Cell depletion with CRISPR nucleases

Abstract

The present invention relates to a method for performing genome editing of cells at a target locus by homology directed repair (HDR) in a cell population and simultaneously enriching cells in the cell population that have been genome edited by HDR. The present invention also relates to a method for selectively depleting cells containing a target locus in a cell population.

Description

[Technical field]

[0001] The present invention relates to a method for performing genome editing of cells at a target locus by homology-directed repair (HDR) in a cell population and simultaneously enriching cells genome-edited by HDR in the cell population, the method comprising: (A) introducing one or more nucleic acid molecules into cells in the cell population, the one or more nucleic acid molecules comprising: (i) (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). (a) a nucleic acid molecule encoding, in an expressible form, a nucleic acid molecule comprising, consisting of, or encoding a CRISPR nuclease; (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (a) a second segment that interacts with the CRISPR nuclease of (i); and (iii) a donor template having homology to the target locus; or (A') a nucleic acid molecule encoding, in an expressible form, a nucleic acid molecule comprising, consisting of, or encoding a nucleic acid molecule comprising (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c (a) a CRISPR nuclease comprising or consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); (ii) a nucleic acid molecule encoding a guide RNA in an expressible form, the guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (a) a second segment that interacts with the CRISPR nuclease of (i); and introducing a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus;or (A'') administering to a cell in the cell population (i) a CRISPR nuclease complex comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease, in a complex with a guide RNA, the CRISPR nuclease comprising: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence that is at least 80% identical to the nucleotide sequence of (b). (ii) introducing a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus, into cells of the cell population; (A) subjecting the cells to genome editing by homology-directed repair (HDR) at the target locus, and (B) culturing the cells under conditions in which the CRISPR nuclease enriches for HDR-edited cells in the cell population;

[0002] Numerous documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all documents referenced are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] The genome editing technology CRISPR allows genome editing in a wide range of cells and organisms. CRISPR-based techniques have enabled highly efficient targeted genetic modification in a variety of prokaryotic and eukaryotic cells. However, depending on the cell type and the targeted DNA region within the genome, CRISPR can be inefficient, resulting in less than 1% of mammalian cells being edited.

[0004] Successful genome editing with CRISPR (or other programmable nucleases) requires three consecutive prerequisites: (1) efficient delivery of the CRISPR-encoding gene into the target cell (transfection / transduction efficiency); (2) efficient expression of the CRISPR components (CRISPR nuclease and CRISPR-RNA); and (3) targeting of the gene of interest (GOI) by the CRISPR ribonucleoprotein complex and repair of the DNA by the cell's own repair pathways.

[0005] The overall success of genome editing depends on the efficiency of each of these single steps: thus, the frequency of successful editing events within a transfected cell population correlates with efficient gene delivery (step 1), high nuclease expression and formation of functional ribonucleoprotein complexes (step 2), and finally the introduction of double-stranded DNA breaks (DSBs) followed by repair of the DSBs within the cell (step 3).

[0006] Several approaches have been developed to isolate subpopulations of cells expressing nucleases, such as CRISPR vectors that allow for fluorescence or magnetic activated cell sorting (FACS and MACS). These and other available methods allow for enrichment of either transfected cells or cells expressing CRISPR nucleases, which is necessary but not sufficient to obtain edited cells. This is due to the fact that expression of nucleases in a given cell does not necessarily lead to the formation of functional CRISPR ribonucleoprotein complexes and the introduction of mutations at the targeted DNA site. Thus, genome editing remains challenging, especially in cells that are difficult to transfect (e.g., human primary cell lines), as these FACS or MACS methods do not allow for immediate enrichment of edited cells.

[0007] A genome co-editing approach to enrich for CRISPR-edited cells, including genome editing of a gene of interest and the LRRC8 gene, is described in WO 2019 / 202099 along with the use of blasticidin as a selection marker. A related approach of co-editing is described in Liao et al., Nucleic Acids Res. 2015;43(20):e134, using 6-thioguanine (6TG) as the selection agent and knocking out the HPRT gene (which encodes hypoxanthine phosphoribosyltransferase) to render cells resistant to 6TG. These approaches efficiently enrich for genome-edited cells, but are based on the co-editing of a second gene and a selection marker. Summary of the Invention

[0008] The present invention therefore aims to provide technically improved methods and means that allow enrichment of CRISPR-edited cells (step 3 above).As an additional advantage, the present invention allows for genomic targeted specific depletion of cells within a cell population by CRISPR nucleases.

[0009] Accordingly, in a first aspect, the present invention provides a method for performing genome editing of cells at a target locus by homology directed repair (HDR) in a cell population and simultaneously enriching cells genome-edited by HDR in the cell population, the method comprising: (A) introducing into cells in the cell population one or more nucleic acid molecules, the one or more nucleic acid molecules comprising: (i) a CRISPR nuclease comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to the sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); and (iii) a nucleic acid sequence that is complementary to the target locus. or (A') introducing into cells within the cell population (i) a CRISPR nuclease comprising or consisting of: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), (ii) a nucleic acid molecule encoding a guide RNA in an expressible form, the nucleic acid molecule comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease of (i), and (iii) a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus;or (A'') administering to a cell within the cell population: (i) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus, and (a) a second segment that interacts with the CRISPR nuclease, in a complex with a guide RNA, the guide RNA comprising: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). (ii) introducing a ribonucleoprotein complex (RNP) comprising or consisting of a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by the target locus; and (b) culturing the cells of the cell population under conditions in which the target locus carried by the cells of the cell population is edited by homology directed repair (HDR), and the CRISPR nuclease enriches for HDR-edited cells in the cell population. [Brief description of the drawings]

[0010] The figure shows.

[0011] [Figure 1] Schematic diagram visualizing the strategy of co-transfecting two vectors into Escherichia coli (E. coli) to compare the DNA targeting mechanism of BEC10 nuclease in comparison to FnCpf1. [Diagram 2] An exemplary culture plate showing E. coli colonies 48 hours after transformation to visualize the different molecular mechanisms of BEC10 compared to FnCpf1. [Diagram 3] An exemplary culture plate showing P. pastoris colonies 48 hours after transformation to visualize the different genome editing mechanism of BEC10 compared to SpCas9. [Figure 4]Target-specific cell depletion A: Negative control: 50 / 50 mix of HEK-EGFP and HEK-DsRed cells transfected with BEC10 combined with a non-targeting spacer. B: Depletion of EGFP-positive cells using an EGFP-specific spacer sequence to activate BEC10 nuclease without affecting viability of EGFP-negative (DsRed-positive) cells. C: FACS analysis showing distribution of approximately 55% EGFP-negative cells (left peak) and approximately 45% EGFP-positive cells (right peak) in the negative control (top panel), and >98.5% EGFP-negative cells (left peak) and <1.5% EGFP-positive cells (right peak) using a spacer matching the EGFP sequence (bottom panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A cell population refers to a group of cells. A cell population can be heterogeneous or homogeneous, and is preferably homogeneous. A heterogeneous cell population comprises cells of different origins, such as different species or sources and / or different cell types of one species or source (e.g., body part). In contrast, a homogeneous cell population comprises only cells of one species or source, and preferably only cells of one cell type or one body part.

[0013] Genome editing (also known as genome engineering) is a type of genetic engineering that inserts, deletes, modifies, or replaces a gene of interest in the genome of a cell. According to a first aspect of the present invention, genome editing uses the homology directed recombination (HDR) pathway. Compared to non-homologous end joining (NHEJ), HDR is a more accurate mechanism for DSB repair because it requires higher sequence homology between the damaged strand of DNA and the intact donor strand. This process is error-free if the DNA template used for repair is identical to the original DNA sequence at the DSB or can introduce specific mutations into the damaged DNA. Therefore, it should be understood that the cells in the population of cells carrying the target locus to be genome edited are capable of HDR.

[0014] Genome editing can result in loss-of-function mutations in the genome of a cell, or in gain-of-function mutations. Loss-of-function mutations (also called inactivating mutations) result in a gene of interest that is less functional or completely nonfunctional (partially or totally inactivated). When an allele loses function completely (completely inactivated), this is also referred to herein as (gene) knockout. Genome editing of a gene of interest is preferably knockout. Knocking out a gene can be achieved by inserting, deleting, modifying, or substituting one or more nucleotides of the gene. In gain-of-function mutations (also called activating mutations), the gene of interest can be altered so that its effect is stronger (enhanced activation) or even replaced by a different (e.g., abnormal) function. Gain-of-function mutations can also introduce a new function or effect into a cell that the cell did not have before. In this situation, a new gene can be added to the genome of the cell (insertion) or can replace a gene in the genome. Such gain-of-function mutations that introduce a new function or effect are also called gene knock-ins.

[0015] HDR-mediated genome editing according to the first aspect of the present invention relies on (i) a clustered regularly interspaced short palindromic repeats (CRISPR / Cas) based nuclease, also referred to herein as a "CRISPR nuclease", (ii) a guide RNA, also referred to herein as a "spacer / protospacer", that mediates the interaction between the CRISPR nuclease and the target locus (note that the spacer binds to the target locus, while the protospacer (or protospacer adjacent motif (PAM)) binds to the CRISPR nuclease), and (iii) a donor template having homology to the target locus, also referred to herein as an "HDR template".

[0016] CRISPR nucleases (or CRISPR-Cas nucleases, or Cas nucleases) are a particular type of programmable nuclease. The CRISPR nucleases used according to the present invention are so-called BRAIN Engineered Cas proteins (BEC) nucleases. More specifically, the amino acid sequences of SEQ ID NO: 1, 2, or 3 and the nucleotide sequences of SEQ ID NO: 4, 5, or 6 are the amino acid and nucleotide sequences of BEC85, BEC67, and BEC10 class 2, type V RNA-guided DNA nucleases disclosed in International Application No. PCT / EP2021 / 000081. Among the three BEC nucleases, BEC10 is the most preferred. SEQ ID NO: 7 is BEC10 optimized for expression in E. coli. SEQ ID NO: 7 is therefore sometimes referred to as E. coli BEC10.

[0017] CRISPR-Cas systems have been used for genome editing in prokaryotes and eukaryotes. A small piece of RNA is created that contains a short "guide" sequence that attaches (binds) to a specific target sequence of DNA in the genome (so-called guide RNA (gRNA) or single guide (sgRNA)). The genomic target site of the gRNA can be any DNA sequence of about 20 nucleotides, provided that two conditions are met: (i) the sequence is unique compared to the rest of the genome, and (ii) the target is immediately adjacent to a protospacer adjacent motif (PAM). The PAM sequence is essential for target binding, but the exact sequence depends on which CRISPR nuclease is used. CRISPR nucleases and their respective PAM sequences are known in the art (see https: / / www.addgene.org / crispr / guide / #pam-table). Thus, the gRNA also binds to the CRISPR nuclease (BEC enzyme). Similar to bacteria, gRNA is used to recognize DNA sequences and CRISPR nuclease cuts the DNA at the target location. Once the DNA is cut, the cell's own DNA repair mechanisms (NHEJ or HDR) alter the DNA by adding or deleting pieces of genetic material or replacing existing segments with customized DNA sequences. Thus, in the CRISPR-Cas system, CRISPR nuclease makes double-stranded breaks in DNA at sites determined by short (approximately 20 nucleotide) gRNAs, and the breaks are then repaired in the cell by NHEJ or HDR. The CRISPR-Cas system can be multiplexed by adding multiple gRNAs. For example, it has been demonstrated that five different mutations can be introduced simultaneously into mouse embryonic stem cells by using five different gRNA molecules and one CRISPR nuclease.

[0018] The design and construction of donor templates suitable for HDR are known in the art. HDR is error-free if the repair template is identical to the original DNA sequence at the double-strand break (DSB) or if highly specific mutations can be introduced into the DNA. The three central steps of the HDR pathway are: (1) the 5'-end DNA strand is excised at the break to create a 3' overhang, which serves both as a substrate for the proteins required for strand invasion and as a primer for DNA repair synthesis. (2) The invaded strand then displaces one strand of a homologous DNA duplex and can pair with the other strand. This results in the formation of a hybrid DNA called a displacement loop (D-loop). (3) The recombination intermediates can then be degraded to complete the DNA repair process.

