processing portion is located 5' of the crRNA sequence portion, often just 5' of the processing portion. According to this embodiment, the processing sequence of the nucleic acid provided herein comprises at least one fragment of the processing portion of the direct repeat sufficient to achieve Cpf1 cleavage. For example, the processing sequence may comprise a fragment of at least 5 adjacent nucleotides of the processing portion of the direct repeat sequence, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17nt of the processing portion of the direct repeat sequence (or the entire processing portion of the direct repeat sequence), the length of which depends on the type from which the direct repeat originates. In some embodiments, the processing sequence comprises the entire processing portion of the direct repeat sequence. Direct repeats can be from the Cpf1 array of any microorganism. In Fig. 9 Examples of direct repeat sequences, and processing portions of direct repeat sequences, are provided in . The processing sequence of the nucleic acid of the invention may comprise Fig. 9A fragment of any processed sequence or the entire sequence (eg, SEQ ID NOs: 2-20).

[0083] Donor nucleic acid

[0084] The nucleic acid construct provided herein can further include a donor nucleic acid (also referred to as a donor polynucleotide). A donor polynucleotide is a nucleic acid inserted into a cleavage site induced by an RNA-guided endonuclease (e.g., Cpf1). The nucleic acid of a donor polynucleotide can be any type of nucleic acid known in the art. For example, the nucleic acid can be a DNA, RNA, a DNA / RNA hybrid, an artificial nucleic acid, or any combination thereof. In one embodiment, the nucleic acid of a donor polynucleotide is a DNA, also referred to herein as a "donor DNA".

[0085] Donor polynucleotide is usually single-stranded, and serves as a template for producing a double-stranded DNA containing a desired sequence. Donor polynucleotide and the genomic sequence region (about 50 bases or less, about 30 bases or less, about 15 bases or less, or about 10 bases or less, about 5 bases or less, or next to the cleavage site) of the genomic sequence of the lateral cleavage site contain enough identity (e.g., 85%, 90%, 95% or 100% sequence identity) to support the homology directional repeat between the donor sequence and the genomic sequence (donor sequence has enough sequence identity with it) of the lateral cleavage site. Donor polynucleotide sequence can have any length, but must have a sufficient number of nucleotides with sequence identity on both sides of the cleavage site to promote HDR. These regions of donor polynucleotide are referred to as homology arms. Homologous arms can have the same number of bases or different numbers of bases, and are generally at least 5 nucleotides in length (e.g., 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, or even 200 nucleotides or more) respectively. Donor polynucleotides also contain a central region flanked by homologous arms, which contain mutations or other target dna sequences. Therefore, the overall length of donor polynucleotides is generally greater than the total length of two homologous arms (e.g., about 15 nucleotides or more, about 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, or even 200 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more).

[0086] The donor polynucleotide sequence is usually different from the target genomic sequence. In contrast, the donor polynucleotide sequence may contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, as long as the homology arms have sufficient sequence identity to support HDR. The donor polynucleotide sequence may further comprise a sequence that facilitates detection of successful insertion of the donor polynucleotide.

[0087] Can be by method well known to persons skilled in the art, protect the end of donor polynucleotide (for example, avoid exonucleolytic degradation).For example, one or more dideoxynucleotide residues are added to the 3 ' end of linear molecule, and / or self-complementary oligonucleotide is connected to one or two ends.Be used to protect exogenous polynucleotide from degrading other method and include but not limited to, add terminal amino and use modified internucleotide bonding, for example phosphorothioate, phosphoramidate and O-methyl ribose or deoxyribose residue.

[0088] In some embodiments, the donor polynucleotide (e.g., donor DNA) is covalently linked to the 5' end of the Cpf1 crRNA, the 5' end of the processing sequence, or the 5' end of the extension sequence. In a preferred embodiment, the donor polynucleotide is linked to the 5' end of the extension sequence. In some embodiments, the bonding between the donor DNA and the nucleic acid is reversible (e.g., a disulfide bond).

[0089] In some embodiments, the donor DNA is covalently linked to the nucleic acid construct. For example, the donor polynucleotide can be linked to the processing sequence and serve as an extension sequence located 5' of the processing sequence. In another embodiment, the donor polynucleotide can be linked at the 5' of the extension sequence.

[0090] Nucleic acid and donor DNA can be connected or conjugated by any method known in the art. In some embodiments, the 3' end of the donor DNA and the 5' end of the nucleic acid are modified to promote bonding. For example, the 5' end of the nucleic acid can be activated by thiopyridine, and the donor DNA can be sulfhydryl-terminated, thereby allowing disulfide bonds to form between the two molecules. In some embodiments, a bridge DNA complementary to both the nucleic acid and the donor DNA allows the two molecules to hybridize and approach to promote the reaction. Figures 5A-5C Non-limiting examples of the synthesis of donor DNA conjugated to nucleic acids are provided.

[0091] In other embodiments, nucleic acid and donor DNA can be conjugated via functional groups, such as functional groups involved in bioorthogonal or "click" chemistry. For example, the 5' end of the nucleic acid can be chemically modified with functional groups such as azide, tetrazine, alkyne, strained olefin or strained alkyne, and the 3' end of the donor DNA can be chemically modified with appropriately paired functional groups. For example, if the nucleic acid contains an azide, the azide will react with the alkynyl group of the donor DNA via an azide-alkyne cycloaddition reaction (copper catalyzed), or will react with the strained alkynyl group of the donor DNA via an azide-strained alkyne cycloaddition reaction (without a catalyst). Similarly, if the nucleic acid contains tetrazine, it will react with the strained olefin via a tetrazine / olefin cycloaddition reaction. Similarly, the opposite configuration can be used, for example, if the nucleic acid contains an alkyne, strained alkyne or strained olefin, it will react with the azide or tetrazine group of the donor DNA by the same cycloaddition reaction.

[0092] In some embodiments, the nucleic acid and the donor DNA are conjugated via a linker. For example, the nucleic acid and the donor DNA can be conjugated by a suicide linker. As used herein, a "suicide linker" is a linker that hydrolyzes under specific conditions (e.g., a specific pH value), which allows the donor DNA to be released from the nucleic acid.

[0093] The joint of the nucleic acid donor DNA conjugate encompasses any joint known in the art, which can covalently connect the donor DNA to the nucleic acid. The joint can be attached to the donor DNA and the nucleic acid at either end. However, in some embodiments, the joint is attached to the 5' end of the nucleic acid (e.g., the 5' end of the crRNA, processing sequence or extension sequence) and the 3' end of the donor DNA. The joint can be attached to the nucleic acid and the donor DNA by any method known in the art, such as those described herein for the conjugation of the donor DNA to the nucleic acid.

[0094] In another embodiment, the donor polynucleotide can hybridize to the extension sequence and / or the processing sequence. Thus, for example, the extension sequence can comprise a sequence that is sufficiently complementary to the donor polynucleotide to promote hybridization.

[0095] When the donor nucleic acid is covalently or non-covalently linked to the extension sequence, it is sometimes desirable that the donor nucleic acid is linked to the extension sequence or a portion thereof that is not cut by the Cpf1 crRNA, so that when the target gene is edited by Cpf1, the donor nucleic acid is tightly bound to the crRNA. It is believed that in some cases, improved gene editing can be achieved by such constructs. As described above, the extension sequence that is not cut by Cpf1 includes, for example, an extension sequence comprising one or more modified internucleotide bonds or synthetic nucleotides.

