Methods for altering a target sequence of a nucleic acid molecule

Abstract

The CRISPR-Cas9 system induces DNA double-strand breaks (DSBs) at targeted sites to activate mainly two competitive DNA repair pathways: homology-directed repair (HDR) which allows precise editing with a homologous DNA template, and non-conservative NHEJ / MMEJ pathways which connect two ends of the broken DNA and are often accompanied by random insertions and deletions (InDels). The relatively low efficiency of HDR compared to NHEJ / MMEJ has been a major bottleneck in achieving precise gene editing at desired loci. Here, the inventors describe the development of new efficient CRISPR-Cas9 tools, named ZIP- editors, that carry a donor repair template annealed to a modified gRNA allowing RNP stability. This specific heteroduplex gRNA-donor repair template is very easy to design. It does not require any specialized and / or expensive chemical modifications nor modified Cas9. The simplicity of using unmodified Cas9, donor repair template and easily-available modified gRNA allows its easy transferability to all laboratories. Importantly, it is versatile, allowing increased HDR for many targets, in cell lines and primary cells, with nuclease and unlocking HDR using safer single nickase.

Description

[0001] METHODS FOR ALTERING A TARGET SEQUENCE OF A NUCLEIC ACID MOLECULE

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular gene editing.

[0004] BACKGROUND OF THE INVENTION:

[0005] Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR / Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbours in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. To increase the precision of gene editing and reduce off-target cuts, other CRISPR genome editing tools are continuously developed, with enhanced targeting scope and improved editing specificity. Two main classes of CRISPR-based genome editing agents are available today, namely, base editors and prime editors. Base editors generate single-nucleotide changes in DNA, while prime editors use Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing gRNA that both specifies the target site and encodes the desired sequence edit template (Choi, Peter S., and Matthew Meyerson. " Targeted genomic rearrangements using CRISPR / Cas technology. " Nature communications 5.1 (2014): 3728). Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides (Anzalone, Andrew V., et al. " Search-and-replace genome editing without double-strand breaks or donor DNA. " Nature 576.7785 (2019): 149-157). Prime editing mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. However, prime editing is challenging and quite complex to implement due to the fact that the technology rely on several components, including a fusion protein with Cas9 and a RT and a pegRNA difficult to design. Prime editors may not be able to confer the large DNA insertions or deletions thatconventional CRISPR / Cas9 systems are capable of. Also, the fact that the desired sequence has to be encoded in an extensive RNA molecule, raises concerns regarding its stability; the longer the RNA strand gets, the more likely it is to be affected by intracellular RNA-degrading enzymes or interferon cellular response. In addition, due to presence of the RT in the molecular device, random cDNAs could be potentially incorporated in the genome. Furthermore, as the protein constructs involved are too large, PE-RNP are not commercially available and this might affect the delivery of a full-length therapeutic protein by a single adeno-associated viral vector. There is thus a need for searching for more simple tool for allowing prime editing.

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to a method of altering a target sequence of a nucleic acid molecule comprising the step consisting of contacting the target nucleic acid sequence with: (a) a nuclease (b) a guide RNA (gRNA); and (c) one or more donor repair template(s) wherein:

[0008] - the gRNA comprises in a 5’ to 3’ orientation:

[0009] o (i) a CRISPR RNA (crRNA) complementary to a portion of the target nucleic acid molecule;

[0010] o (ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and

[0011] o (iii) a 3 ’ extension arm that is capable of hybridizing to one or more donor repair template(s), and,

[0012] - the donor repair template comprises:

[0013] o (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and o (ii) a complementary sequence for hybridization to the 3’ extension arm of gRNA.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] Main definitions:

[0016] As used herein, the term “comprises” or a variant thereof (e.g “comprise”, “comprising”) refers to according to common patent application drafting terminology. Any occurrence of “comprise” or a variant thereof also encompasses narrower expression “substantially consit of’, further narrow expression “consist of’ and any variants thereof (e.g. “consist of’, “consisting of’). As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeablyherein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

[0017] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0018] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0019] As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

[0020] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). " A general method applicableto the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0021] As used herein, the term "encode", or "encoding" or "encoded" refers to a nucleic acid sequence that codes for a polypeptide sequence.