[0019] For example, HDR templates used to introduce mutations or insert new nucleotides or nucleotide sequences into genes require some homology around the target sequence to be modified. Homology arms originating from a CRISPR-induced DSB can be used. In general, the insertion site of the modification should be very close to the DSB, ideally less than 10 bp away if possible. One important point to keep in mind is that the CRISPR enzyme may continue to cut DNA even after the DSB has been introduced and repaired. As long as the gRNA target site / PAM site remains intact, the CRISPR nuclease will continue to cut and repair DNA. This repeated editing can be problematic when introducing very specific mutations or sequences into a gene of interest. To avoid this, the repair template can be designed to eventually block further targeting of the CRISPR nuclease after the initial DSB has been repaired. Two common methods to block further editing are to mutate the PAM sequence or the gRNA seed sequence. The size of the intended edit is taken into consideration when designing the repair template. For smaller mutations, ssDNA templates (also referred to as ssODNs) are commonly used. Small insertions / edits may require as little as 30-50 bases per homology arm, with the exact number best varying based on the gene of interest. Homology arms of 50-80 bases are commonly used. For example, Richardson et al. (Nat Biotechnol. 2016 Mar;34(3):339-44) found that asymmetric homology arms (36 bases distal to the PAM and 91 bases proximal to the PAM) supported HDR efficiencies of up to 60%. Due to the difficulties that may be associated with creating ssODNs longer than 200 bases, it is preferable to use dsDNA plasmid repair templates to insert large inserts such as fluorescent proteins or selection cassettes into genes of interest. These templates can have homology arms of at least 800 bp. To increase the frequency of HDR edits based on plasmid repair templates, self-cleaving plasmids containing gRNA target sites flanking the template may be used.In the presence of CRISPR nuclease and the appropriate gRNA(s), the template is released from the vector. To avoid plasmid cloning, it is possible to use long dsDNA templates generated by PCR. Furthermore, Quadros et al. (Genome Biol. 2017 May 17;18(1):92) developed Easi-CRISPR, a technique that allows for large mutations and can exploit the advantages of ssODNs. To create ssODNs longer than 200 bases, RNA encoding the repair template is transcribed in vitro and then a complementary ssDNA is created using reverse transcriptase. Easi-CRISPR works well in mouse knock-in models, increasing the editing efficiency from 1-10% for dsDNA to 25-50% for ssODNs. The efficiency of HDR varies depending on the locus and experimental system, but in general, the highest HDR editing frequency is obtained with ssODN templates.

[0020] The target site is not particularly limited and refers to a genomic site of interest present in the genome of the cell to be genome-edited. The target site in the genome refers to mitochondrial DNA or genomic DNA, preferably genomic DNA. The target site is preferably, but not necessarily, a gene of interest (or a target gene). The target site may be, for example, a gene regulatory element such as a promoter region or a cis-regulatory element.

[0021] According to step (A) of the method of the present invention, one or more nucleic acid molecules encoding the CRISPR nuclease and the guide RNA in an expressible form, as well as the HDR template, are introduced into the cells of the cell population directly (in the form of single-stranded or double-stranded DNA) or in an expressible form encoded by a nucleic acid molecule (preferably an expression vector), while according to step (A') of the method of the present invention, the CRISPR nuclease itself (i.e. in proteinaceous form), the nucleic acid molecule encoding the guide RNA, as well as the HDR template, are introduced into the cells of the cell population directly or in an expressible form encoded by a nucleic acid molecule. Also according to step (A''), the CRISPR nuclease itself (i.e. in proteinaceous form) is introduced into the cells of the cell population, in this case in the form of a ribonucleoprotein complex (RNP) together with the guide RNA. The RNP can be assembled in vitro and delivered into the cells by methods known in the art, such as, for example, electroporation or lipofection. RNPs can cleave target sites with efficacy comparable to nucleic acid-based (e.g., vector-based) CRISPR nucleases (Kim et al. (2014), Genome Research 24(6):1012-1019). Also, according to step (A''), the HDR template is either directly introduced into cells in the cell population or is in an expressible form encoded by a nucleic acid molecule that is introduced into cells in the cell population.

[0022] When HDR template is in expressible form encoded by nucleic acid molecule, said nucleic acid molecule can be another nucleic acid molecule, or can be the same nucleic acid molecule that encodes CRISPR nuclease and / or guide RNA.When HDR template is directly introduced into cell, HDR template can be a linear double-stranded or single-stranded DNA molecule.Linear double-stranded DNA molecule is preferably linearized PCR product, and linear single-stranded DNA molecule is preferably ssODN as described herein above.

[0023] The nucleic acid molecules used according to the present invention can be inserted into several commercially available vectors. A single vector is commercially available that contains both CRISPR nuclease and gRNA, and optionally HDR template, and acts as an all-in-one vector. Alternatively, the method of the present invention can be carried out by using two or three vectors that contain CRISPR nuclease, gRNA, and HDR template. It is also possible to use vectors with only gRNA and / or HDR template, and to use cells with CRISPR nuclease integrated into the genome. It is preferable to use an all-in-one vector that expresses gRNA, CRISPR nuclease, and optionally HDR template, since only one vector is introduced into the cell. For example, Sakuma et al., Sci Rep. 2014; 4: 5400, describes a vector that can express CRISPR nuclease and up to seven gRNAs.

[0024] Many single gRNA empty vectors (both with and without CRISPR nuclease) are available in the art. Similarly, some empty multiplex gRNA vectors are also available (with or without CRISPR nuclease expression) that can be used to express multiple gRNAs from a single plasmid. Finally, vectors that express only CRISPR nuclease are also available (see https: / / www.addgene.org / crispr / empty-grna-vectors / ).

[0025] Vector modification techniques are known in the art and are described, for example, in Sambrook and Russel, 2001. In general, a vector may contain one or more origins of replication (ori) for cloning or expression and an inheritance system, one or more markers for selection in a host, such as antibiotic resistance, and one or more expression cassettes. Suitable origins of replication include, for example, Col E1, SV40 virus, and M13 origins of replication. Nucleic acid sequences inserted into a vector may, for example, be synthesized by standard methods or isolated from natural sources. Ligation of the coding sequence to transcriptional control elements and / or other amino acid coding sequences can be performed using established methods. Such control sequences are well known to those skilled in the art and include, but are not limited to, control sequences ensuring transcription initiation, translation initiation, an internal ribosome entry site (IRES), or a 2A linker (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476), as well as, optionally, control elements ensuring transcription termination and stabilizing the transcript. Non-limiting examples of regulatory elements that ensure transcription initiation include the translation initiation codon, enhancers such as the SV40-enhancer, insulators and / or promoters such as the cytomegalovirus (CMV) promoter, the elongation factor-1 alpha (EF1 alpha), promoter, the SV40 promoter, the RSV promoter (Rous sarcoma virus), the lacZ promoter, the chicken β-actin promoter, the CAG promoter (the chicken β-actin promoter in combination with the cytomegalovirus immediate early enhancer), the gai10 promoter, the human elongation factor 1a promoter, the AOX1 promoter, the GAL1 promoter, the CaM-kinase promoter, the lac, trp promoter, or the tac promoter, the lacUV5 promoter, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedron promoter, or globin introns in mammalian and other animal cells.Non-limiting examples of regulatory elements that ensure transcription termination include the V40-polyA site, the tk-polyA site, or the polyhedral polyadenylation signals of SV40, lacZ, or AcMNPV, which are included downstream of the nucleic acid sequence of the invention. Further regulatory elements may include translational enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences capable of directing the expressed polypeptide to a cellular compartment, depending on the expression system used. Additionally, elements such as origins of replication, drug resistance genes, regulatory elements (as part of an inducible promoter), etc. may be included.

[0026] Means and methods for introducing nucleic acid molecule(s) expressing a CRISPR nuclease and / or gRNA, and optionally (further) an HDR template into a cell are known in the art and include transducing or transfecting the cell.

[0027] Transduction is the process by which foreign DNA is introduced into cells by a virus or viral vector. Transduction is a common tool used by molecular biologists to stably introduce foreign genes into the genome of a host cell. Generally, a plasmid is constructed in which the gene to be transferred is flanked by viral sequences that are used by viral proteins to recognize the viral genome and package it into viral particles. This plasmid is inserted (usually by transfection) into producer cells along with other plasmids (DNA constructs) carrying the viral genes necessary for the formation of infectious virions. In these producer cells, the viral proteins expressed by these packaging constructs bind to sequences on the transferred DNA / RNA (depending on the type of viral vector) and are inserted into viral particles. For safety, none of the plasmids used contain all the sequences necessary for virus formation, so multiple plasmids must be transfected simultaneously to obtain infectious virions. Furthermore, only the plasmid carrying the transferred sequences contains the signals that allow the genetic material to be packaged into virions, so none of the genes encoding the viral proteins are packaged. The viruses recovered from these cells are then applied to the cells to be transformed. These initial stages of infection mimic infection by natural viruses, resulting in expression of the transferred genes and (in the case of lentiviral / retroviral vectors) insertion of the transferred DNA into the cellular genome. However, because the transferred genetic material does not code for any viral genes, these infections do not produce new viruses (the viruses are "replication-deficient"). In this example, transduction may be used to generate cells that contain CRISPR nucleases in their genome in an expressible form.

[0028] Transfection is the process of deliberately introducing naked or purified nucleic acid, or purified protein, or assembled ribonucleoprotein complexes into a cell. Transfection is generally a non-viral based method.

[0029] Transfection may be chemical-based transfection. Chemical-based transfection can be divided into several types, such as transfection using cyclodextrins, polymers, liposomes, or nanoparticles. One of the cheapest methods uses calcium phosphate. A HEPES-buffered saline solution (HeBS), which contains phosphate ions, is combined with a calcium chloride solution, which contains the DNA to be transfected. When the two are combined, a fine precipitate of positively charged calcium and negatively charged phosphate is formed, and the DNA to be transfected binds to its surface. A suspension of the precipitate is then added to the cells to be transfected (usually cell cultures grown in monolayers). By this process, which is not completely understood, the cells take up part of the precipitate, taking the DNA with them. This process is the preferred method for identifying many cancer genes. Other methods use highly branched organic compounds, so-called dendrimers, to attach the DNA and import it into the cells. Another method is to use cationic polymers such as DEAE-dextran or polyethyleneimine (PEI). Negatively charged DNA binds to polycations and the complex is taken up by cells via endocytosis. Lipofection (or liposomal transfection) is a technique used to inject genetic material into cells using liposomes, which are vesicles that can easily fuse with cell membranes because they are both made of a phospholipid bilayer. Lipofection generally uses positively charged (cationic) lipids (cationic liposomes or mixtures) to form aggregates with negatively charged (anionic) genetic material. In terms of transfer into cells, this transfection technique performs the same task as other biochemical procedures that utilize polymers, DEAE-dextran, calcium phosphate, and electroporation. The efficiency of lipofection can be improved by treating the transfected cells with a mild heat shock. Fugene is a series of widely used proprietary non-liposomal transfection reagents that can directly transfect a wide variety of cells with high efficiency and low toxicity.

[0030] Transfection can also be a non-chemical method. Electroporation is a common method in which exposure of cells to a short pulse of strong electric field transiently increases the permeability of the cell membrane. Cell squeezing allows the delivery of molecules into cells via the deformation of the cell membrane. Sonoporation uses high-intensity ultrasound to induce pore formation in the cell membrane. This pore formation is primarily due to the cavitation of air bubbles interacting with nearby cell membranes, as facilitated by the addition of ultrasound contrast agents, which are the source of cavitation nuclei. Phototransfection is a method in which a highly focused laser is used to transiently create tiny (approximately 1 μm in diameter) holes in the plasma membrane of cells. Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme to remove the cell wall. This is followed by the use of fusogens (e.g., Sendai virus, PEG, electroporation) to fuse protoplasts carrying the gene of interest with recipient target cells.

[0031] Finally, transfection can be a particle-based method. A direct approach to transfection is the gene gun, where DNA is attached to nanoparticles of an inert solid (usually gold) and then "fired" (or particle bombarded) directly into the nucleus of the target cell. Thus, nucleic acids, usually attached to microprojectiles, are delivered by penetrating the membrane at high speed. Magnetofection or magnetically assisted transfection is a transfection method that utilizes magnetic forces to deliver DNA into the target cell. Impalefection is performed by impaling cells with elongated nanostructures and arrays of such nanostructures, such as carbon nanofibers or silicon nanowires, functionalized with plasmid DNA.