[0096] Composition and carrier

[0097] The present invention also includes compositions comprising any nucleic acid molecules and carriers as described herein. Any suitable carrier for nucleic acid delivery can be used. In some embodiments, the carrier can include molecules that can interact with any nucleic acid as described herein and promote nucleic acid entry into cells.

[0098] In some embodiments, the carrier comprises a cationic lipid. A cationic lipid is an amphiphilic molecule having a positively charged polar head group connected via an anchor to a non-polar hydrophobic domain generally comprising two alkyl chains. In some embodiments, the cationic lipid forms a liposome (e.g., a lipid vesicle) around the nucleic acid construct and optionally the Cpf1 protein. Thus, in a related aspect, a liposome comprising a nucleic acid construct and optionally the Cpf1 protein is provided.

[0099] In yet another embodiment, the carrier comprises a cationic polymer. Examples of cationic polymers of the compositions of the present invention include polyethyleneimine (PEI), poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-asparagine] (pAsp(DET)), block copolymers of polyethylene glycol (PEG) and polyarginine, block copolymers of PEG and polylysine, block copolymers of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]asparagine} (PEG-pAsp[DET], ({2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1,3-dioxane-5-yl}methyl)dimethylamine, (3aR,5s,6aS)-N ,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (3aR,5r,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (3aR,5R,7aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-amine, (3aS,5R,7aR)-N,N- dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-amine, (2-{2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1,3-dioxane-4-yl}ethyl)dimethylamine, (3aR,6aS)-5-methyl-2-((6Z,9Z)-octadec-6,9-dien-1-yl)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydro-3aH-[1,3]dioxol-[4,5-c]pyrrole, (3aS,7aR)-5-methyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-amine, (2-{2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1,3-dioxane-4-yl}ethyl)dimethylamine, 2-(dimethylamino)acetate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 2-(dimethylamino)acetate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propionate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propionate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propionate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propionate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl31-tetraen-19-yl-4-(dimethylamino)butyrate], (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-5-(dimethylamino)pentanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-6-(dimethylamino)hexanoate, (3-{2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1,3-dioxane-4-yl}propyl)dimethylamine, 1-((3aR,5r,6aS)-2,2-bis((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydro-3aH-cyclopenta[d][1,3]di 1-((3aR,5s,6aS)-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydro-3aHcyclopenta[d][1,3]dioxol-5-yl)-N,N-dimethylmethylamine, 8-methyl-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxa-8-azaspiro[4.5]decane, 2-(2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N-methyl-N-(pyridin-3-ylmethyl)ethylamine, 1,3-bis(9Z,12Z) -octadec-9,12-dien-1-yl 2-[2-(dimethylamino)ethyl]malonate, N,N-dimethyl-1-((3aR,5R,7aS)-2-((8Z,11Z)-octadec-8,11-dien-1-yl)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-yl)methanamine, N,N-dimethyl-1-((3aR,5S,7aS)-2-((8Z,11Z)-octadec-8,11-dien-1-yl)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-yl)methanamine 、(1s,3R,4S)-N,N-dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)cyclopentaneamine、(1s,3R,4S)-N,N-dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)cyclopentaneamine、2-(4,5-bis((8Z,11Z)-heptadeca-8,11-dien-1-yl)-2-methyl-1,3-dioxolan-2-yl)-N,N-dimethylethylamine、2,3-bis((8Z,11Z)-heptadeca-8,11-dien-1-yl)-N,N-dimethyl-1,4-dioxaspiro[4.5]dec-8-amine、(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(diethylamino)butyrate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-[bis(propan-2-yl)amino]butyrate, N-(4-N,N-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-amine, (2-{2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1,3-dioxane-5-yl}ethyl)dimethylamine, (4-{2,2-bis[(9Z,12Z)-octadec-9,12-dien-1-yl]-1, 3-dioxane-5-yl}butyl)dimethylamine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (2-(dimethylamino)ethyl)carbamate, 2-(dimethylamino)ethyl (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbamate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(ethylamino)propionate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(propan-2-ylamino)butyrate, N1,N1,N2-trimethyl-N2 -((11Z,14Z)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicosyl-11,14-dien-1-yl)ethane-1,2-diamine, 3-(dimethylamino)-N-((11Z,14Z)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicosyl-11,14-dien-1-yl)propanamide, (6Z,9Z,28Z,31Z)-heptatriacontria-6,9,28,31-tetraen-19-yl 4-(methylamino)butyrate, dimethyl({4-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-{[(9Z,12Z)-octadec-9,12-dien-1-yloxy] 1-((3aR,4R,6aR)-6-methoxy-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-eicosyl-11,14-dien-1-yl)carbamate, 1-((3aR,4R,6aR)-6-methoxy-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-eicosyl-11,14-dien-1-yl)carbamate, 1-((3aR,4R,6aR)-6-methoxy-2,2-di((9Z,12Z)-octadec-9,(12-dien-1-yl)tetrahydrofurano[3,4-d][1,3]dioxol-4-yl)-N,N-dimethylmethylamine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-[ethyl(methyl)amino]butyrate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-aminobutyrate, 3-(dimethylamino)propyl((11Z,14 Z)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicosyl-11,14-dien-1-yl)carbamate, 1-((3aR,4R,6aS)-2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)tetrahydrofurano[3,4-d][1,3]dioxol-4-yl)-N,N-dimethylmethanamine, (3aR,5R,7aR)-N,N-dimethyl-2,2-di(( 9Z,12Z)-octadec-9,12-dien-1-yl)hexahydrobenzo[d][1,3]dioxol-5-amine, (11Z,14Z)-N,N-dimethyl-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicosyl-11,14-dien-1-amine, (3aS,4S,5R,7R,7aR)-N,N-dimethyl-2-((7Z,10Z)-octadec-7,10-dien-1-yl)-2- ((9Z,12Z)-octadec-9,12-dien-1-yl)hexahydro-4,7-methylenebenzo[d][1,3]dioxol-5-amine, N,N-dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)butan-1-amine, and 3-(4,5-di((8Z,11Z)-heptadeca-8,11-dien-1-yl)-1,3-dioxolan-2-yl)-N,N-dimethylpropan-1-amine. Any combination of the foregoing polymers may also be used. ,

[0100] In other embodiments, the carrier comprises polymer nanoparticles.For example, the compositions of the present invention can be administered as nanoparticles as described in International Patent Application No. PCT / US2016 / 052690, the entire disclosure of which is expressly incorporated by reference.

[0101] Cpf1 polypeptide or nucleic acid encoding the same

[0102] In some embodiments, including the liposome embodiments described above, the composition further comprises a Cpf1 polypeptide or a nucleic acid encoding the same. Any Cpf1 polypeptide may be used in the compositions of the invention, although the selected Cpf1 should be appropriately selected so as to act in combination with the crRNA of the nucleic acid construct in the composition to cleave the target nucleic acid and / or cleave the processing sequence of the nucleic acid construct, where applicable. The Cpf1 of the composition may be a naturally occurring Cpf1 or a variant or mutant Cpf1 polypeptide. In some embodiments, the Cpf1 polypeptide is enzymatically active, e.g., when bound to a guide RNA, the Cpf1 polypeptide cleaves the target nucleic acid. In some embodiments, relative to a wild-type Cpf1 polypeptide (e.g., relative to a composition comprising Figure 8 The Cpf1 polypeptide has the amino acid sequence shown in (SEQ ID NO: 1), the Cpf1 polypeptide exhibits reduced enzymatic activity and retains DNA binding activity. Mutations that alter the enzymatic activity of Cpf1 are known in the art.