[0022] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Any method known in the art can be used to measure the expression of the gene (e. g. HPLC analysis of protein and RT-qPCR analysis of mRNA.) Typically, said methods are described in the EXAMPLE.

[0023] As used herein, the terms "decrease", "reduced", "reduction" "repress" are all used generally to mean a decrease by a statistically significant amount, for example, a decrease by at least 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level. As used herein, the term "knock-down" as used herein refers to reduction in the expression of a gene or its gene product(s).

[0024] As used herein, the terms "increased", "increase" or "enhance" or "activate" are all used to generally mean an increase by a statically significant amount, for example, an increase of at least 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0025] As used herein, the term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick basepairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” or “fully complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0026] “Substantially complementary” or “Partially complementary” as used herein refers to a degree of complementarity that is at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0027] As used herein, the term “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N. Y.

[0028] As used herein, the term “hybridization” or “hybridizing” refers to a process where completely or partially complementary nucleic acid strands come together under specified hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. Although hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs may form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0029] “Fully hybridization” or “perfectly hybridization” refers to a process where perfectly complementary nucleic acid strands come together under specified hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. “Partial hybridization” or “substantial hybridization” refers to a process in which two nucleic acid strands are partially complementary. Under specified hybridization conditions, the complementary regions of the nucleic acids come together to form a doublestranded structure or region in which the two constituent strands are joined by hydrogen bonds. The non-complementary regions, however, remain single-stranded and / or not joined by hydrogen bonding. “

[0030] As used herein, the term “nuclease” includes an enzyme that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.

[0031] As used herein, the term “CRISPR / Cas nuclease” has its general meaning in the art and refers to segments of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes. In bacteria the CRISPR / Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR / Cas nucleases Cas9 and Cpfl belong to the type II and type V CRISPR / Cas system and have strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a trans-activating small RNA(tracrRNA) that also serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG for S. Pyogenes Cas9) protospacer adjacent motif (PAM) to specify the cut site (the 3rdor the 4thnucleotide upstream from PAM).

[0032] As used herein, the term " Cas9" or " Cas9 endonuclease" (also known as COG3513, Csxl2, Cas5, or Csnl) refers to a CRISPR-associated protein with two nuclease domains that uses a crRNA:tracRNA duplex for site-specific double- stranded cleavage of DNA. According to the present invention, the terms may refer to a wild-type Cas9 protein, or any variant, including mutants, homologs, orthologs, that mediate RNA-guided double-stranded or single- stranded cleavage of DNA. In particular, the term “Cas9” refers wild type Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1), Corynebacterium ulcerans (NCBIRefs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria. (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBIRef: NP_472073.1); Campylobacter jejuni (NCBIRef: YP_002344900.1); or Neisseria, meningitidis (NCBIRef: YP_002342100.1).

[0033] As used herein, the term “nickase” has its general meaning in the art and refers to a nuclease which cleaves only a single strand of a DNA duplex (“nicking”). Accordingly, the term “Cas9 nickase” refers to a nickase derived from a Cas9 protein, typically by inactivating one nuclease domain of Cas9 protein.

[0034] As used herein, the term “guide RNA” or “gRNA” has its general meaning in the art and is a particular type of guide nucleic acid which associates with a CRISPR / Cas nuclease (e.g. Cas9), directing the nuclease to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA.

[0035] As used herein, the term “protospacer adjacent motif sequence” or “PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5’ to 3’direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes is 5’-NGG-3’ wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.

[0036] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target sequence). In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.

[0037] As used herein, the term “crRNA” refers to the portion of the guide RNA of about 20 nucleotides which contains a nucleotide sequence that is complementary to the protospacer sequence in the target nucleic sequence. The crRNA sequence anneals to the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer sequence.