[0032] Means of introducing proteins (or peptides) into living cells are known in the art and include, but are not limited to, microinjection, electroporation, lipofection (using liposomes), nanoparticle-based delivery, and protein transduction. Any one of these methods can be used in relation to step (a'). In this regard, the CRISPR nuclease to be introduced can be isolated from its natural environment or can be recombinantly produced.

[0033] Liposomes used for lipofection are vesicles composed of the same material as the cell membrane (i.e., lipid bilayer, usually made of, for example, phospholipids), which can be loaded with one or more protein(s) (e.g., Torchilin VP. (2006), Adv Drug Deliv Rev., 58(14): 1532-55). To deliver proteins into cells, the lipid bilayer of the liposome can be fused with the lipid bilayer of the cell membrane, thereby delivering the contained protein into the cell. Liposomes used according to the present invention are preferably composed of cationic lipids. The cationic liposome strategy has been successfully applied to protein delivery (Zelphati et al. (2001). J. Biol. Chem. 276, 35103-35110). As is known in the art, the exact composition and / or formulation of cationic lipids used can vary depending on the protein(s) of interest and the cell type used (Felgner et al. (1994). J. Biol. Chem. 269, 2550-2561). Nanoparticle-based delivery of Cas9 ribonucleoprotein and donor DNA to induce homology-directed DNA repair is described, for example, in Lee et al. (2017), Nature Biomedical Engineering, 1:889-90.

[0034] Protein transduction directs the internalization of proteins from the external environment into cells (Ford et al. (2001), Gene Therapy, 8:1-4). This method relies on the inherent property of a small number of proteins and peptides (preferably 10-16 amino acids in length) to penetrate cell membranes. The transduction properties of these molecules can be conferred to proteins expressed as fusions with the molecules, thus providing an alternative to gene therapy, for example, for delivering therapeutic proteins to target cells. Commonly used proteins or peptides capable of penetrating cell membranes are, for example, the antennapedia peptide, the herpes simplex virus VP22 protein, the HIV TAT protein transduction domain, peptides derived from neurotransmitters or hormones, or the 9xArg tag.

[0035] Microinjection and electroporation are well known in the art, and those skilled in the art understand how to perform these methods. Microinjection refers to the process of introducing a substance into a single living cell at a microscopic or submicroscopic level using a glass micropipette. Electroporation is the dramatic increase in electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electric field. By increasing the permeability, proteins (or peptides or nucleic acid sequences) can be introduced into living cells.

[0036] CRISPR nucleases can be introduced into cells as active enzymes or as proenzymes, in which case they undergo a biochemical change within the cell (e.g., a hydrolysis reaction that exposes an active site or a change in configuration that exposes an active site) such that the proenzyme becomes an active enzyme.

[0037] The term "in an expressible form" means that one or more nucleic acid molecules may encode the components in a form that ensures that the guide RNA and / or HDR template (if encoded) is transcribed and that the CRISPR nuclease (if encoded) is transcribed and translated into an active enzyme within the cell.

[0038] Example 3 illustrates the method of the first aspect of the invention. Here, the BEC10 nuclease of the invention and the prior art nuclease spCas9 are used in a comparison in a method of genome editing at a target locus in P. pastoris cells by HDR, noting that P. pastoris cells can repair dsDNA breaks by NHEJ. The results using the prior art nuclease spCas9 were as expected. The results were a mixture of cells in which genome editing of the target locus was successful by HDR and cells in which the target locus after the dsDNA break introduced by spCas9 at the target locus was repaired by NHEJ instead of integration of the HDR template. On the other hand, the results using the nuclease BEC10 of the invention were completely unexpected and technically advantageous. As a result, in all 20 clones obtained, cells were obtained in which the target locus was successfully HDR genome edited as desired. No clones were obtained that showed repair of the dsDNA break by NHEJ. Thus, the method according to the first aspect of the invention provides a significant increase in the efficiency of obtaining cells genome-edited by HDR. In Example 3, the efficiency was 100%.

[0039] The discussed results of Example 3, together with the further results of Examples 2, 3, and 5 described herein below in relation to the second aspect of the invention, demonstrate that BEC nucleases exhibit a novel mechanism of action that, to the best of the inventors' knowledge, is not known from any prior art CRISPR nucleases, which enables and forms the basis for the various embodiments described herein.

[0040] This novel mechanism of action can be described as "double-stranded DNA collateral cleavage activity". It is known from the prior art that certain CRISPR nucleases can exhibit single-stranded DNA collateral cleavage activity (ssDNA; Cas12) or single-stranded RNA collateral cleavage activity (ssRNA; Cas13); see Shashital (2018), Genome medicine; 10:32. These prior art CRISPR nucleases bind to their target loci via guide RNA, resulting in collateral activity of collateral ssDNA or ssRNA. However, since the genomes of almost all organisms are composed of dsDNA, the prior art CRISPR nucleases cannot target genomic DNA and are not suitable for genome editing.

[0041] The results of Examples 2, 3, and 5 show that the BEC nuclease of the present invention exhibits double-stranded DNA collateral cleavage activity. Thus, when BEC nuclease is activated by binding to its target locus, essentially all dsDNA in the cell whose genome is edited can be cleaved by BEC nuclease. To the best of the inventors' knowledge, this disclosure is the first report of a CRISPR nuclease with double-stranded DNA collateral cleavage activity.

[0042] Thus, as used herein, a CRISPR nuclease is preferably not only structurally defined by exhibiting the requisite sequence homology to the exact sequence of a BEC nuclease, but furthermore functionally defined as exhibiting double-stranded DNA collateral cleavage activity and / or capable of depleting cells in which the dsDNA breaks introduced by the active BEC nuclease at the target locus have been repaired by NHEJ.

[0043] In relation to the method of the first aspect of the present invention, it should be noted that cells that have successfully undergone genome editing of the target locus by HDR no longer exhibit the target locus because the target locus has been replaced by the donor template. These cells are protected from dsDNA collateral cleavage activity. On the other hand, if the target locus is not eliminated by the introduction of the HDR template, BEC nuclease is activated by the matching spacer / protospacer sequence and induces collateral cleavage of dsDNA. The dsDNA cleavage of the genomic DNA provided in trans is expected to deplete undesirable cells without the introduction of the HDR template, and due to collateral activity, the endogenous NHEJ repair mechanism of the cell cannot prevent cell death. Non-specific dsDNA cleavage in the genome kills the cells.

[0044] According to a preferred embodiment of the first aspect of the present invention, the method further comprises (C) isolating one or more cells in which the target locus has been genome edited by homology directed repair (HDR).

[0045] In Example 3, it is emphasized that 20 of the 20 clones, i.e., 100% of the clones, are composed of cells in which the target locus has been genome-edited by HDR rather than NHEJ. Thus, in the case of the method of the first aspect of the present invention, step (C) is as simple as recovering one or more cells from the culture plate.

[0046] In general, means and methods for isolation steps from heterogeneous cell populations are known in the art, non-limiting examples are single cell dilution, laser capture microdissection, manual or automated cell picking, FACS, and MACS.

[0047] In single cell dilution, a solution containing cells is diluted in more steps until a solution containing only a single cell is obtained. Laser capture microdissection is a method to isolate specific cells of interest from a microscopic region of a tissue, cell, or organism. A laser is coupled to a microscope and focused on a selected cell in a cell population. The movement of the laser by the optics or stage causes the focal point to follow a trajectory predefined by the user. This trajectory containing the selected cell, also called an element, is then excised and separated from the neighboring cells. Manual cell picking is a simple, convenient, and efficient method to isolate single cells. A manual cell picking micromanipulator consists of an inverted microscope combined with a micropipette that is movable via a motorized mechanical stage. Cell picking can also be equipped in an automated device. Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry with sorting capabilities and is the most sophisticated and user-friendly technique to characterize and define different cell types in a heterogeneous cell population based on size, granularity, and fluorescence. FACS allows for simultaneous quantitative and qualitative multiparametric analysis of single cells. Magnetic-activated cell sorting (MACS) is another passive separation technique commonly used to isolate different cell types according to their differentiation clusters. It has been reported that MACS can isolate specific cell populations with a purity of over 90%.

[0048] The present invention also relates to isolated cells obtainable by the above method and to compositions comprising said cells, said compositions being preferably industrial, diagnostic or pharmaceutical compositions, with pharmaceutical compositions being preferred.

[0049] Industrial compositions are intended for use in industry, including agriculture, for example, cells into which a particular enzyme has been introduced as a gene of interest may be used in the production of chemicals, biofuels, food and beverages, animal feed, cosmetics, and consumer products.

[0050] The diagnostic composition is intended to be used in the diagnosis of a disease or condition, for example, cells into which a particular fluorescent protein has been introduced as a gene of interest can be detected within an organism or tissue sample and thus used for diagnosis.

[0051] The term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention comprises the cells described above. Optionally, it may comprise additional molecules capable of altering the properties of the cells of the present invention, thereby, for example, stabilizing, regulating and / or activating their function. The composition is preferably in liquid form, such as (a) solution(s). The pharmaceutical composition of the present invention may optionally further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents, etc. Compositions containing such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject in a suitable dose. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs being administered at the same time. The therapeutically effective amount in a given situation is readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician or physician. In general, the regimen for regular administration of the pharmaceutical composition is from 1×10 4 ~1×10 8 However, a more preferred dosage is 1 x 10 cells per day. 5 ~1×10 7 cells, most preferably 5 x 10 5 ~5×10 6 The range may be in the range of cells.

[0052] The length of treatment needed to observe changes and the interval after treatment for a response to occur will vary depending on the desired effect. The specific amounts can be determined by conventional testing well known to those skilled in the art.

[0053] According to a preferred embodiment of the first aspect of the invention, the donor template (i) inserts one or more nucleotides at the target locus, (ii) deletes one or more nucleotides at the target locus, and / or (ii) substitutes one or more nucleotides at the target locus.

[0054] This can be achieved by appropriately designing the donor template. As mentioned above, the donor template generally contains homology arms that can ensure that the donor template replaces the target locus by HDR. The sequence between the homology arms can contain additional nucleotides or stretch(es) of nucleotide(s) compared to the target site (insertion); can lack one or more nucleotides or stretch(es) of nucleotide(s) compared to the target site (deletion); and / or can contain one or more different nucleotides or different stretch(es) of nucleotide(s) compared to the target site (substitution). The stretch of nucleotides of the insertion can contain or consist of one or more genes, optionally together with one or more expression control sequences such as promoters.

[0055] Thus, HDR templates can be used to specifically modify target loci by adding, deleting, and / or substituting one or more nucleotides or stretch(es) of nucleotide(s) as needed.

[0056] According to a further preferred embodiment of the first aspect of the invention, the donor template comprises or consists of a nucleotide sequence carrying one or more intended mutations flanking nucleotide sequences homologous to the target locus.

[0057] The nucleotide sequence that is homologous to the target locus is also referred to herein as a homology arm. In the case of a double-stranded donor template, the homology arm is preferably 30-800 bp, more preferably 300-500 bp. In the case of a single-stranded donor template, the homology arm is preferably at least 300 bp, more preferably at least 800 bp.

[0058] As discussed, sequences having one or more intended mutations between the arms may include additions, deletions, and / or substitutions of one or more nucleotides or stretch(es) of nucleotide(s).

[0059] According to another preferred embodiment of the first aspect of the invention, the method further comprises synchronizing and capturing cells in S and G2 phase in step (B).

[0060] Synchronizing and capturing cells in S and G2 phases in step (B) can increase the efficiency of HDR-mediated genome editing; see, e.g., Lin et al. (2014), eLife;3:e04766. Synchronizing and capturing cells in S phase is preferred.

[0061] Cell cycle synchronization techniques are well established and are reviewed, for example, in Jackman and O'Connor (2011), Curr Protoc Cell Biol; Chapter 8: Unit 8.3.

[0062] In a second aspect, the present invention provides a method for selectively depleting cells comprising a target locus in a cell population, comprising: (A) introducing into cells in the cell population one or more nucleic acid molecules, wherein the one or more nucleic acid molecules have: (i) (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence that is at least 80% identical to the nucleotide sequence of (b). or (A') encoding in an expressible form to cells within the cell population: (i) a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is a complement to a sequence of the target locus; and (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease of (i); or (A') encoding in an expressible form to cells within the cell population: (i) (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), and (ii) introducing into cells in the cell population one or more nucleic acid molecules encoding a guide RNA in an expressible form, the guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus, and (b) a second segment that interacts with the CRISPR nuclease of (i); or (A'') introducing into cells in the cell population one or more nucleic acid molecules encoding a guide RNA in an expressible form, the guide RNA comprising (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus, and (b) a second segment that interacts with the CRISPR nuclease, the guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus, and (b) a second segment that interacts with the CRISPR nuclease, in complex with (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a);or (d) introducing a ribonucleoprotein complex (RNP) comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); and (B) culturing the cells under conditions where the CRISPR nuclease selectively depletes cells in the cell population that contain the target locus;

[0063] The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect of the invention insofar as they are amendable for combination with the second aspect of the invention.