[0103] For example, Cpf1 may be derived from a bacterium of the genus Acidaminococcus or from the genus Lachnospiraceae, or from Fig. 9 Any genus or species identified in. Figure 8 Examples of Cpf1 protein sequences are provided in . In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence similar to Figure 8 , at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity. In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence that is identical to Figure 8 A contiguous stretch of 100 amino acids to 200 amino acids (aa), 200aa to 400aa, 400aa to 600aa, 600aa to 800aa, 800aa to 1000aa, 1000aa to 1100aa, 1100aa to 1200aa, or 1200aa to 1300aa of the amino acid sequence shown in has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity.

[0104] In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence similar to Figure 8In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence identical to that of the RuvCI domain of the Cpf1 polypeptide. Figure 8 In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence identical to that of the RuvCII domain of the Cpf1 polypeptide of the amino acid sequence shown in . In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence identical to that of the RuvCII domain of the Cpf1 polypeptide of the amino acid sequence shown in . Figure 8 The RuvCIII domain of a Cpf1 polypeptide having the amino acid sequence shown in has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity.

[0105] In some embodiments, the Cpf1 polypeptide is FnCpf1, Lb3Cpf1, BpCpf1, PeCpf1, SsCpf1, AsCpf1, Lb2Cpf1, CMtCpf1, EeCpf1, MbCpf1, LiCpf1, LbCpf1, PcCpf1, PdCpf1, or PmCPf1; or a Cpf1 polypeptide comprising an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity thereto.

[0106] In some embodiments, the Cpf1 polypeptide is included in a region corresponding to Figure 8 an amino acid substitution (e.g., a D→A substitution) at the amino acid residue at position 917 of the amino acid sequence shown in ; and / or a substitution (e.g., a D→A substitution) at the position corresponding to Figure 8 an amino acid substitution at the amino acid residue at position 1006 of the amino acid sequence shown in (e.g., an E→A substitution); and / or a substitution contained in a residue corresponding to Figure 8 An amino acid substitution (e.g., a D→A substitution) at the amino acid residue at position 1255 of the amino acid sequence shown in .

[0107] The Cpf1 polypeptide can also be an RNase-inactivated Cpf1, such as a modified Cpf1 comprising H800A, K809A, K860A, F864A or R790A of Acidococcus Cpf1 (AsCpf1), or corresponding positions of different Cpf1 orthologs. Examples of mutant Cpf1 proteins include proteins disclosed in Zetsche et al., "Multiplex Gene Editing by CRISPR-Cpf1 Through Autonomous Processing of a Single crRNAArray," Nat. Biotechnol. 2017, 35(1), 31-34. The Cpf1 polypeptide can also be a dCpf1 base editor (e.g., Cpf1-cytosine deaminase fusion protein). Examples include, for example, proteins disclosed in Li et al., Nature Biotechnology, 36324-327 (2018), and Mahfouz et al., Biochem J., 475 (11), 1955-1964 (2018). An example of a synthetic variant Cpf1 is the MAD7 Cpf1 ortholog by Inscripta, Inc. (CO, USA). Additional examples of Cpf1 proteins include any of those Cpf1 proteins disclosed in International Patent Application No. PCT / US2016 / 052690, including chimeric or mutant proteins, the entire disclosure of which is expressly incorporated herein by reference.

[0108] Other nucleic acids

[0109] In addition to crRNA, the composition may further include other nucleic acids. For example, as described herein, the composition may include donor polynucleotides. Alternatively or in addition, the composition may include one or more other nucleic acids that are not donor polynucleotides (for example, nucleic acids that have no significant sequence identity with the target sequence to be edited or any endogenous nucleic acid sequence of the editing cell (for example, not enough to allow homologous recombination sequence identity level). These other nucleic acids can be RNA or DNA, such as single-stranded RNA or DNA molecules (or hybrid molecules comprising both RNA and DNA, optionally with synthetic nucleic acid residues). Other nucleic acids can be any length, such as at least 5 nucleotides in length (for example, 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, or even 200 nucleotides or more). In some embodiments, the nucleic acid may include 500 nucleotides or more, 1000 nucleotides or more, or even 5000 nucleotides or more). However, in most cases, the nucleic acid comprises about 5000 nucleotides or fewer, such as about 1000 nucleotides or fewer, or even 500 nucleotides or fewer (eg, 200 nucleotides or fewer).

[0110] The composition may further comprise a nucleic acid encoding a protein of particular interest, for example, an RNA-guided endonuclease (eg, a Cpf1 polypeptide). The RNA-guided endonuclease may be any as described herein with respect to other aspects of the invention.

[0111] Divalent metal ions

[0112] In some embodiments, the composition is substantially or completely free of divalent metal ions (e.g., magnesium) that activate the particular Cpf1 protein used so as to reduce or prevent premature cleavage of the processing sequence prior to delivery. The composition is considered to be substantially free of magnesium at a concentration that does not allow Cpf1 self-processing enzymatic activity. In some embodiments, the composition comprises about 20 mM or less NaCl and is substantially or completely free of magnesium or other divalent ions that activate the Cpf1 protein.

[0113] Methods for genetically modifying eukaryotic cells

[0114] The present invention also provides a method for genetically modifying a eukaryotic target cell, comprising contacting the eukaryotic target cell with any nucleic acid or composition described herein (e.g., comprising Cpf1 crRNA, an extension sequence of crRNA 5', and optionally a processing sequence between crRNA and the extension sequence) to genetically modify the target nucleic acid. In some embodiments, the Cpf1 crRNA of the nucleic acid comprises a targeting sequence (e.g., 3' of the stem-loop domain) that hybridizes with a target sequence in the target cell. In some embodiments, the Cpf1 crRNA comprises a processing sequence that is cleaved after entering the cell, thereby releasing the Cpf1 crRNA from the processing sequence and the extension sequence. In other embodiments, the Cpf1 crRNA does not comprise a processing sequence.

[0115] The target nucleic acid is a polynucleotide (e.g., RNA, DNA) to which the targeting sequence of the crRNA binds and induces cleavage by Cpf1. The target nucleic acid comprises a "target site" or "target sequence," which is a sequence present in the target nucleic acid to which the crRNA hybridizes, which in turn guides the endonuclease to the target nucleic acid.

[0116] A "eukaryotic target cell" can be any eukaryotic cell known in the art, and includes both in vivo and in vitro cells. In one embodiment, the target cell is a mammalian cell.

[0117] Any route of administration can be used to deliver the composition to mammals. In fact, although more than one approach can be used to administer the composition, a specific approach can provide a more immediate and more effective reaction than another approach. When applied to cells in vitro or in vitro, nucleic acid or composition can be contacted with cells by any suitable method. For example, nucleic acid can be in liposomes, encapsulated by cationic polymers and / or introduced by electroporation. When applied to an object such as a mammal or a person, the composition can be administered by any of the various approaches. For example, a dose of the composition can also be applied to or instilled into the body cavity, absorbed by the skin (for example, via a transdermal patch), sucked, taken in, applied topically to tissue, or administered parenterally via, for example, intravenous, intraperitoneal, intraoral, intradermal, subcutaneous or intraarterial administration.