[0038] As used herein, the term “tracrRNA” refers to the sequence within the gRNA that is responsible for the nuclease binding, it does not include the 20 bp spacer / targeting sequence that is used to guide the nuclease to its target nucleic sequence.

[0039] As used herein, the term “extension arm” refers to a nucleotide sequence component of a gRNA which hybridizes (partially or fully) one or more donor repair templates.

[0040] As used herein, the term “complementary extension sequence of donor repair templates” refers to a nucleotide sequence component of a donor repair templates which hybridizes (partially or fully) one or more gRNA.As used herein, the term “donor repair template” refers to a nucleic acid molecule comprising a sequence which is to be incorporated into the target nucleic acid molecule. The sequence to be incorporated is introduced into the target nucleic acid molecule via homology directed repair at the target sequence, thereby causing an alteration of the target sequence, from the original target sequence to the sequence comprised by the template nucleic acid. Accordingly, the sequence comprised by the template nucleic acid can be, relative to the target sequence, an insertion, a deletion, an indel, a point mutation, a repair of a mutation, etc.

[0041] As used herein, the term “target sequence” or “target” refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid may be single-stranded or double-stranded, and often is double-stranded DNA. A “target nucleic acid sequence,” “target sequence” or “target region” as used herein, means a specific sequence or the complement thereof that one wishes to bind to using the CRISPR system as disclosed herein. As used herein, the term “target nucleic acid strand” refers to a strand of a target nucleic acid that is subject to base-pairing with a guide RNA as disclosed herein. That is, the strand of a target nucleic acid that hybridizes with the crRNA and guide sequence is referred to as the “target nucleic acid strand.” The other strand of the target nucleic acid, which is not complementary to the guide sequence, is referred to as the “non-complementary strand.” In the case of double-stranded target nucleic acid (e.g., DNA), each strand can be a “target nucleic acid strand” to design crRNA and guide RNAs and used to practice the method of this invention as long as there is a suitable PAM site.

[0042] As used herein, the term “editing” refers to a type of genetic engineering in which a nucleic acid sequence is inserted, replaced, or removed from a target nucleic acid sequence, e.g., the genome of a cell, using one or more nucleases according to the present invention. The nucleases create specific double-strand breaks (DSBs) or single-strand breaks (SSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) (e.g., homologous recombination). Thus the term "editing" refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., a naturally-occurring wild type nucleic acid sequence or a naturally-occurring mutated nucleic acid sequence by introducing a change to a specific genomic target; the genomic target may include a chromosomal region, a coding polynucleotide (e.g., a gene), a promotor, a non-coding polynucleotide, or any nucleic acid sequence. The changes to a nucleic acid may include deletion, addition and other changes to the nucleic acid sequence in the genome.As used herein, the term “recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, "homologous recombination” or “HR" refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule to template repair of a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion," because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and / or "synthesisdependent strand annealing," in which the donor repair template is used to re-synthesize genetic information that will become part of the target, and or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0043] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. The term "deletion" means that a specific amino acid residue is removed. The term "insertion" means that one or more amino acid residues are inserted before or after a specific amino acid residue. As used herein, the term "point mutation" refers to a substitution that replaces one of the nucleotides in a target polynucleotide.

[0044] As used herein, the term “mutagenesis” refers to the introduction of mutations into a polynucleotide sequence.

[0045] As used herein, the term “variant” refers to a first composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. A variant molecule can have entire sequence identity with the original parent molecule, or alternatively, can have less than 100% sequence identity with the parent molecule. For example, a variant of a sequence can be a second sequence that is at least 50; 51; 52; 53;54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100% identical in sequence compare to the original sequence.