[0064] In this regard, it is noted that the second aspect of the invention uses a CRISPR nuclease and guide RNA as defined in relation to the first aspect of the invention, but does not use a donor template.

[0065] According to the second aspect of the present invention, the above-mentioned novel mechanism of action of BEC nuclease is used to selectively deplete cells containing a specific target locus in a cell population. In Example 2, this is illustrated based on a guide RNA targeting ampicillin resistance of a vector DNA in E. coli cells, which also carries kanamycin resistance. E. coli cells cannot repair double-stranded DNA breaks by NHEJ, and therefore cannot repair dsDNA breaks when introduced into ampicillin resistance without HDR template. The results using the prior art Cpf1 nuclease were as expected. The resulting E. coli cells were no longer resistant to ampicillin, but remained resistant to kanamycin. The results using BEC10 nuclease were again unexpected and technically advantageous. The resulting E. coli cells were no longer resistant to ampicillin and kanamycin. These results are explained by the dsDNA collateral cleavage activity described herein above, which adds dsDNA breaks to kanamycin resistance as well. The results of Example 2 are confirmed by those of Example 3, where no donor template was used, in which NHEJ-repaired cells were eliminated by BEC10 nuclease, and no clones were obtained, since production of HDR-repaired cells is not possible in the absence of a donor template. Example 5 also illustrates the method of the second aspect of the invention by showing that BEC10 nuclease can be used to selectively deplete mammalian cells, i.e., in a target cell-specific manner.

[0066] Thus, as used herein, CRISPR nucleases are not only structurally defined by exhibiting the requisite sequence homology to the exact sequence of a BEC nuclease, but are also preferably functionally defined herein as exhibiting double-stranded DNA collateral cleavage activity and / or capable of depleting cells containing a target locus.

[0067] According to preferred embodiments of the first and second aspects of the invention, the cells in the population are capable of repairing double-stranded DNA breaks by non-homologous end joining (NHEJ).

[0068] As shown in Example 3, cells capable of repairing double-stranded DNA breaks by NHEJ can repair double-stranded DNA breaks introduced by CRISPR nucleases by NHEJ. This is undesirable, particularly when genome editing by HDR template is desired. It has been demonstrated that the BEC nuclease of the present invention can eliminate or prevent the occurrence of cells in which double-stranded DNA is repaired by NHEJ in the presence and, notably, in the absence of HDR template. This is believed to be achieved by the above-mentioned dsDNA collateral activity of the BEC nuclease of the present invention.

[0069] According to a further preferred embodiment of the first and second aspects of the invention, the cells in the population are prokaryotic cells, or preferably eukaryotic cells, more preferably vertebrate cells, and most preferably mammalian cells.

[0070] The prokaryotic cell is preferably a bacterial cell, such as an E. coli or B. subtilis cell.

[0071] Eukaryotic cells are preferred compared to prokaryotic cells. Eukaryotic cells may be chordate or vertebrate cells, as well as yeast cells such as P. pastoris or A. niger cells.

[0072] The vertebrate cells are preferably cells of a vertebrate (particularly mammalian) cell line, an organoid, a primary cell, a cell from a primary cell line, or a pluripotent stem cell.

[0073] Mammalian cell lines are populations of cells derived from mammals that do not normally proliferate indefinitely, but due to mutations (e.g., occurring naturally in tumors or through artificial mutagenesis) are able to avoid normal cellular senescence and instead continue to divide, thus allowing the cells to grow for long periods in vitro.

[0074] Organoid is a miniaturized and simplified version of organs that is produced in vitro in three dimensions and shows realistic microanatomy.Organoid originates from one or a few cells obtained from tissue, embryonic stem cell, or induced pluripotent stem cell, and can self-organize in three-dimensional culture due to its self-renewal and differentiation ability.

[0075] Primary cells are cells taken directly from living tissue (e.g., biopsy material) and established for growth in vitro. These cells are more representative of the main functional components of the tissue from which they are derived compared to serially passaged (tumor or artificially immortalized) cell lines, since they have undergone few population doublings, and therefore generally represent a more representative model of the in vivo condition. Primary cell lines are cell lines established from primary cells.

[0076] Pluripotent stem cells are cells that have the ability to self-replicate by dividing and develop into the three main germ cell layers of the early embryo and eventually into all cells of the adult body, but not into extraembryonic tissues such as the placenta. Embryonic stem cells and induced pluripotent stem cells are multipotent stem cells. Embryonic stem cells are derived from the inner cell mass of the blastocyst, an early stage preimplantation embryo. A human embryo reaches the blastocyst stage 4-5 days after fertilization, at which point it consists of 50-150 cells. They are preferably isolated from the embryo without destroying the embryo. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. iPSC technology was pioneered by Shinya Yamanaka's laboratory in Kyoto, Japan. He showed in 2006 that adult cells could be converted into pluripotent stem cells by introducing four specific genes that code for transcription factors. The generation of iPSCs using Oct3 / 4 and / or factors belonging to the Myc, KIf, and Sox families of factors is described in WO 2009 / 144008.

[0077] According to another preferred embodiment of the first and second aspects of the invention, the method is an in vitro method or an ex vivo method.

[0078] Ex vivo methods are methods carried out in the context of a living organism. Similarly, in vitro methods are carried out using microorganisms, cells, or biomolecules outside of their normal biological context.

[0079] Both in vitro and ex vivo methods exclude methods of treatment of the human or animal body by surgery or therapy and diagnostic methods performed on the human or animal body.

[0080] In a third aspect, the present invention relates to a nucleic acid sequence comprising: (A)(i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence which is at least 80% identical to the nucleotide sequence of (b), for use in treating a disease by selectively depleting cells which comprise a target locus associated with the disease to be treated. and (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); or (A')(i)(a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; or (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a). or (d) a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), and (ii) one or more nucleic acid molecules encoding a guide RNA in an expressible form, the guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); or (A″)(a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i). and (a) a second segment that interacts with the CRISPR nuclease, in a complex with a guide RNA, the complex comprising: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b).

[0081] Also provided is a method of treating a disease by selectively depleting cells comprising a target locus associated with the disease being treated, comprising: one or more nucleic acid molecules encoding, in an expressible form, a therapeutically effective amount of (A)(i) a CRISPR nuclease comprising or consisting of: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); and (ii) a guide RNA comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); or (A')(i)(a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), and (ii) one or more nucleic acid molecules encoding a guide RNA in an expressible form, the guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); or (A″) a complex with a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (a) a second segment that interacts with the CRISPR nuclease, the complex comprising (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a);or (d) administering to a subject in need thereof a ribonucleoprotein complex (RNP) comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b);

[0082] The definitions and preferred embodiments of the first and second aspects of the invention apply mutatis mutandis to the third aspect of the invention insofar as they are amendable for combination with the third aspect of the invention.

[0083] Diseases that would benefit from the selective depletion of cells containing a target locus associated with the disease being treated are generally diseases that are candidates for gene knockdown or knockout therapy.

[0084] The disease that would benefit from selective depletion of cells containing a target locus associated with the disease being treated is preferably selected from the group consisting of cystic fibrosis, hemophilia A or hemophilia B with or without an inhibitor, thalassemia, anemia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases, Wilson's disease or Menkes disease, lysosomal acid lipase deficiency, cancer, type 1 or type 2 diabetes, Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases), RPE65 deficiency, choroideremia, viral infections, hepatitis B, hepatitis C, HIV, or bacterial or fungal infections.

[0085] In a related aspect, the invention provides a nucleic acid molecule comprising, consisting of, or encoding (A)(i) a CRISPR nuclease comprising or consisting of (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), (ii) one or more nucleic acid molecules encoding, in an expressible form, a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease of (i), and (iii) a donor template having homology to the target locus; or (A')(i) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, 2, or 3; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); (ii) a nucleic acid molecule encoding a guide RNA in an expressible form, the nucleic acid molecule comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease of (i); and (iii) a nucleic acid molecule having homology to the target locus. or (A'') a nucleic acid molecule comprising, consisting of, or encoding a donor template having (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a);or (d) a ribonucleoprotein complex (RNP) comprising or consisting of a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b), and (ii) a nucleic acid molecule comprising, consisting of, or encoding a nucleic acid molecule encoding in an expressible form a donor template having homology to the target locus;

[0086] Similarly, the present invention provides a therapeutically effective amount of (A)(i) (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). (A') a nucleic acid molecule comprising, consisting of, or encoding one or more nucleic acid molecules encoding, in an expressible form, a CRISPR nuclease, (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease of (i), and (iii) a donor template having homology to the target locus; or (A') a nucleic acid molecule comprising, consisting of, or encoding one or more nucleic acid molecules encoding (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a nuclease, (ii) a nucleic acid molecule encoding a guide RNA in an expressible form, the nucleic acid molecule comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (a) a second segment that interacts with a CRISPR nuclease of (i), and (iii) a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus; or (A″) a nucleic acid molecule comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and (b) a second segment that interacts with the CRISPR nuclease in a complex with a guide RNA, the nucleic acid molecule comprising (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a);or (d) a ribonucleoprotein complex (RNP) comprising or consisting of a CRISPR nuclease comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b) and (ii) a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to said target locus in an expressible form, to a subject in need thereof;

[0087] Gene therapy involves replacing mutated genes or adding new genes to the genome of a subject in need of the gene to treat a disease. Gene therapy holds promise for treating a wide range of diseases. Non-limiting examples include cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and AIDS.

[0088] The desired therapeutic effect can be achieved through the properties of the donor template, which can be designed, for example, to replace or repair a mutated gene or to render the cells more susceptible to attack by the immune system or therapeutic antibodies.

[0089] In a fourth aspect, the present invention relates to a vector comprising, in an expressible form, (i) a CRISPR nuclease comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b); (ii) a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of a target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); and (iii) optionally, a vector comprising a donor template having homology to the target locus in an expressible form.

[0090] The definitions and preferred embodiments of the first to third aspects of the present invention apply mutatis mutandis to the fourth aspect of the present invention insofar as they are amendable for combination with the fourth aspect of the present invention.

[0091] Vectors comprising a donor template are suitable for the method according to the first aspect of the invention, whilst vectors not comprising a donor template are suitable for the methods according to the first and second aspects of the invention, however it should be noted that the donor template may also be supplied via a separate expression vector or as a nucleic acid molecule such as a PCR product or ssODN.

[0092] The vector or CRSPR nuclease of the present invention may be included in a kit, which preferably further comprises one or more of: (i) a population of cells as defined herein; (ii) a medium for culturing these cells; and (iii) instructions for using the kit to perform genome editing of cells at a target locus by homology directed repair (HDR) in the cell population and simultaneously enrich for HDR-edited cells in the cell population and / or selectively deplete cells containing the target locus in the cell population.

[0093] The components of the kit may be packaged separately or in different combinations, taking into consideration the intended use for modifying the nucleotide sequence of the target site in the genome of a cell. The components of the kit may be packaged, for example, in vials, tubes, bags, or the like.

[0094] The instructions may be in the form of a leaflet inside the packaging, or in the form of a web link, bar code, or QR code on the packaging.

[0095] In a fifth aspect, the present invention relates to the use of a CRISPR nuclease comprising or consisting of: (a) an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b) for genome editing of cells at a target locus by homology directed repair (HDR) in a cell population and simultaneously enriching cells in the cell population that have been genome edited by HDR and / or selectively depleting cells comprising the target locus in the cell population.

[0096] The definitions and preferred embodiments of the first to fourth aspects of the present invention apply mutatis mutandis to the fifth aspect of the present invention insofar as they are amendable for combination with the fifth aspect of the present invention.

[0097] The use is preferably an ex vivo or in vitro use.

[0098] For use of the fifth aspect of the invention - genome editing purposes by HDR and cell depletion applications - As described herein above in relation to the first and second aspects of the invention, the CRISPR nuclease according to the invention must be used in combination with a guide RNA as defined herein above. For genome editing applications by HDR, a donor template as described herein above is further used.