[0118] Composition can be used in the device of allowing control or sustained release or thereon, the device such as sponge, biocompatible mesh, mechanical reservoir or mechanical implant.Implant (see, for example, United States Patent (USP) 5,443,505), device (see, for example, United States Patent (USP) 4,863,457), such as implantable device, such as mechanical reservoir or implant or the device being made up of polymer composition, is particularly useful for the use of composition.Composition can also be used in the form of sustained release preparation (see, for example, United States Patent (USP) 5,378,475), it comprises such as gel foam, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphate such as bis-2-hydroxyethyl-ethylene terephthalate (BHET) and / or polylactic glycolic acid.

[0119] Example

[0120] The following examples further illustrate the invention but, of course, should not be construed in any way as limiting its scope. Table 2 below provides the nucleic acid sequences used in these experiments.

[0121] Table 2

[0122] Supplementary Table 1

[0123]

[0124]

[0125]

[0126]

[0127] *: Phosphorothioate

[0128] U: 2' deoxy

[0129] Underline : Pre-crRNA sequence

[0130] Example 1

[0131] This example illustrates that unmodified Cpf1 crRNA tends to provide lower gene editing efficiency compared to Cas9 sgRNA.

[0132] Using a green fluorescent protein (GFP) reporter system, SpCas9 and AsCpf1 were compared, both without the extension of the guide RNA. Matching pre-spacer DNA sequences in the GFP gene that could be recognized by both nucleases were selected to allow for a direct comparison of AsCpf1 and SpCas9. Fig.13AThe RNP complex was introduced into HEK293T cells expressing the GFP gene under the control of a doxycycline-inducible promoter (GFP-HEK) using electroporation and cationic lipids. Editing activity was determined by measuring the population of GFP-negative cells in which GFP was destroyed by NHEJ-mediated indel mutations.

[0133] AsCpf1 RNP showed lower gene editing than SpCas9 in both electroporated and cationic liposome-treated cells ( Fig. 13B and 13C ). Cells electroporated with AsCpf1 RNP were 31% GFP negative, whereas cells electroporated with SpCas9 RNP were 41% GFP negative ( Fig. 13B Using cationic liposomes and the RNAiMax delivery system, AsCpf1-treated cells were approximately 8% GFP-negative, while SpCas9-treated cells were approximately 30% GFP-negative ( Fig. 13C ).

[0134] Example 2

[0135] This example demonstrates that a nucleic acid comprising Cpf1 crRNA, a processing sequence 5' to Cpf1 crRNA, and an extension sequence 5' to the processing sequence (a reversibly supercharged crRNA) enhances Cpf1 delivery by cationic lipids in vitro.

[0136] Cationic materials such as cationic liposomes and polycations are the most commonly used delivery vehicles for nucleic acids in cells and living animals. Therefore, the efficiency of Cpf1 / crRNA complex transfection into HEK cells (GFP-HEK) expressing green fluorescent protein by cationic lipid cationic liposomes was analyzed. In short, the crRNA designed to knock down the GFP gene via insertion and deletion is compounded with Cpf1, and or electroporated (nucleofection) is intracellular or transfected with cationic liposomes. Then via flow cytometry, the gene editing efficiency is determined by measuring the number of GFP knockout cells, wherein the cell no longer expressing GFP indicates that the cell has been transfected by Cpf1 / crRNA complexes.

[0137] Results from these experiments showed that cationic liposomes were not able to efficiently transfect unmodified Cpf1 crRNA complexes ( Figure 1A Specifically, cells treated with cationic liposomes and Cpf1 / crRNA complexes had only 8% NHEJ efficiency, whereas cells electroporated with Cpf1 / crRNA complexes had 40% NHEJ efficiency, confirming that delivery limitations were the main reason for the low NHEJ efficiency using cationic liposomes.

[0138] To examine the effect of sequence extension, the 41-nucleotide crRNA was extended by 9 nucleotides (crRNA + 9), or extended by 59 nucleotides (crRNA +59 ). The extended portion includes a self-processing sequence that self-cleaves into active crRNA. Unmodified crRNA, crRNA + 9 and crRNA +59 Each was individually complexed with Cpf1 and transfected into GFP-HEK cells using cationic liposomes. RNPs were formed under low salt conditions to prevent potential processing of 5' end extensions. The level of NHEJ, which is an indicator of transfection efficiency, was determined by measuring the percentage of GFP-negative cells.

[0139] like Figure 1B As shown in , extending crRNA with self-processing sequences significantly enhanced the transfection efficiency using cationic media. Cells treated with unextended crRNA via cationic liposomes showed 8% GFP negative, while cells treated with 9-base extended crRNA were 18% GFP-, and cells treated with 59-base extended crRNA were 37%, which was about 4 times higher than the control unmodified crRNA. In addition, crRNA+9, crRNA+15, and crRNA+25 were tested with cationic liposomes, and a length-dependent increase in gene editing efficiency was shown ( Figure 1C ).

[0140] To determine if there is a specific 5' extension sequence requirement for this enhancement, three different 59-nucleotide 5' extensions were compared. The first and original 59-nucleotide extended crRNAs were described above and contained an AsCpf1 pre-crRNA (crRNA+59), the second 59-nucleotide extended crRNA contained four AsCpf1 pre-crRNA sites in tandem (crRNA+59-D2), and the third 59-nucleotide extended crRNA contained the FnCpf1 pre-crRNA followed by a scrambled DNA sequence with no homology to any sequence in the human genome (crRNA+59-D3) ( Figure 1D ). These crRNAs were delivered using Lipofectamine 2000, similar to the above paragraph. All three 5' extensions showed equivalent editing activity: crRNA+59 cells were 32% GFP negative, crRNA+59_D2 cells were 30% GFP negative, and crRNA+59_D3 cells were 27% GFP negative ( Figure 1E). This suggests that there is no strict sequence requirement for 5' extension enhancement, similar to previous findings with 9-nucleotide extended crRNA and electroporated cells. In addition, these results provide evidence supporting the above hypothesis that increasing the negative charge density on crRNA, and thereby also on the AsCpf1 RNP complex, can enhance the delivery of AsCpf1 to cells by cationic lipids.

[0141] The extended crRNAs were tested in an in vitro DNA cleavage assay to determine whether the extended crRNAs enhanced the intrinsic nuclease activity of Cpf1. With incubation times of 15 and 60 minutes, no differences in activity were observed between the three crRNAs tested: wild-type crRNA, crRNA +9 and crRNA +59 When the incubation time was only 5 minutes, crRNA +59 Even has slower DNA cleavage than wild-type crRNA.

[0142] 5' extended crRNAs were also studied to determine if there was enhanced gene editing activity in Cpf1 if delivered via a plasmid rather than as RNP. Cpf1 plasmids were transfected 24 hours prior to electroporation of crRNA and gene editing activity was determined. No improvement in gene editing efficiency was observed when Cpf1 was produced from a plasmid.

[0143] Further, the crRNA was labeled with a fluorescent dye to determine whether the extended crRNA had enhanced uptake in cells after delivery via electroporation or cationic liposomes. Electroporation of Cpf1 RNP resulted in more than 90% of cells being positive for dye-crRNA and showed highly efficient delivery regardless of the length of the crRNA. The delivery efficiency of Cpf1 RNP with cationic liposomes depended on the length of the crRNA, and the extended crRNA was more efficiently delivered into HEK 293T cells than the wild-type crRNA ( Figure 1F ).