[0046] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0047] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of population of cells to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total usage the gene editing platform will be decided by the attending physician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment, drugs used in combination or coincidental with the population of cells, and like factors well known in the medical arts. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is dependent on the relative representation of the cells with the desired specificity, on the age and weight of the recipient, and on the severity of the targeted condition. This number of cells can be as low as approximately 103 / kg, preferably 5xl03 / kg; and as high as 107 / kg, preferably 108 / kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. Typically, the minimal dose is 2 millions of cells per kg. Usually 2 to 20 millions of cells are injected in the subject. The desired purity can be achieved by introducing a sorting step. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

[0048] Methods of the present invention:

[0049] The present invention relates to a method of altering a target sequence of a nucleic acid molecule comprising the step consisting in contacting the target nucleic acid sequence with: (a) a nuclease (b) a guide RNA (gRNA); and (c) a donor repair template wherein:

[0050] - the gRNA comprises in a 5’ to 3’ orientation:

[0051] o (i) a CRISPR RNA (crRNA) complementary to a portion of the target nucleic acid molecule;

[0052] o (ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and

[0053] o (iii) a 3 ’ extension arm that is capable of hybridizing to the donor repair template, and,- the donor repair template comprises:

[0054] o (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and o (ii) a complementary sequence for hybridization to the 3’ extension arm of gRNA.

[0055] In other words, the present invention relates to a method of altering a target sequence of a nucleic acid molecule comprising the step consisting in contacting the target nucleic acid sequence with: (a) a nuclease (b) a guide RNA (gRNA); and (c) a donor repair template wherein:

[0056] - the gRNA comprises in a 5’ to 3’ orientation:

[0057] o (i) a CRISPR RNA (crRNA) complementary to a portion of the target nucleic acid molecule;

[0058] o (ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and

[0059] o (iii) a 3 ’ extension arm that is capable of hybridizing to the donor repair template, and,

[0060] - the donor repair template comprises:

[0061] o (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and o (ii) an extension sequence comprising a complementary sequence (or “complementary extension sequence”) for hybridization to the 3’ extension arm of gRNA.

[0062] According to the present invention, the contacting results in producing one or more doublestrand breaks or single-strand breaks in the target nucleic acid molecule, thereby triggering DNA repair pathways to repair the breaks, whereby repair of the cleaved target nucleic acid double-stranded target DNA sequence proceeds substantially by homology directed repair (HDR) in presence of the donor repair template, and, thereby modifying the sequence of the target nucleic acid molecule.

[0063] Nuclease:In some embodiments, the nuclease is a CRISPR / Cas nuclease. In some embodiments, the CRISPR / Cas nuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In some embodiments, the nuclease domains of the protein can be modified. In some embodiments, the protein can be truncated to remove domains that are not essential for the function of the protein. In some embodiments, the protein is truncated or modified to optimize the activity of the RNA binding domain.

[0064] In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease i.e. a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has the RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites. In some embodiments, the mutant comprises an amino acid sequence having at least 50% of identity with the wild type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in this invention. Non-limiting examples of suitable CRISPR / CRISPR / Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csfl, Csf2, Csf3, Csf4, and Cul966. See e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0065] In some embodiments, the CRISPR / Cas nuclease is derived from a type II CRISPR-Cas system.

[0066] In some embodiments, the CRISPR / Cas nuclease is a Cas9 nuclease.

[0067] The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocar diopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa,Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidates Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalter omonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina, inter alia.

[0068] In some embodiments, the CRISPR / Cas nuclease is a mutant of a wild type CRISPR / Cas nuclease (such as Cas9) or a fragment thereof. In some embodiments, the CRISPR / Cas nuclease is a mutant Cas9 protein from S. pyogenes.

[0069] In some embodiments, the Cas9 nuclease comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

[0070] SEQ ID NO: 1: Cas9 sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0071] In some embodiments, the CRISPR / Cas nuclease of the present invention is a nickase, particularly a Cas9 nickase. This nickase, derived from S. pyogenes Cas9, contains one mutation in either the RuvC or HNH domains to inactivate one of its nuclease active sites. For example, mutations such as D10A and H840A can be utilized. These modifications result in anickase that retains the ability to bind to DNA but introduces a single-strand break instead of a double-strand break, making it useful for applications requiring high precision and reduced off-target effects.