[0099] According to preferred embodiments of all aspects of the invention, the sequence identity of at least 80% is preferably at least 85%, more preferably at least 90% and most preferably at least 95%.

[0100] The above mentioned at least 80% sequence identity with respect to these SEQ ID NOs is increasingly preferred as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, and 100%.

[0101] Analysis and alignment of amino acid and nucleotide sequences relevant to the present invention is preferably performed using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Those skilled in the art will be aware of additional programs suitable for aligning nucleic acid sequences.

[0102] Also, in relation to this preferred embodiment, CRISPR nuclease as used herein is preferably not only structurally defined, but also functionally defined as exhibiting double-stranded DNA collateral cleavage activity.Alternatively or additionally, CRISPR nuclease can be defined as being capable of depleting the cells that the dsDNA break introduced by active BEC nuclease at the target locus is repaired by NHEJ (when using HDR template), or capable of depleting the cells that contain the target locus (when not using HDR template).

[0103] With regard to the embodiments characterized in this specification, and in particular in the claims, each embodiment mentioned in a dependent claim is intended to be combined with each embodiment of each claim (independent claim or dependent claim) from which the dependent claim depends. For example, in the case of independent claim 1 reciting three options A, B, and C, dependent claim 2 reciting three options D, E, and F, and claim 3 dependent on claims 1 and 2 and reciting three options G, H, and I, it will be understood that the specification, unless otherwise specified, expressly discloses embodiments corresponding to combinations of A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0104] Similarly, even if an independent claim and / or dependent claim does not recite alternatives, it is understood that if a dependent claim refers to more than one preceding claim, any combination of the subject matter covered thereby is considered to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to both claims 2 and 1, the combination of the subject matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject matter of claims 3, 2, and 1. If there is further dependent claim 4 referring to any one of claims 1 to 3, the combination of the subject matter of claim 4 and claim 1, the subject matter of claims 4, 2, and 1, the subject matter of claims 4, 3, and 1, and the subject matter of claims 4, 3, 2, and 1 is clearly and unambiguously disclosed. EXAMPLES

[0105] The examples illustrate the invention.

[0106] Example 1: Construction of functional genome editing systems for E. coli (BEC10 and FnCpf1) and Pichia pastoris (BEC10 and SpCas9) 1.1 CRISPR / BEC-Ec and FnCpf1-Ec vector systems for genome editing in E. coli BW25113 The genetic elements required for inducible expression of BEC10 or FnCpf1 nuclease and constitutive expression of guide RNA (gRNA) transcription were provided on three separate vectors (CRISPR / BEC10-Ec, CRISPR / FnCpf1-Ec, and CRISPR / gRNA-Ec).

[0107] The construction of the CRISPR / BEC10-Ec vector and the CRISPR / gRNA-Ec system is described below. The CRISPR / FnCpf1-Ec vector system was constructed using the same approach as the CRISPR / BEC10-Ec vector.

[0108] Design of BEC10_Coli protein expression vector The synthetic 3696 bp BEC10 nucleotide sequence was codon-optimized for expression in E. coli BW25113 using bioinformatics applications provided by the gene synthesis provider GeneArt (Thermo Fisher Scientific, Regensburg, Germany), SEQ ID NO: 7. For protein expression, the resulting synthetic gene was fused to the inducible araC-ParaBAD inducible promoter system (SEQ ID NO: 8) and the fdT terminator (SEQ ID NO: 9) (Otsuka & Kunisawa, Journal of Theoretical Biology 97 (1982), 415-436). The final BEC10_E. coli protein expression cassette was inserted by Gibson Assembly Cloning (NEB, Frankfurt, Germany) into an E. coli shuttle vector containing all the genetic elements required for episomal propagation and selection of recombinant E. coli cells.

[0109] CRISPR / BEC10-Ec vector system The complete nucleotide sequence of the constructed CRISPR / BEC10-Ec vector system is provided as SEQ ID NO:10.

[0110] CRISPR / FnCpf1-Ec vector system The complete nucleotide sequence of the constructed CRISPR / fnCpf1-Ec vector system is provided as SEQ ID NO:11.

[0111] Design of guide RNA (gRNA) expression vector Expression of chimeric gRNA for specific kanamycin gene targeting by BEC10 or FnCpf1 DNA nuclease was driven by the SacB RNA polymerase II promoter (SEQ ID NO: 12) from Bacillus megaterium (Richhardt et al., Applied Microbiology Biotechnology 86 (2010), 1959-1965) and terminated using the transcriptional T1 and T2 termination regions of the E. coli rrnB gene (SEQ ID NO: 13) (Orosz et al., European Journal of Biochemistry 201 (1991), 653-659). The chimeric gRNA consisted of a constant 19 bp BEC family stem-loop sequence (SEQ ID NO: 14, which also functions for FnCpf1 nuclease) fused to a kanamycin target-specific 24 bp spacer sequence (SEQ ID NO: 15) located inside the kanamycin resistance gene of the CRISPR / BEC10-Ec or CRISPR / FnCpf1-Ec vectors.

[0112] The final gRNA expression cassette was inserted by Gibson Assembly Cloning (NEB, Frankfurt, Germany) into an E. coli shuttle vector containing all the genetic elements required for episomal propagation and selection of recombinant E. coli cells.

[0113] Construction of the final CRISPR / gRNA-Ec vector system was mediated by Gibson Assembly Cloning (NEB, Frankfurt, Germany).

[0114] The identity of all cloned DNA elements was confirmed by Sanger sequencing at LGC Genomics (Berlin, Germany).

[0115] The complete nucleotide sequence of the constructed CRISPR / gRNA-Ec vector system is provided as SEQ ID NO:16.

[0116] 1.2 Cultivation and transformation of E. coli Transformation of competent E. coli BW25113 cells Briefly, a single colony of E. coli BW25113 was inoculated into 5 mL of LB-Kan medium and incubated at 37 °C and 200 rpm in a horizontal shaker for 12-14 h. An overnight grown preculture was diluted into 60 mL of fresh LB medium to an optical density at 600 nm (OD600) of 0.06. The inoculated medium was incubated at 30 °C and 200 rpm in a horizontal shaker until the culture reached an optical density at OD600 of 0.2. 600 μL of 20% arabinose was added and the cells were incubated at 30 °C and 200 rpm until the culture reached an optical density at OD600 of 0.5. The cells were transferred to a 50 mL conical tube and harvested by centrifugation at 4000 × g for 5 min at 4 °C. Pelleted cells from the 50 mL culture were resuspended in 60 mL of water and centrifuged at 4000 × g for 5 min at 4 °C.

[0117] A washing step was performed and the cells were resuspended in 30 mL of 10% glycerol followed by centrifugation at 4000×g for 5 min at 4° C. In a second washing step, the cells were resuspended in 6 mL of 10% glycerol followed by centrifugation at 4000×g for 5 min at 4° C. In a final step, the cells were resuspended in 150 μL of 10% glycerol. An aliquot of 25 μL of competent cells was stored at −80° C. until use. For the transformation step, an aliquot of competent cells was thawed and 50 ng of plasmid DNA was added. The prepared cells were electroporated at 1800 V, 25 μF, 200 ohms for 5 ms. Then, 975 μL of NEB® 10-beta / Stable Outgrowth Medium was added and 100 μL of the suspension was plated on selective agar plates.

[0118] 1.3 CRISPR / BEC10-Pp vector system for genome editing in Pichia pastoris The genetic elements required for constitutive expression of BEC10 and transcription of guide RNA (gRNA) were provided in an all-in-one CRISPR / BEC10-Pp vector system.

[0119] Design of BEC10 protein expression cassette The synthetic 3696 bp BEC10 nucleotide sequence was codon-optimized for expression in yeast cells using a bioinformatics application provided by the gene synthesis provider GeneArt (Thermo Fisher Scientific, Regensburg, Germany), SEQ ID NO: 6. Furthermore, the DNA nuclease coding sequence was 5'-extended by a sequence encoding the SV40 nuclear localization signal (NLS), SEQ ID NO: 17 (Kalderon et al., Cell 39 (1984), 499-509). For protein expression, the resulting synthetic 3723 bp gene was fused to the constitutive and bidirectional P. pastoris HTX1 promoter (SEQ ID NO: 18) (Weninger et al., Journal of Biotechnology 235 (2016), 139-149) and the P. pastoris AOX1TT terminator (SEQ ID NO: 19) (Weninger et al., Journal of Biotechnology 235 (2016), 139-149). The final BEC10 protein expression cassette was inserted by Gibson Assembly Cloning (NEB, Frankfurt, Germany) into the E. coli / P. pastoris shuttle vector pPpT6e (EP3572512A1), which contains all the genetic elements required for episomal propagation and selection of recombinant E. coli and recombinant P. pastoris cells.

[0120] For vector propagation and selection of recombinant E. coli cells, the plasmid contained a high copy ColE1 origin of replication from pUC and the kanMX6 marker gene (SEQ ID NO: 20) under the control of a bifunctional ILV5 / synthetic Em72 promoter (SEQ ID NO: 21) (Weninger et al., Journal of Biotechnology 235 (2016), 139-149), conferring kanamycin resistance in P. pastoris and E. coli, respectively. The Pichia autonomously replicating sequence 1 (PARS1) (SEQ ID NO: 22) allowed episomal replication of the pPpT6e shuttle plasmid in P. pastoris cells.

[0121] Design of guide RNA (gRNA) expression cassette For specific Ade2 gene targeting by BEC10 DNA nuclease in P. pastoris, a ribozyme-based technique was applied to liberate chimeric gRNAs from RNA polymerase II transcripts (Gao & Zhao, Journal of Integrative Plant Biology 56 (2014), 343-349). The bidirectional HTX1 RNA polymerase II promoter was used to express gRNAs flanked by 5' truncated hammerhead (HH)-type and 3' truncated hepatitis delta virus (HDV)-type ribozymes (Weninger et al., Journal of Biotechnology 235 (2016), 139-149).

[0122] The chimeric gRNA consisted of a constant 19 bp BEC family stem-loop sequence (SEQ ID NO: 14) fused to an Ade2 target-specific 24 bp spacer sequence (SEQ ID NO: 23) identified in the P. pastoris Ade2 gene (SEQ ID NO: 24) downstream of the nuclease BEC10-specific PAM transporter 5'-TTN-3'.

[0123] The complete RNA expression cassette, consisting of the HTX1 RNA polymerase II promoter and a chimeric gRNA flanked by the designed HH / HDV-type ribozyme, was provided as a synthetic gene fragment by GeneArt (Thermo Fisher Scientific, Regensburg, Germany).

[0124] The construction of the all-in-one CRISPR / BEC10-Pp vector system was completed by cloning the synthetic RNA expression cassette into the prepared E. coli / P. pastoris pPpT6e shuttle vector containing the BEC10 DNA nuclease expression cassette. The construction of the final CRISPR / BEC10-Pp vector system was mediated by Gibson Assembly Cloning (NEB, Frankfurt, Germany).

[0125] The identity of all cloned DNA elements was confirmed by Sanger sequencing at LGC Genomics (Berlin, Germany).

[0126] CRISPR / BEC10-Pp all-in-one vector system The complete nucleotide sequence of the constructed CRISPR / BEC10-Pp vector system is provided as SEQ ID NO:25.

[0127] 1.4 CRISPR / SpCas9-Pp vector system for genome editing in P. pastoris The genetic elements required for constitutive expression of SpCas9 (S. pyogenes Cas9) DNA nuclease and transcription of single guide RNA were provided in an all-in-one CRISPR / SpCas9_Pp vector system.

[0128] Design of SpCas9 protein expression cassettes Based on the published SpCas9 nucleotide sequence from Streptococcus pyogenes (Deltcheva et al., Nature 471 (2011), 602-607), DNA synthesis of a yeast codon-optimized SpCas9 coding sequence for expression in P. pastoris was ordered from GeneArt (Thermo Fisher Scientific, Regensburg, Germany) (SEQ ID NO: 26). For nuclear import, the SpCas9 DNA nuclease coding sequence was 5'-extended by a sequence encoding the SV40 nuclear localization signal (NLS) (SEQ ID NO: 17). The resulting synthetic 4134 bp SpCac9 gene was fused to the constitutive and bidirectional P. pastoris HTX1 promoter (SEQ ID NO: 18) and the P. pastoris AOX1TT terminator (SEQ ID NO: 19) following the protein expression strategy described for the BEC10 DNA nuclease (Weninger et al., Journal of Biotechnology 235 (2016), 139-149). The final SpCas9 protein expression cassette was inserted into the E. coli / P. pastoris pPpT6e shuttle vector carrying identical genetic elements for propagation and selection by Gibson Assembly Cloning (NEB, Frankfurt, Germany) as previously described for the CRISPR / BEC10-Pp vector system.