[0144] The results show that the extended crRNA as provided herein enhances delivery and gene editing efficiency.

[0145] Example 3

[0146] This example demonstrates that the activity of Cpf1 is enhanced by 5' end extension in HEK cells using electroporation.

[0147] GFP-targeted crRNAs with 5' end extensions of 4, 9, 15, 25, and 59 nucleotides were introduced into GFP-HEK cells as RNP complexes with AsCpf1 by electroporation. The sequences of the 4 to 25 nucleotide extensions were scrambled, and the 59 nucleotide extension consisted of the AsCpf1 pre-crRNA followed by a scrambled RNA sequence with no homology to the human genomic sequence.

[0148] All crRNAs with 4 to 25 nucleotide 5' extensions showed dramatically increased gene editing over crRNAs without extensions. Cells electroporated with unextended cRNA were 30% GFP negative (crRNA), with crRNAs with 4 to 25 nucleotide extensions were 55 to 60% GFP negative, and with crRNAs with 59 nucleotide extensions were 37% GFP negative ( Figure 2 ). The gene editing levels of crRNA with a 5' extension of 4 to 25 nucleotides were comparable to those of cells electroporated with SpCas9 RNP.

[0149] The results confirmed that 5' extension of Cpf1 crRNA increased gene editing efficiency.

[0150] Example 4

[0151] This example demonstrates that a nucleic acid comprising Cpf1 crRNA and a 5' extension sequence enhances the delivery of Cpf1 by cationic lipids in vivo and the activity of Cpf1 in cells.

[0152] Three different chemical modifications were investigated for crRNAs with extensions: 2'O-methyl modification, phosphorothioate linkage, and deoxyribose moiety ( Fig.15A ). The first 3 of the 4 nucleotides were extended with: 2'-O-methyl nucleotides and 3' phosphorothioate linkages (MS), a deoxynucleotide at the 9th position of the 9-nucleotide 5'-extended crRNA (9dU), 3' phosphorothioate linkages for all 9 nucleotides plus a deoxynucleotide at the 9th position of the 9-nucleotide 5'-extended crRNA (9s).

[0153] Cpf1 RNPs containing crRNAs with extensions and chemical modifications were electroporated into GFP-HEK cells, and gene editing activity was determined by flow cytometry. The crRNAs with chemically modified extensions had similar activity to the unmodified crRNAs (41% to 46% of GFP-negative cells) ( Fig.15D ).

[0154] In addition, these crRNAs were examined using a HEK293T cell line (BFP-HEK) expressing blue fluorescent protein (BFP). The results are presented in Fig.12 middle.

[0155] The results from this experiment showed that 5' extension increased the gene editing efficiency of AsCpf1, and the tolerance of the 5' end of crRNA to chemical modification. Further, if the 5' end of crRNA is extended, 5' chemical modification of crRNA is possible without compromising activity.

[0156] The key benefit of using chemically modified crRNAs is that they are more stable to hydrolysis by serum nucleases. Therefore, the serum stability of 5' chemically modified crRNAs was investigated.

[0157] 5' chemically modified crRNA was incubated in diluted fetal bovine serum and its degradation was analyzed via gel electrophoresis. Fig. 15B The quantification of the remaining crRNA after 15 minutes of incubation in serum is provided. The results show that unmodified crRNA is rapidly degraded in serum, while crRNA containing a phosphorothioate backbone +9S Significantly more stable to hydrolysis in serum.

[0158] 5' modified crRNAs were also investigated to determine whether they could enhance the ability of cationic liposomes to transfect Cpf1 RNPs due to their ability to protect crRNA from nucleases in cells and serum. +9S Cpf1 is more effective than crRNA in editing genes in cells +9 40% more effective, suggesting that 5'crRNA chemical modification achieved by 5'crRNA extension has many applications in gene editing ( Fig. 15C ).

[0159] The crystal structure of Cpf1 RNP has recently been solved and confirmed that the AsCpf1 protein forms numerous interactions with the phosphodiester backbone of crRNA. Therefore, 5'-chemical modification of the unextended crRNA has a high probability of disrupting important interactions between crRNA and Cpf1, leading to the disruption of AsCpf1 gene editing activity.

[0160] In contrast, crRNAs with 5' extensions appear to be resistant to chemical modification because the nucleotides that interact with the AsCpf1 protein are unmodified. These results provide methods for introducing chemical modifications at the 5' end of crRNA, which can potentially enhance delivery for ex vivo and in vivo therapeutic applications. Such constructs also allow other molecules, such as targeting ligands, endosomal escape moieties, or other functional molecules, to be conjugated to the extended crRNA and retained with the Cpf1 molecule. For example, biotin or avidin (or streptavidin) can be conjugated to the crRNA extension, allowing the modified crRNA to bind to another molecule (e.g., a targeting molecule) conjugated to streptavidin or biotin, as appropriate (e.g., Fig.14 ). In addition, crRNA can be conjugated to Cpf1 mRNA by extension, allowing Cpf1 to be delivered through translation of mRNA. Once Cpf1 mRNA is translated in the cytoplasm and Cpf1 protein is produced, Cpf1 protein recognizes the crRNA portion of the construct and optionally processes the connecting RNA sequence to separate Cpf1 mRNA and crRNA.

[0161] Example 5

[0162] Experiments were also performed to determine whether crRNA with extended sequences could enhance the ability of cationic polymers to transfect Cpf1. In particular, crRNAs of various lengths (crRNAs without extension, with 9nt extension, and with 59nt extension) as used in Example 2 were complexed with Cpf1, mixed with the cationic polymer PAsp(DET), and added to GFP-HEK cells. The NHEJ efficiency of the preparations was determined by measuring the frequency of GFP-negative cells via flow cytometry.

[0163] like Fig.16 As shown in , the 5' extension of 59 bases enhanced the delivery of PAsp(DET)-mediated AsCpf1 RNP to cells by 2-fold. The unextended crRNA (crRNA) was 8% GFP negative, the 9-nucleotide extended crRNA (crRNA+9) was 10% GFP negative, and the 59-nucleotide extended crRNA (crRNA+59) was 18% GFP negative. These results confirm that the extended sequence can improve the delivery of crRNA to cells using cationic polymers.

[0164] Example 6

[0165] This example demonstrates that extended crRNA enhances Cpf1 delivery in vivo.

[0166] Experiments were performed to determine the extended crRNA (crRNA +59) can enhance cationic lipid-mediated Cpf1 delivery in vivo. A schematic diagram of the experiment is in Figure 3A Available in.

[0167] Studies were performed in Ai9 mice using previously validated spacers. Ai9 mice were given intramuscular injections of cationic lipids or PAsp(DET) in combination with either: AsCpf1-crRNA complexes or AsCpf1-crRNA +59 Two weeks after injection, the expression of tdTomato (red fluorescence) was imaged in 10 μm sections of gastrocnemius muscle ( Figure 3B ). Comparison of images collected for unextended and extended crRNAs showed that extended crRNAs dramatically enhanced the ability of PAsp(DET) to deliver AsCpf1 RNPs in vivo. In addition, RNPs with extended crRNAs (Cpf1 RNP+59) complexed with PAsp(DET) induced tdTomato expression several millimeters away from the injection site and throughout the gastrocnemius muscle. The high range of tdTomato expression in muscle is likely due to the unique multinucleated nature of muscle fibers. This allows TdTomato to be expressed over the entire length of the muscle fiber and can therefore be observed over several millimeters in length.

[0168] The ability of the 59-nucleotide extended crRNA to enhance delivery, and the editing efficiency of the AsCpf1 RNP in vivo by the extension, supports the value of Cpf1 as a tool for animal studies and as a potential therapeutic agent for the treatment of human disease, particularly genetic muscular dystrophy.

[0169] Example 7

[0170] 5.1: Extended crRNA increases HDR and NHEJ rates

[0171] To examine whether 5′-extensions could increase HDR rates in addition to NHEJ levels, AsCpf1 RNPs with crRNAs containing various extensions were introduced into GFP-HEK cells along with a single-stranded oligonucleotide donor (ssODN). NHEJ levels were determined by measuring the population of GFP-negative cells (similar to the first section), while HDR rates were quantified using restriction enzyme digestion assays. A 2-fold improvement in HDR was observed for both 4- and 9-nucleotide extended crRNAs (in Figure 4A , 17% HDR frequency for crRNA+4, and 18% HDR frequency for crRNA+9, compared to 9% for unmodified crRNA). A smaller increase in HDR was observed for the 59-base stretch (in Figure 4A , 13% HDR rate for crRNA+59).

[0172] Interestingly, ssODN for HDR also dramatically increased the NHEJ efficiency of AsCpf1. ssODN increased the percentage of GFP-negative cells from 30% to 46% for unextended crRNA (crRNA), from 55% to 95% for 4-base extended crRNA (crRNA+4), from 58% to 93% for 9-base extended crRNA (crRNA+9), and from 37% to 58% for 59-base extended crRNA (crRNA+59). Figure 4B ). The finding that exogenously added DNA enhanced AsCpf1 RNP-mediated editing was further validated in the BFP reporter system. Single-stranded DNA (ssDNA) without any homology to the human genome was electroporated into BFP-HEK cells with AsCpf1 RNP. Similarly, the addition of ssDNA increased AsCpf1 editing activity by 2-fold for both unextended and extended crRNA. The BFP-negative population increased from 31% to 50% for unextended crRNA (crRNA), from 59% to 91% for 4-nucleotide extended crRNA (crRNA+4), and from 60% to 95% for 9-nucleotide extended crRNA (crRNA+9) ( Figure 4C ). Additional experiments were performed to determine whether exogenously added DNA must have homology to the Cpf1 RNP target site in order to enhance gene editing. AsCpf1 RNP was electroporated into cells along with single-stranded DNA (ssDNA) without any homology to the target sequence, and the gene editing efficiency was measured. Similarly, for both extended crRNAs, the addition of ssDNA without homology also increased AsCpf1 editing activity to approximately 90% ( Figure 4D ). The results confirmed that ssDNA can enhance editing with AsCpf1. In addition, activity enhancement with 5' end extension synergized with exogenous ssDNA, and together they induced gene editing close to 100%. It was observed that if cationic liposomes were used as a delivery method instead of electroporation, the addition of ssDNA did not enhance Cpf1 gene editing efficiency.

[0173] It is sometimes preferred to use RNA rather than DNA for gene editing because ssRNA cannot integrate into the genome and can be safer to use. To test the effect of ssRNA rather than DNA, GFP-HEK cells were electroporated with Cpf1 RNP and two different ssRNAs (9nt and 100nt), and the resulting gene editing levels were determined. Both 100nt ssRNAs with slight sequence variations dramatically increased the gene editing efficiency of Cpf1, resulting in a 2-fold improvement, while the 9nt ssRNA induced a 10% enhancement in gene editing efficiency ( Figure 4E ).

[0174] These results demonstrate that single-stranded nucleic acids can be used to enhance Cpf1 editing activity in cells.

[0175] 5.2: Single-molecule extension of crRNA and donor DNA.

[0176] Part 2 of this example demonstrates that extended crRNA combined with donor DNA in a single molecule can enhance HDR.

[0177] The vast majority of genetic diseases require gene correction rather than knockout, and therefore, there is great interest in developing Cpf1-based therapeutics that can correct gene mutations via HDR. In order to solve the problem of generating nanoparticles that effectively encapsulate both donor DNA and Cpf1-crRNA, experiments were performed to determine whether crRNA and donor DNA can be combined into a single molecule via reversible disulfide bonds. As previously reported, one challenge is that the 5' end of crRNA is quite sensitive to chemical modification. Therefore, chemical modifications at the 5' end of the self-processing sequence extended by crRNA were tested.

[0178] The crRNA is extended with four additional nucleotides at its 5' end, and chemical modifications (i.e. 5' DBCO, 5' thiol or 5' azide) are added to the ends of the nucleotides. The activity of the crRNA with and without chemically modified 4nt extensions at the 5' end was tested. In short, the super-charged crRNA (designed to knock down the GFP gene via insertion and deletion formation) of 5' modification is compounded with Cpf1, and electroporated (nucleofection) is added to the cell. Then, via flow cytometry, the gene editing efficiency is determined by measuring the number of GFP knockout cells, wherein the cells that no longer express GFP indicate that the cells have been transfected by Cpf1 / crRNA complexes.

[0179] Figure 4F It was shown that neither chemical modification nor nucleotide extension affected Cpf1-crRNA activity, as all crRNA complexes showed approximately 40% GFP knockdown, a level similar to that of unmodified control crRNA.

[0180] Since chemistries can be added to the 5' end of crRNA without loss of activity, the 5' end of crRNA is activated with thiopyridine to react with thiol-terminated donor DNA (see Figures 5A-5C ). The reaction between the two macromolecules has slow kinetics. Therefore, a method using a bridge DNA complementary to both crRNA and donor DNA was used. The bridge hybridizes and brings the two macromolecules close to promote the reaction so as to enhance the conjugation yield between crRNA and donor DNA. The conjugation yield is as high as 40%, and the product is purified via gel extraction. The conjugate is named "homologous DNA-crRNA" (HD-RNA). HD-RNA contains disulfide bonds, which should be reduced in the cytoplasm. Thiol-mediated HD-RNA cleavage is determined by incubating it in DTT for 6 hours and analyzing its molecular weight via gel electrophoresis. Figure 5C Gel comparison in shows that DTT reduces HD-RNA and regenerates donor DNA and crRNA.

[0181] HD-RNA complexed with Cpf1 was electroporated into GFP-HEK cells, and the levels of HDR and NHEJ were compared to cells electroporated with Cpf1-crRNA and donor DNA, respectively. Similar NHEJ and HDR levels were observed from cells electroporated with HD-RNA compared to cells electroporated with Cpf1-crRNA and donor DNA, which confirmed that the conjugation of crRNA to donor DNA via disulfide bonds did not affect the functionality of crRNA or donor DNA.

[0182] Experiments were performed to determine whether HD-RNA enhanced the HDR efficiency of Cpf1 following transfection with cationic lipids (i.e., cationic liposomes) or cationic polymers (i.e., PAsp(DET)). For these experiments, HD-RNA complexed with Cpf1 was transfected into GFP-HEK cells using cationic liposomes or PAsp(DET), and the levels of HDR and NHEJ were compared. NHEJ was determined by measuring the frequency of GFP-negative cells and confirmed by performing a Surveyor assay with a PCR amplicon of a targeted region of the BFP gene. HDR efficiency was determined by isolating cellular DNA and analyzing the presence of restriction enzyme sites embedded in the donor DNA. The results are presented in Figure 6 and 7 middle.