[0129] Design of guide RNA expression (gRNA) cassette For specific Ade2 gene targeting by SpCas9 DNA nuclease in P. pastoris, a ribozyme-based technique was applied to liberate chimeric gRNAs from RNA polymerase II transcripts (Gao & Zhao, Journal of Integrative Plant Biology 56 (2014), 343-349). The bidirectional HTX1 RNA polymerase II promoter was used to express gRNAs flanked by 5' truncated hammerhead (HH)-type and 3' truncated hepatitis delta virus (HDV)-type ribozymes (Weninger et al., Journal of Biotechnology 235 (2016), 139-149).

[0130] The chimeric gRNA consisted of an Ade2 target-specific 20 bp spacer sequence (SEQ ID NO: 27) fused to an 80 bp SpCas9-specific sgRNA sequence (SEQ ID NO: 28). The target spacer sequence was identified in the P. pastoris Ade2 gene (SEQ ID NO: 24) downstream of the nuclease SpCa9-specific PAM transporter 5'-NGG-3'.

[0131] The complete RNA expression cassette, consisting of the HTX1 RNA polymerase II promoter and a chimeric gRNA flanked by the designed HH / HDV-type ribozyme, was provided as a synthetic gene fragment by GeneArt (Thermo Fisher Scientific, Regensburg, Germany).

[0132] To generate the final CRISPR / SpCas9-Pp vector system, the synthetic RNA transcription cassette was cloned into a prepared E. coli / P. pastoris pPpT6e shuttle vector containing the SpCas9 DNA nuclease expression cassette by Gibson Assembly Cloning (NEB, Frankfurt, Germany). The identity of all cloned DNA elements was confirmed by Sanger sequencing at LGC Genomics (Berlin, Germany).

[0133] CRISPR / SpCas9-Pp all-in-one vector system The complete nucleotide sequence of the constructed CRISPR / SpCas9-Pp vector system is provided as SEQ ID NO:29.

[0134] 1.5 Design of homology-directed repair template (HDR-template) The 1998 bp Ade2 BEC10 and spCas9 HDR-template were designed to create a 1692 bp site-specific deletion in the P. pastoris Ade2 gene on the chromosome by homologous recombination. Successful homologous recombination resulted in a complete deletion of the Ade2 gene. In the HDR template, the introduced Ade2 gene deletion was flanked by 1022 bp and 976 bp sequences that were homologous to the target region on the chromosome. Successful recombination events mediated by the designed HDR-template eliminated the previously described PAM and protospacer regions of the Ade2 gene on the chromosome, preventing the programmed gRNA / BEC10 or gRNA / spCas9 DNA nuclease complex from targeting the P. pastoris genome again. Furthermore, gene deletions were introduced resulting in Ade2 mutant clones that were easily recognized by their red colony color, because mutant cells lacking adenine accumulated red purine precursors in the vacuole (Ugolini et al., Curr Genet (2006), 485-92).

[0135] The complete sequence of the Ade2 HDR-template for BEC10 and SpCas9 is provided as SEQ ID NO:30.

[0136] 1.6 Cultivation and transformation of Pichia pastoris Preparation of competent P. pastoris CBS7435 cells Preparation and transformation of competent P. pastoris CBS7435 cells was performed as described in Wu & Letchworth, Biotechniques (2014), 36, 152-154. Briefly, a single colony of P. pastoris was inoculated into 5 mL of YPD medium and incubated at 30°C and 250 rpm in a horizontal shaker for 6-8 hours. Cells from the preculture were diluted in 100 mL of YPD medium to an optical density at 600 nm (OD600nm) of 0.0025.

[0137] The inoculated medium was incubated overnight at 30 °C and 250 rpm in a horizontal shaker until the optical density at OD600nm of the culture reached 1.0-2.0. Cells were harvested by centrifugation at 4302 × g for 5 min at room temperature (RT). The cell density (cells / mL) of the culture was estimated from the optical density at OD600nm according to the following relationship: 1 × OD600nm = 5 × 10 7 Cells / mL. 8 x 10 for each transformation 8 The cells were suspended in 8 mL of 100 mM LiAc, 10 mM DTT, 0.6 M sorbitol, and 10 mM Tris-HCI. The resuspended cells were incubated at RT for 30 min.

[0138] The cells were then pelleted by centrifugation at 3999×g for 5 min at RT and resuspended in 1.5 mL of ice-cold 1 M sorbitol. The cells were transferred to a 1.5 mL microcentrifuge tube, washed three times with 1.5 mL of ice-cold 1 M sorbitol, and finally diluted to 100% for a final cell density of approximately 10 10 The cells were resuspended in 80 μL of 1 M ice-cold sorbitol to give cells / mL.

[0139] Transformation of competent P. pastoris cells One aliquot of prepared competent cells was mixed with 1 μg of supercoiled plasmid DNA in 5 μL of water. To repair the DNA damage induced by CRISPR / BEC10 or CRISPR / Cas9 by homologous recombination, 1.5 μg of linear double-stranded DNA fragments were added to the cells. The total volume of DNA solution did not exceed 5 μL. The DNA-cell mixture was transferred to a pre-chilled 0.2 cm gap vial and incubated on ice for 5 min. An electroporation pulse was applied at 2 kV, 25 μF, 200 Ω using a Bio-Rad Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Munich, Germany). The electroporated cells were immediately diluted with 1 mL of ice-cold regeneration medium (0.5×YPD, 0.5 M sorbitol) and transferred to a 2 mL microcentrifuge tube. After incubation for 1 h without agitation and 2 h on a horizontal shaker at 250 rpm, the transformed cells were plated onto appropriate selective agar plates depending on the experimental setup.

[0140] Plating of transformed P. pastoris cells To analyze the efficiency of different gRNA spacers, in the absence of any homologous repair template, transformed cells were plated onto YPD agar plates containing 200 μg / mL geneticin (G418) and incubated at 30°C for at least 2 days.

[0141] Disruption of the Ade2 gene and the induced red phenotype of mutant P. pastoris cells by integration of the co-transformed homologous repair template into the Ade2 target gene mediated by BEC10-gRNA and Cas9-gRNA were visualized using a simple colony color filter assay: transformed cells were plated on NC transfer membrane filters (Merck Chemicals, Darmstadt, Germany) and directly applied to the surface of YPD agar plates supplemented with 200 μg / mL geneticin (G418) and 50 μg / mL adenine. After incubation at 30°C for at least 2 days, the filters containing grown cells were transferred to minimal medium agar plates supplemented with 200 μg / mL geneticin (G418) and 5 μg / mL adenine.

[0142] Example 2: Vector targeting in E. coli to demonstrate a novel DNA targeting mechanism of BEC family nucleases compared to FnCpf1 To demonstrate the novel DNA targeting mechanism of BEC family nucleases, experiments were performed with two different vectors co-transformed into E. coli. In the two different approaches, the CRISPR / BEC10-Ec vector or the CRISPR / FnCpf1-Ec vector was co-transformed with the CRISPR / gRNA-Ec vector containing a spacer sequence targeting the ampicillin resistance gene located on the CRISPR / BEC10-Ec vector or the CRISPR / FnCpf1-Ec vector (a schematic diagram of the experimental setup is shown in Figure 1). Using a nuclease with classical DNA targeting activity (double-strand break), the activation of the CRISPR nuclease linearizes the CRISPR / BEC10-Ec or CRISPR / FnCpf1-Ec vector, disrupting the open reading frame of the ampicillin resistance gene and preventing the vector from propagating. Thus, deletion of the ampicillin resistance gene located on the CRISPR / BEC10-Ec or CRISPR / FnCpf1-Ec vector renders the cells sensitive to ampicillin, but the kanamycin resistance gene located on the CRISPR / gRNA-Ec vector remains intact and therefore resistant to kanamycin.

[0143] To directly compare the DNA targeting mechanisms of BEC family (BEC10) and classical (FnCpf1) nucleases, the following experiment was performed: 1. CRISPR / BEC10-Ec and CRISPR / gRNA-Ec vectors were co-transformed into E. coli cells and grown / selected on plates containing ampicillin and kanamycin as well as on plates containing kanamycin only. 2. The CRISPR / FnCpf1-Ec and CRISPR / gRNA-Ec vectors were co-transformed into E. coli cells and grown / selected on plates containing ampicillin and kanamycin as well as on plates containing kanamycin only. 3. Negative control (NC) experiments of setups 1 and 2 were performed using the same experimental approach except that a nonsense spacer sequence (not matching a sequence in the vector or one of the E. coli genomes) was used in the CRISPR / gRNA-Ec vector.

[0144] All plates were visually assessed by counting the number of colonies that grew.

[0145] result All experiments were performed in five biological replicates, and the results obtained from these replicates were combined to evaluate the DNA targeting mechanism of BEC10 in comparison to FnCpf1 (an exemplary plate is shown in Figure 2).

[0146] FnCpf1 The results obtained with FnCpf1 nuclease showed the expected outcome: plates containing kanamycin antibiotic showed a similar number of grown E. coli colonies as the negative control (NC), because the cells were still resistant to kanamycin since the resistance gene was located on the CRISPR / gRNA-Ec vector, which was not targeted by the gRNA. In contrast, plates containing ampicillin and kanamycin showed a strong colony reduction (>99%) in contrast to the negative control, because the vector containing the ampicillin resistance gene (CRISPR / FnCpf1-Ec) was targeted and depleted by FnCpf1 nuclease.

[0147] BEC10 Surprisingly, the results obtained with BEC10 nuclease showed a different growth pattern. Plates containing kanamycin antibiotics and plates containing ampicillin and kanamycin antibiotics showed a strong colony reduction (>99%) compared to the negative control. As described for the FnCpf1 experiment, plates containing ampicillin and kanamycin were expected to show a reduction in colonies (targeting / depletion of vectors with ampicillin). In addition to this, plates containing only kanamycin showed no colony reduction with FnCpf1 nuclease, but a strong colony reduction with BEC10 nuclease.

[0148] conclusion This striking difference in results between BEC10 and FnCpf1 is explained by the novel mechanism of action of BEC family nucleases: the initial sequence-specific binding of the spacer sequence (incorporated into the gRNA) to the protospacer region (target region) on the vector activates the BEC nuclease, triggering non-specific targeting of double-stranded DNA. Thus, the DNA targeting activity of the BEC nuclease (once specifically activated) can degrade dsDNA and "jump" to other DNA strands in the same cell. This is the kind of reaction that can be described as collateral activity, since even DNA that does not have a target region is degraded when the nuclease is activated.

[0149] In the experiments shown, the BEC nuclease was activated by a protospacer sequence located on the CRISPR / BEC10-Ec vector, and once activated, its activity "jumped" and targeted CRISPR / gRNA-Ec and / or the E. coli genome, resulting in a robust reduction of colonies on plates containing kanamycin alone.

[0150] To the best of our knowledge, no nuclease has been reported that is specifically activated by spacer / protospacer pairing and has collateral dsDNA cleavage activity upon activation. This novel activity pattern can be used in a variety of applications where classical CRISPR nucleases do not function or have limited scope of application (e.g., Example 3 and Example 5).

[0151] Example 3: Precise genome editing in P. pastoris using BEC10 nuclease compared with SpCas9 To demonstrate one of the advantages of the novel mechanism of action of BEC family nucleases, genome editing experiments were performed in organisms that are natively capable of non-homologous end joining (NHEJ) DNA repair.

[0152] Experimental setup: In this example, the Ade2 gene was knocked out in P. pastoris using the CRISPR / BEC10-Pp or CRISPR / SpCas9-Pp vector system and HDR template.

[0153] Ade2 is a nonessential gene in P. pastoris, but when knocked out it produces colonies with a red phenotype, as mutant cells accumulate red purine precursors in their vacuoles (Ugolini et al., Curr Genet (2006), 485-92). Because of this easy readout, knocking out the Ade2 gene can be used as a screening system to monitor the ability of CRISPR Cas proteins to function as genome editing tools.