[0183] Figure 6 and 7It was demonstrated that HD-RNA enhanced both NHEJ and HDR efficiency of Cpf1 after delivery with PAsp(DET). Specifically, HDR was detected in up to 60% of cells treated with HD-RNA / Cpf1 complexes delivered with PAsp(DET), which was significantly higher than the HDR rate of cells treated with Cpf1 / crRNA and donor DNA complexed with PAsp(DET). In addition, the 60% HDR rate observed with HD-RNA / Cpf1 complexes delivered with PAsp(DET) was even higher than the HDR rate observed with electroporation of Cpf1 / crRNA complexes, and suggested that having donor DNA in the vicinity of the Cpf1 cleavage site can contribute to HDR.

[0184] These results confirm that extended crRNA can enhance HDR.

[0185] Example 9

[0186] This example demonstrates the use of extended crRNA in different cell types, and the utility of this approach in treating genetic disorders.

[0187] HD-RNA has numerous potential applications due to its ability to enhance Cpf1 to generate HDR in cells after delivery with cationic lipids. Duchenne muscular dystrophy (DMD) is being tested as an initial medical application for HD-RNA. DMD is an early-onset fatal disease caused by mutations in the dystrophin gene; it is the most common congenital myopathy, and approximately 30% of DMD patients have single base mutations or small deletions that can potentially be treated with HDR-based therapeutics.

[0188] Therefore, an HD-RNA designed to target the dystrophin gene was tested for its ability to correct the dystrophin mutation in myoblasts from mdx mice via HDR. The HD-RNA was designed to cleave the dystrophin gene and also contained donor DNA designed to correct the C to T mutation present in its dystrophin gene (see Fig. 10A and 10B ). The HDR rate in mdx myoblasts treated with Cpf1 / HD-RNA+cationic liposomes was determined and compared to mdx myoblasts treated with Cpf1-crRNA, donor DNA, and cationic liposomes. It was found that Cpf1 complexed with HD-RNA was more efficient in generating HDR in mdx myoblasts compared to cells treated with Cpf1 RNP and donor DNA. For example, HD-RNA treated cells had 5-10% HDR rate, while control cells had only 1% HDR rate.

[0189] In another experiment, primary myoblasts isolated from Ai9 mice were electroporated with AsCpf1RNPs complexed with crRNA with and without 5' extensions. The Ai9 mice are transgenic mouse strains containing stop codons in all 3 reading frames, which are coupled to the triple poly (A) signal upstream of the tdTomato reporter gene. Ai9 mice are transgenic mouse strains containing tdTomato reporter genes, which have stop codons in all three reading frames coupled to the triple poly (A) signal. The AsCpf1 spacer is designed to introduce multiple breaks into DNA, which results in the removal of termination sequences deleted by the genome. Successful gene editing is indicated by the expression of tdTomato (red fluorescent protein, RFP), which can be visualized by fluorescence microscopy and quantified using flow cytometry. Extended crRNA increases gene editing by 40-50% over unextended crRNA. Myoblasts treated with unextended crRNA were 12% RFP positive; myoblasts treated with 2-nucleotide extended crRNA were 15% RFP positive; myoblasts treated with 9-nucleotide extended crRNA were 18% RFP positive, and myoblasts treated with 59-nucleotide extended crRNA were 16% RFP positive ( Fig. 10C In addition, the efficiency of gene editing was tested using crRNA together with ssDNA ssRNA (100 nt) that had no sequence homology to the target DNA primary myoblasts. Both ssDNA and ssRNA enhanced gene editing efficiency ( Fig. 10D ).

[0190] Overall, the deletion of target sequences in primary myoblasts suggests that extended crRNA-enhanced gene editing can be applied broadly across genetic targets and cell types. These results demonstrate that HD-RNA can be used as a therapeutic agent for genetic diseases.

[0191] Example 10

[0192] The following examples demonstrate that the gene editing effects enhanced by 5'crRNA extension can be broadly applied across genetic targets and cell types. Using Serpina1 as a test platform, these crRNAs were tested to see if they could enhance the ability of Cpf1 RNP to edit endogenous genes.

[0193] Using electroporation, the Serpina1 gene-targeted construct with crRNA or crRNA +9Cpf1 was transfected into HepG2 cells. Serpina1 was chosen for further investigation because mutations in the Serpina1 gene cause α1-antitrypsin deficiency, making it a target for therapeutic gene editing. Droplet digital PCR was performed on genomic DNA from HepG2 cells to quantify NHEJ efficiency.

[0194] like Fig. 10E As shown in , compared with wild-type crRNA, +9 Cpf1 RNP has enhanced NHEJ efficiency. These results further indicate that the gene editing effect of 5'crRNA extension can be widely applied across genetic targets.

[0195] 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.

[0196] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "one" and "a kind of", as well as "the / said" and "at least one / kind", and similar indicators, should be interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be understood to mean an item selected from the listed items (A or B), or any combination of two or more listed items (A and B), unless otherwise specified herein or clearly contradicted by the context. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the description of the numerical range herein is intended to serve only as a shorthand method of individually referring to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually described herein. Unless otherwise specified herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the present invention.

[0197] 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 in those preferred embodiments will become apparent to those of ordinary skill in the art. The inventors expect that skilled technicians will appropriately adopt such changes, and the inventors wish to practice the present invention in a manner different from that specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter described in the claims attached thereto as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, any combination of the above-mentioned elements in all possible variations thereof is encompassed by the present invention. Sequence Listing <110> Gene Editing Ltd. <120> Modified CPF1 guide RNA <130> 512701 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 1300 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys 20 25 30 Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys 35 40 45 Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu 50 55 60 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser 65 70 75 80 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys 85 90 95 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr 100 105 110 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile 115 120 125 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln 130 135 140 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 145 150 155 160 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr 165 170 175 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser 180 185 190 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu 195 200 205 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys 210 215 220 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 225 230 235 240 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg 245 250 255 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr 260 265 270 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys 275 280 285 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile 290 295 300 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 305 310 315 320 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser 325 330 335 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met 340 345 350 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys 355 360 365 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln 370 375 380 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 385 390 395 400 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala 405 410 415 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn 420 425 430 Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala 435 440 445 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn 450 455 460 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 465 470 475 480 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys 485 490 495 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys 500 505 510 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp 515 520 525 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His 530 535 540 Ser Gln Ser Glu Asp Lys Ala Asn Ileu Asp Lys Asp Glu His 545 550 555 560 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val 565,570,575 Pro Leu Tyr Asn With Arg Asn Tyr With Thr Gln Lys Pro Tyr Ser 580,585,590 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly 595,600,605 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys 610 615 620 Asp Asp Lys Tyr Leu Gly Val Met Lys Lys Asn Asn Lys Ile 625 630 635 640 Phe Asp Asp Lys Ala Ile Lys Glu Asn Gly Glu Gly Tyr Lys Lys 645,650,655 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val 660,665,670 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile 675,680,685 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln 690,695,700 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 705 710 715 720 Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp 725 730 735 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu 740,745,750 Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn 755,760,765 Ser Glu Ser Tyr and Asp Ser Val Val Asn Gln Gly Lys Tyr 770,775,780 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 785,790,795,800 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn 805 810 815 Gln Asp Val Val Tyr Lys Gln Gl Glu Ala Glu Phe Tyr 820 825 830 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala 835 840 845 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu 850 855 860 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 865 870 875 880 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe 885 890 895 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His 900 905 910 Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu 915 920 925 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile 930 935 940 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 945 950 955 960 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn 965 970 975 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile 980 985 990 Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu 995 1000 1005 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val 1010 1015 1020 Tyr Gln Lys Leu Glue Met Leu And Leu Glue Asn Tyr Leu 1025 1030 1035 Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg 1040 1045 1050 Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly 1055 1060 1065 Lys Gln Thr Gly Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser 1070 1075 1080 Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys 1085 1090 1095 Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp 1100 1105 1110 Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe 1115 1120 1125 Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr 1130 1135 1140 Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Ser Asn Ser Asp 1145 1150 1155 Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu 1160 1165 1170 Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly 1175 1180 1185 Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1190 1195 1200 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg 1205 1210 1215 Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val 1220 1225 1230 Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys 1235 1240 1245 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly 1250 1255 1260 Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu 1265 1270 1275 Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu 1280 1285 1290 Phe Val Gln Asn Arg Asn Asn 1295 1300 <210> 2 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 2 gucuaagaac uuuaaau 17 <210> 3 <211> 16 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 3 gucaaaagac cuuuuu 16 <210> 4 <211> 16 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 4 guuucaaaga uuaaau 16 <210> 5 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 5 cucuagcagg ccuggca 17 <210> 6 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 6 auuugaaagc aucuuuu 17 <210> 7 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 7 ggcuauaaag cuuauuu 17 <210> 8 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 8 gccaaauacc ucuauaa 17 <210> 9 <211> 15 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 9 gucuaacuac cuuuu 15 <210> 10 <211> 15 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 10 gucuaacuac cuuuu 15 <210> 11 <211> 15 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 11 gucuaacuac cuuuu 15 <210> 12 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 12 cucuaauaag agauaug 17 <210> 13 <211> 18 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 13 ugcuuagaac auuuaaag 18 <210> 14 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 14 guuuaaaacc acuuuaa 17 <210> 15 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 15 cucuacaacu gauaaag 17 <210> 16 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 16 guuuaaaagu ccuauug 17 <210> 17 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 17 gucuaaaacu cauucag 17 <210> 18 <211> 18 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 18 ugcuuaguac uuauaaag 18 <210> 19 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 19 gccaagaacc uauagau 17 <210> 20 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 20 gucuauaaga cauuuau 17 <210> twenty one <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> twenty one aauuucuacu guuguagau 19 <210> twenty two <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> twenty two aauuucuacu cuuguagau 19 <210> twenty three <211> 20 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> twenty three aauuucuacu aaguguagau 20 <210> twenty four <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> twenty four aauuucuacu guuguagau 19 <210> 25 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 25 aauuucuacu auuguagau 19 <210> 26 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 26 aauuucuacu auuguagau 19 <210> 27 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 27 aauuucuacu uuuguagau 19 <210> 28 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 28 aauuucuacu guuuguagau 20 <210> 29 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 29 aauuucuacu guuuguagau 20 <210> 30 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 30 aauuucuacu guuuguagau 20 <210> 31 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 31 aauuucuacu guuguagau 19 <210> 32 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 32 aauuucuacu auuguagau 19 <210> 33 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 33 aauuucuacu auuguagau 19 <210> 34 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 34 aauuucuacu uuuguagau 19 <210> 35 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 35 gauuucuacu uuuguagau 19 <210> 36 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 36 aauuucuacu aguguagau 19 <210> 37 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 37 aauuucuacu auuguagau 19 <210> 38 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 38 aauuucuacu guuguagau 19 <210> 39 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 39 aauuucuacu auuguagau 19

Claims

1. A nucleic acid comprising a Cpf1 crRNA, an extension sequence of at least 6 nucleotides and no more than 60 nucleotides located 5' of the crRNA, and optionally a processing sequence between the crRNA and the extension sequence, wherein the processing sequence is a sequence that is self-cleaved by Cpf1, wherein the Cpf1 crRNA comprises a stem-loop domain 5' of the targeting sequence, And the extension does not contain the processing sequence, the aptamer or the sequence of the Cpf1 crRNA.

2. The nucleic acid of claim 1, wherein the nucleic acid comprises a processing sequence, and the processing sequence comprises a fragment of a direct repeat sequence of a Cpf1 array, wherein the direct repeat sequence comprises a crRNA sequence portion and a processing portion located 5' of the crRNA sequence portion, and the fragment comprises at least 5 contiguous nucleotides of the processing portion of the direct repeat sequence.

3. The nucleic acid of claim 2, wherein the processing sequence comprises a fragment of at least 10 nucleotides of the processed portion of the direct repeat sequence.

4. The nucleic acid of claim 2, wherein said processing sequence comprises the entire processing portion of said direct repeat sequence.

5. The nucleic acid of claim 1, wherein the extension sequence comprises 10 to 60 nucleotides.

6. The nucleic acid of claim 1, wherein the nucleic acid contains only a single Cpf1 crRNA sequence.

7. The nucleic acid of claim 1, further comprising a second processing sequence 5' to said extension sequence and a second extension sequence 5' to said second processing sequence.

8. The nucleic acid of claim 1, further comprising a donor nucleic acid covalently linked to the 5' portion of the processing sequence or to the 5' portion of the extension sequence.

9. The nucleic acid of claim 1, further comprising a donor nucleic acid hybridized to the extension sequence and / or processing sequence.

10. The nucleic acid of claim 1, wherein the nucleic acid does not comprise a processing sequence.

11. The nucleic acid of claim 1, wherein the extension sequence comprises a self-hybridizing sequence, or wherein the extension sequence comprises a repetitive trinucleotide motif.

12. The nucleic acid of claim 1, wherein the extension sequence or a portion thereof is resistant to nuclease degradation, or wherein the extension sequence comprises one or more modified internucleotide bonds, or wherein the extension sequence comprises one or more xenogeneic nucleic acids (XNA).

13. The nucleic acid of claim 1, wherein the nucleic acid further comprises a biotin and / or an avidin or streptavidin molecule attached to the 5' end of the extension sequence.

14. A composition comprising the nucleic acid of any one of claims 1 to 13 and a vector.

15. The composition of claim 14, wherein the nucleic acid is in a liposome, or the nucleic acid is partially or fully encapsulated by a polymer nanoparticle, or attached to a metal or polymer nanoparticle.

16. The composition of claim 14 or 15, further comprising Cpf1 protein.

17. The composition of claim 16, wherein the Cpf1 protein is complexed with the nucleic acid.

18. A method for genetically modifying a eukaryotic target cell in vitro or ex vivo, comprising contacting the eukaryotic target cell with Cpf1 and a nucleic acid according to any one of claims 1 to 13 or a composition according to any one of claims 14 to 17 to genetically modify the target nucleic acid.

19. Use of a nucleic acid according to any one of claims 1 to 13 or a composition according to any one of claims 14 to 17 in the preparation of a medicament for treating a genetic disease or disorder in a human subject.

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