[0154] In this approach, knockout of the Ade2 gene was used to monitor the genome editing activity of BEC10 nuclease compared to SpCas9 in two experimental setups. 1. Ade2 knockout by NHEJ (without HDR template) 2. Specific Ade2 knockout using homology-directed repair (Introduction of an HDR template that site-specifically deletes the Ade2 gene, eliminating the PAM and protospacer sequences)

[0155] Briefly, CRISPR / BEC10-Pp or CRISPR / SpCas9-Pp expression constructs with or without homology-directed repair templates were transformed into P. pastoris cells and plated as described in Example 1.6.

[0156] In parallel, negative control experiments using expression constructs of CRISPR / BEC10-Pp or CRISPR / SpCas9-Pp lacking the spacer sequence targeting the Ade2 gene were performed to demonstrate the dependency of Cas proteins to be guided to the target DNA region by a specific spacer.

[0157] After transformation and incubation at 30° C. for 48 hours, the culture plates were analyzed by counting the number of grown colonies and assessing their phenotype (red or white).

[0158] result: All experiments were performed in five biological replicates, and the results from these replicates were combined to assess the genome editing activity of BEC10 in comparison to SpCas9 (an exemplary plate is shown in FIG. 3).

[0159] In the first experimental setup, expression constructs of CRISPR / BEC10-Pp or CRISPR / SpCas9-Pp were transformed into P. pastoris cells without the use of HDR template (-HDR). Compared to the negative control experiment, active SpCas9 and BEC10 nuclease strongly reduced colonies (>99%), and even more so when BEC10 nuclease was used. Furthermore, in the experimental approach with Cas9 nuclease, almost 40% of the remaining cells showed a red (Ade2 knockout) phenotype, in contrast to cells treated with BEC10 nuclease, where 0% of cells showed a red phenotype.

[0160] In the second experimental setup, expression constructs of CRISPR / BEC10-Pp or CRISPR / SpCas9-Pp were transformed into P. pastoris cells together with the HDR template (+HDR). Compared to the negative control experiment, active SpCas9 and BEC10 nucleases strongly reduced colonies (>98%), but the overall cell number was slightly less dramatically reduced compared to the experiment without the HDR template. Furthermore, the majority of surviving cells showed an Ade2 knockout phenotype for SpCas9 and BEC10 (SpCas9 ≈ 73% / BEC10 ≈ 90%). Furthermore, 40 colonies showing the Ade2 knockout phenotype (20 treated with SpCas9 and 20 treated with BEC10) were further analyzed by Sanger sequencing, which showed that 14 of the 20 colonies treated with Cas9 had integrated the HDR template into the genome, and 6 had been edited by NHEJ. In contrast, 20 of 20 colonies analyzed after BEC10 treatment showed that the HDR template had been introduced into the genomic Ade2 gene.

[0161] Conclusion: Although both SpCas9 and BEC nucleases significantly reduced overall colony size after activation, Ade2 gene editing showed distinctly different results between the two nucleases.

[0162] Since P. pastoris is an organism that can naturally repair DNA double-strand breaks using NHEJ, targeting the Ade2 gene with SpCas9 results in Ade2 knockout colonies due to frameshift mutations caused by defects in the NHEJ mechanism. In contrast, cells treated with BEC10 nuclease did not show Ade2 knockout colonies, which can be explained by the novel mechanism of action of BEC family nucleases. BEC family nucleases show a completely different mechanism of action that prevents NHEJ, because activation of BEC nucleases induces collateral dsDNA activity that causes cell death without giving the cell an opportunity to prevent death by NHEJ-derived DNA repair.

[0163] In contrast, Ade2 knockout colonies were present with SpCas9 and BEC10 when the gene was knocked out using HDR template. In the case of SpCas9, a dsDNA break introduced in the Ade2 gene causes the cell to repair this break. To do so, the cell has two options: A: introduce a HDR template, or B: repair the DNA break using NHEJ. Because P. pastoris prefers homologous recombination repair over NHEJ, nearly 70% of edited cells had the HDR template introduced into their genome, and nearly 30% of edited cells had NHEJ-mediated DNA repair.

[0164] In contrast, BEC10 nuclease prevents cells from performing NHEJ-mediated DNA repair by killing the cells when BEC nuclease is activated. Thus, BEC10 nuclease forces cells to introduce HDR template into the genome before BEC nuclease is activated. Since the integration of HDR template deletes the PAM and protospacer sequences in the genome, the protospacer sequence that matches the spacer sequence of BEC gRNA is no longer present in the genome, and BEC nuclease remains inactive. Cells in which the HDR template is integrated into the genome and BEC nuclease remains inactive can survive the treatment, which explains why 100% of BEC-edited cells have integrated the HDR template into their genome.

[0165] View: Most higher organisms (eukaryotes) are capable of performing NHEJ. Using genome editing tools, this mechanism can be used to knock out genes of interest in a fairly non-specific manner (introduction of indels by a defective NHEJ repair mechanism). However, as soon as precise knock-outs or knock-ins are performed using HDR templates, the NHEJ mechanism causes many technical problems, leading to a mixture of cell populations with undesired edits, as some of the edited cells will repair their DNA using the NHEJ pathway instead of introducing the HDR template. The use of BEC family nucleases overcomes this limitation, since they display a novel mechanism of action that inhibits the ability of cells to target dsDNA and perform NHEJ to survive the DNA double-strand break. Moreover, to prevent cell death, cells are forced to introduce HDR templates into the genome, making it a very efficient way to perform precise gene knock-outs or knock-ins using BEC nucleases.

[0166] Example 4: Expression and purification of BEC10 RNP for HEK cell transfection 4.1 CRISPR / BEC-FLAG-Ec and CRISPR / gRNA-Ec vector systems for BEC expression (E. coli) and RNP purification The genetic elements required for inducible expression and FLAG-tagged purification of BEC10 nuclease, as well as constitutive expression of guide RNA (gRNA) transcription, were provided in three separate vectors (CRISPR / BEC10-FLAG-Ec, CRISPR / gRNA-EGFP-Ec, and CRISPR / gRNA-NC-Ec).

[0167] Design of CRISPR / BEC-FLAG-Ec protein expression vector The synthetic 3696 bp BEC10 nucleotide sequence was codon-optimized for expression in E. coli BW25113 using bioinformatics applications provided by the gene synthesis provider GeneArt (Thermo Fisher Scientific, Regensburg, Germany). For protein expression, the resulting synthetic gene was fused to an inducible araC-ParaBAD inducible promoter system and an fdT terminator (Otsuka & Kunisawa, Journal of Theoretical Biology 97 (1982), 415-436). Furthermore, the DNA nuclease coding sequence was 3'-extended by a sequence encoding a nucleoplasmin nuclear localization signal (NLS) and two SV40 NLSs. At the 5'-end of the DNA nuclease coding sequence, the sequence was extended by a sequence encoding a myc NLS. To purify the DNA nuclease, a FLAG tag was linked to the 5'-coding sequence of the DNA nuclease by a linker to the myc NLS.

[0168] The final BEC10_E. coli protein expression cassette was inserted by Gibson assembly cloning (NEB, Frankfurt, Germany) into an E. coli shuttle vector containing all the genetic elements required for episomal propagation and selection of recombinant E. coli cells.

[0169] CRISPR / BEC10-FLAG-Ec vector system The complete nucleotide sequence of the constructed CRISPR / BEC10-FLAG-Ec vector system is provided as SEQ ID NO:31.

[0170] Design of guide RNA (gRNA) expression vectors for EGFP targeting (CRISPR / gRNA-EGFP-Ec) and negative control (CRISPR / gRNA-NC-Ec) The design of the gRNA expression vector is described in Example 1.1. In contrast to the vector described in 1.1, the spacer sequence targeting the kanamycin gene is replaced with a spacer sequence targeting the EGFP gene (SEQ ID NO: 32) or a negative control (NC) sequence (SEQ ID NO: 33) that does not match any of the target regions of the HEK cells used.

[0171] The complete nucleotide sequence of the constructed CRISPR / gRNA-EGFP-Ec vector system is provided as SEQ ID NO:34.

[0172] The complete nucleotide sequence of the constructed CRISPR / gRNA-NC-Ec vector system is provided as SEQ ID NO:35.

[0173] Nuclease expression Briefly, a single colony of E. coli BW25113+CRISPR / BEC10-FLAG-Ec+CRISPR / gRNA-EGFP-Ec for BEC10-EGFP RNP expression or E. coli BW25113+CRISPR / BEC10-FLAG-Ec+CRISPR / gRNA-NC-Ec for BEC10-NC RNP expression was inoculated into 5 mL of LB-Kan-Amp medium and incubated at 37 °C and 200 rpm in a horizontal shaker for 12-14 h. The overnight grown preculture was diluted in 60 mL of fresh LB medium to an optical density at 600 nm (OD600) of 0.05. The inoculated medium was incubated at 30 °C and 200 rpm in a horizontal shaker until the culture reached an optical density at OD600 of 0.2. The incubation temperature was reduced to 21 °C. When the optical density OD600 reached 0.5, 600 μL of 20% L-arabinose was added and the cells were incubated at 21° C. and 200 rpm for approximately 21 hours until the culture reached an optical density at OD600 of 4. The culture was transferred to a 50 mL conical tube and the cells were harvested by centrifugation at 4000×g for 10 minutes at 4° C. Pelleted cells from the 90 ODV culture were stored at −20° C.

[0174] Cell disruption and purification of FLAG tag 90 ODV of pelleted cells were resuspended in 3 mL of TBS (50 mM TRIS-HCl, 150 mM NaCl, pH 7.4) and disrupted by ultrasonic cell disruption using a Branson Sonifier 250 at 50% duty cycle, power output 2.5, for four 30-s sonication cycles. Between each cycle, cells were cooled on ice for 1 min. The soluble fraction was separated from the insoluble fraction by centrifugation at 4500 rpm for 10 min at 4°C.

[0175] Nuclease and gRNA (already bound to each other) were purified with Pierce™ magnetic anti-DYKDDDDK-agarose (ThermoFisher Scientific, Regensburg, Germany). A 120 μL aliquot of magnetic beads was used to purify 1.5 mL of soluble cell fraction. The magnetic beads were equilibrated twice with 1 mL of TBS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl). The supernatant was removed using a magnetic stand. 1.5 mL of the soluble fraction was incubated with the magnetic beads for 18-20 h on a rotator at 4 °C. After binding, the magnetic beads were washed four times with 1 mL of TBS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and once with 1 mL of LC-MS analysis water (TH Geyer, Renningen, Germany) and the supernatant was removed with a magnetic stand. Nuclease / gRNA complexes were eluted from the beads by adding 50 μL of 1.5 mg / mL Pierce™ 3×DYKDDDK-peptide (ThermoFisher Scientific, Regensburg, Germany) dissolved in dPBS (PAN Biotech, Aidenbach, Germany) and incubated for 30 min at room temperature on a rotator. The supernatant containing purified BEC10-EGFP or BEC10-NC ribonucleoproteins (RNPs) was removed from the beads on the magnetic stand and stored at 4°C.

[0176] Generation of HEK-EGFP and HEK-DsRed cells To generate two types of HEK cells, the commercially available Flp-In™ T-REx™ 293 Cell Line (Thermo Fisher) was used in combination with the Flp-In™ T-REx™ Core Kit (Thermo Fisher). To integrate a gene of interest, a DNA fragment containing the coding sequence of EGFP (enhanced green fluorescent protein) (SEQ ID NO: 36) or DsRed (red fluorescent protein) (SEQ ID NO: 37) was integrated locus-specifically into the genome of Flp-In™ T-REx™ 293 cells according to the instructions provided with the Flp-In™ T-REx™ Core Kit, generating two identical cell lines (HEK-EGFP and HEK-DsRed) that differ only in the inserted gene.

[0177] Transfection and culture of HEK cells Cells were seeded and cultured in medium (DMEM, 8% FCS, tetracycline-free, 4 mM L-glutamine; PAN Biotech, Aidenbach, Germany, #P04-03600, #P30-3602, #P04-80100) + 1 μg / mL tetracycline (Merck KGaA, Darmstadt, Germany, #T7660) (to induce expression of EGFP and DsRed genes) to reach 70%-90% confluence on the day of transfection. 2+ and Mg 2 After rinsing with 100 µL of TrypLE (no; PAN Biotech, Aidenbach, Germany, #P04-53500), cells were detached by adding 1 mL of pre-warmed TrypLE (Thermo Fisher Scientific, Regensburg, Germany) to a 10 cm Petri dish and incubated at 37 °C for 2 min. After adding 5 mL of pre-warmed medium, the cell number of the suspension was determined with a CASY cell counter (OLS OMNI Life Science GmbH & Co KG, Bremen, Germany). The cell number was determined by 1 × 10 5 The cells were transferred to a 5 mL conical tube and centrifuged at 200 g for 5 min at room temperature. The supernatant was removed and the pelleted cells were resuspended in 1 mL of 1x DPBS (Ca2+ and Mg 2+ The cells were resuspended in 10 μL of Resuspension Buffer R containing 50 pmol of purified BEC10-EGFP or BEC10-NC RNP and centrifuged at 200 g for 5 min at room temperature. For electroporation using the Neon™ Transfection System (Thermo Fisher Scientific, Regensburg, Germany), the Neon™ 10 μL Kit (Thermo Fisher Scientific, Regensburg, Germany) was applied, the supernatant was removed again, and the cells were resuspended in 10 μL of Resuspension Buffer R containing 50 pmol of purified BEC10-EGFP or BEC10-NC RNP. The cell-RNP suspension was then aspirated with a 10 μL Neon™ Transfection Tip and transferred to a Neon™ Transfection Tube containing 3 mL of Electrolytic Buffer E. The cells were then electroporated with a single pulse of 1600 V and 20 ms pulse width. The transfected cells were cultured in 24-well plates at 37° C., 5% CO2 with 500 μL of pre-warmed (37° C.) medium containing 1× Gibco™ antibiotic / antimycotic solution (Thermo Fisher Scientific, Regensburg, Germany) and 15 μg / mL Gibco™ gentamicin (Thermo Fisher Scientific, Regensburg, Germany). After 24 h of culture, the medium was supplemented with 1 μg / mL tetracycline.

[0178] After reaching approximately 70%-90% confluence, the transfected cells in 24-well plates were detached using pre-warmed (37°C) TrypLE, transferred to 10 cm Petri dishes, and cultured for an additional 5 days in medium + 1 μg / mL tetracycline (medium was changed after 3 days of culture) before visual evaluation by bright field and fluorescence (EGFP = Ex λ 488 nm, Em λ 507 nm / DsRed = Ex λ 558 nm, Em λ 583) microscopy (Leica DM Il LED - Leica Microsystems GmbH, Wetzlar, Germany) and flow cytometric cell counting (CyFlow Space-Sysmex Deutschland GmbH, Norderstedt, Germany).

[0179] FACS (fluorescence-activated cell sorting) analysis Cells were rinsed with 5 mL of 1xDPBS and detached with 1 mL of TrypLE for 10 min at 37°C. To stop the detachment process, 9 mL of DMEM was added and cell counts were determined by CASY cell counter. After transferring the cell suspension to a 50 mL conical tube, cells were pelleted by centrifugation at 200g for 5 min at room temperature and then resuspended in an appropriate volume of 1xDPBS to determine cell counts of 1x10 6 The cell suspension was then visually analyzed by FACS (CyFlow Space-Sysmex Deutschland GmbH, Norderstedt, Germany) to assess EGFP staining of the cells according to the instructions provided by the manufacturer.

[0180] Example 5: Target-specific cell depletion of mammalian cells To demonstrate the use of BEC family nucleases in applications where classical CRISPR nucleases cannot be applied, experiments were performed showing BEC10-induced depletion of mammalian cells based on specific target regions.

[0181] Experimental setup: To demonstrate specific depletion of cells using the novel mechanism of action of BEC family nucleases (collateral activity after initial guide-specific activation), experiments were performed with a 50 / 50 mixture of two types of HEK cells (HEK-EGFP and HEK-DsRed). Both types of HEK cells are Flp-In™ T-REx™ 293 cells that are nearly identical except for the locus-specific integration of the EGFP or DsRed genes. A 50 / 50 mixture of both cell types was cultured as described in Example 4, and the BEC10 nuclease was transfected into the cells as described in Example 4 in combination with a spacer sequence that specifically binds to a region of the EGFP gene (and does not match either the rest of the genome or the DsRed gene). After culturing the cell mixture for 7 days, the cells were visually evaluated using a microscope (bright field and fluorescent) and counted by FACS to demonstrate specific depletion of cells bearing EGFP without affecting the viability of cells bearing DsRed.

[0182] In parallel, negative control experiments using BEC10 nuclease in combination with a spacer sequence that does not bind to either HEK-EGFP or HEK-DsRed cell sequences were performed to demonstrate the dependence of guide-specific BEC10 activation on targeted cell depletion.

[0183] result: All experiments were performed in six biological replicates, and the results from these replicates were combined to assess the cell depletion efficiency of BEC10 nuclease. Exemplary microscopy images and FACS results are shown in FIG. 4.

[0184] After 7 days in culture, a 50 / 50 mixture of HEK-EGFP and HEK-DsRed cells was assessed by microscopy (bright field and fluorescence) and FACS counting.

[0185] Negative control experiments using BEC10-NC RNPs (combination of BEC10 nuclease with a spacer sequence that does not match sequences derived from either HEK-EGFP or HEK-DsRed cells) showed normal cell growth on plates (Figure 4A, top image) (approximately half of the grown cells showed EGFP staining (Figure 4A, center image) and the other half showed DsRed staining (Figure 4A, bottom image). In support of this visual assessment, FACS counting of grown colonies in the negative control (Figure 4C, top image) showed that nearly 55% of the counted cells were identified as EGFP-negative (peak on the left side of the figure) and nearly 45% were identified as EGFP-positive (peak on the right side of the figure) cells, thus confirming that the 50 / 50 mixture of both cell types did not change significantly throughout the 7 days of culture in this negative control setup.

[0186] BEC-specific cell depletion experiments using BEC10-EGFP RNP (combination of BEC10 nuclease and a spacer sequence specifically matching a sequence derived from the EGFP gene) showed a significant reduction in proliferating cells after 7 days compared to the negative control (Figure 4B top image vs. Figure 4A top image), indicating that cells were depleted during the 7-day cell proliferation period. Furthermore, fluorescence images revealed that only two cells showed EGFP staining in the field of view (Figure 4B middle image), indicating that HEK-EGFP-positive cells were largely depleted, whereas the viability of HEK-DsRed cells (Figure 4B bottom image) was not affected by BEC10 treatment (almost all of the proliferating cells showed DsRed staining). FACS counting also supported these results, as the right peak (indicating EGFP-positive cells) was almost completely eliminated, whereas the left peak showed a strong signal of EGFP-negative cells. Further evaluation of the FACS results confirmed specific cell depletion of EGFP-positive cells, as EGFP-positive cells accounted for less than 1.5% of the total cell population, and more than 98.5% were EGFP-negative.

[0187] Conclusion: The Examples demonstrate the utilization of a novel and unique mechanism of action of BEC family nucleases for the targeted depletion of mammalian cells.

[0188] Non-homologous end joining (NHEJ) is the preferred DNA repair mechanism in most higher eukaryotic cells (e.g., mammalian and plant cells), i.e., DNA double-strand breaks introduced by classical CRISPR nucleases are repaired by the NHEJ mechanism (often resulting in short indels (insertions or deletions)), allowing cells to survive the DNA damage.

[0189] In contrast, BEC family nucleases induce collateral dsDNA degradation after initial guide-specific activation, overloading the target cell's repair machinery and causing cell death. Due to the PAM- and spacer sequence-specific activation of the initial BEC family nuclease, this novel mechanism of action can be used to selectively deplete all cells carrying the marker sequence without affecting the viability of surrounding cells that do not carry this specific sequence.

[0190] In the future, this novel and unique mechanism of action can be used in a variety of applications, such as the target-specific depletion of prokaryotic and eukaryotic cells, including, for example, cancer, autoreactive immune, or virus-infected cells, as well as the enrichment of classical genome editing events with the subsequent depletion of all non-edited cells.

Claims

1. 1. A method for performing genome editing of cells at a target locus by homology directed repair (HDR) within a cell population and simultaneously enriching for HDR-edited cells within the cell population, comprising: (A) introducing into cells within the cell population one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are: (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of (ii) a guide RNA comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease of (i); and (iii) a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus. in an expressible form; or (A') injecting cells within the cell population, (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of (ii) a nucleic acid molecule encoding a guide RNA in an expressible form, the nucleic acid molecule comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease of (i); and (iii) a nucleic acid molecule comprising, consisting of, or encoding a donor template having homology to the target locus. introducing; or (A'') injecting cells within the cell population, (i) a complex with a guide RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus, and (b) a second segment that interacts with the CRISPR nuclease; (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a ribonucleoprotein complex (RNP) comprising or consisting of a CRISPR nuclease comprising or consisting of (ii) expressing a donor template having homology to the target locus in an expressible form; a nucleic acid molecule comprising, consisting of, or encoding a nucleic acid molecule and (B) culturing the cells under conditions in which a target locus carried by cells of the cell population is genome edited by homology-directed repair (HDR), and the CRISPR nuclease simultaneously enriches for HDR-genome-edited cells in the cell population; A method comprising:

2. 2. The method of claim 1, further comprising: (C) isolating one or more cells in which the target locus has been genome edited by homology-directed repair (HDR).

3. 3. The method of claim 1 or 2, wherein the donor template introduces one or more mutations into the target locus.

4. 3. The method of claim 1 or 2, wherein the donor template inserts one or more nucleotides into the target locus.

5. 3. The method of claim 1 or 2, wherein the donor template deletes one or more nucleotides at the target locus.

6. 3. The method of claim 1 or 2, wherein the donor template replaces one or more nucleotides at the target locus.

7. 3. The method of claim 1 or 2, wherein the donor template comprises or consists of a nucleotide sequence having one or more intended mutations flanking a nucleotide sequence homologous to the target locus.

8. 1. A method for selectively depleting cells containing a target locus in a cell population, comprising: (A) introducing into cells within the cell population one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are: (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of: (ii) a guide RNA comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease of (i). or (A') injecting cells within the cell population, (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of: (ii) one or more nucleic acid molecules encoding a guide RNA in an expressible form, the nucleic acid molecule comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease of (i). introducing; or (A'') injecting cells within the cell population, (i) a complex with a guide RNA, the complex comprising: (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease. (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). Ribonucleoprotein complexes (RNPs) comprising or consisting of and (B) culturing the cells under conditions in which the CRISPR nuclease selectively depletes cells in the cell population that contain the target locus; A method comprising:

9. 10. The method of any one of claims 1, 2 or 8, wherein cells in the population are capable of repairing double-stranded DNA breaks by non-homologous end joining (NHEJ).

10. 10. The method of any one of claims 1, 2 or 8, wherein the cells in the population are prokaryotic or eukaryotic cells, preferably vertebrate cells, most preferably mammalian cells.

11. 10. The method of any one of claims 1, 2 or 8, which is an in vitro or ex vivo method.

12. For use in treating a disease by selectively depleting cells containing a target locus associated with the disease to be treated, (A) (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of: (ii) (a) a guide RNA comprising a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i). one or more nucleic acid molecules encoding in an expressible form; (A') (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of: (ii) (a) one or more nucleic acid molecules encoding a guide RNA in an expressible form, the guide RNA comprising a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); or (A″) (a) a first segment comprising a nucleotide sequence complementary to a sequence of the target locus; and (b) a second segment that interacts with the CRISPR nuclease, in a complex with a guide RNA. (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). A ribonucleoprotein complex (RNP) comprising or consisting of:

13. (i) (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). a CRISPR nuclease comprising or consisting of (ii) (a) a guide RNA comprising a first segment comprising a nucleotide sequence complementary to a sequence of the target locus and a second segment that interacts with the CRISPR nuclease of (a)(i); and (iii) optionally, a donor template homologous to the target locus; A vector comprising the above in an expressible form.

14. To perform genome editing of cells at a target locus by homology directed repair (HDR) within a cell population and simultaneously enrich for HDR-edited cells within the cell population and / or to selectively deplete cells containing the target locus within the cell population; (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3; (b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7; (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a); or (d) an amino acid sequence encoded by a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of (b). Use of a CRISPR nuclease comprising or consisting of:

15. 15. The method of any one of claims 1, 2 or 8, the one or more nucleic acid molecules, or CRISPR nuclease and guide RNA, or RNP for use as described in claim 12, the vector of claim 13, or the use of claim 14, wherein the at least 80% sequence identity is at least 85%, preferably at least 90%, most preferably at least 95%.

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