[0072] In some embodiments, the nickase of the present invention comprises the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO:3

[0073] SEQ ID NO: 2> S. pyogenes nCas9 Protein Sequence having the D10A mutation MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0074] SEQ ID NO: 3> S. pyogenes nCas9 Protein Sequence having the H840A mutation MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDIn some embodiments, the nickase of the present invention comprises the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3 and further comprises the R221K and N394K mutations that was previously shown to improve Cas9 nuclease activity (Spencer, Jeffrey M., and Xiaoliu Zhang. " Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains. " Scientific reports 7.1 (2017): 1-14).

[0075] Some aspects of the disclosure provide Cas9 proteins that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to of the Cas9 protein to bind to a particular nucleotide sequence within a genome. Accordingly, in some embodiments, any of the Cas proteins provided herein may be capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. For example, Cas9 proteins that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481- 485 (2015),' and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition ” Nature Biotechnology 33, 1293-1298 (2015),' Walton, Russell T., et al. " Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. " Science 368.6488 (2020): 290-296 the entire contents of each are hereby incorporated by reference. In some embodiments, the Cas9 protein of the present invention comprises the following mutations D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R. This variant is capable of targeting an expanded set of NGN PAMs (i.e. the “SpG” variant as described in Walton, Russell T., et al. " Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. " Science 368.6488 (2020): 290-296). In some embodiments, the Ca9 variant SpG can further be optimized to develop a near-PAMless SpCas9 variant named “SpRY” that furthers includes the 5 additional mutations A61R, L1111R, N1317R, A1322R, and R1333P.

[0076] In some embodiments, the nuclease comprises additional features. Other exemplary features that may be present are localization sequences, such as nuclear localization sequences (NLS), cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. In particular, the nuclease incorporates one or more nuclear localization sequence. As used herein, the term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cellnucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT / EP2000 / 011690, filed Nov. 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO:4) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO:5).

[0077] Guide RNA:

[0078] According to the present invention, the gRNA comprises in a 5’ to 3’ orientation:

[0079] (i) a CRISPR RNA (crRNA) region complementary to a portion of the target nucleic acid molecule;

[0080] (ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and (iii) a 3’ extension arm that is capable of hybridizing to the donor repair template,

[0081] The gRNA molecule of the present invention thus comprises a crRNA sequence for providing the targeting specificity. It includes a crRNA sequence that is complementary and capable of hybridization to a pre-selected target site of interest. In some embodiment, this crRNA sequence can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the crRNA sequence and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. In some embodiments, the crRNA sequence is about 17-20 nucleotides in length, such as 20 nucleotides. Typically, a software program is used to identify candidate CRISPR target sequences on both strands of the DNA nucleic acid molecule containing the targeted genes based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme. One requirement for selecting a suitable target nucleic acid is that it has a 3' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred herein as a Cas-targeted site. Type II CRISPR system, one of the most well characterized systems, needs only Cas 9 protein and a guide RNA complementary to a target sequence to affect target cleavage. For example, target sites for Cas9 from S. pyogenes, with PAM sequences NGG, may be identified by searching for 5'-Nx-NGG-3' both on the input sequence and on the reverse-complement of the input. Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant referencegenome. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence, such as the 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed: PAM sequence in the relevant genome. The user may be allowed to choose the length of the seed sequence. The user may also be allowed to specify the number of occurrences of the seed: PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s). Further details of methods and algorithms to optimize sequence selection can be found in U. S. application Ser. No. 61 / 836,080; incorporated herein by reference.

[0082] According to the present invention, the extension arm is located at the 3 ’ end of the guide RNA, and comprises a sequence that is capable of hybridizing to one or more donor repair template(s) (i.e. “hybridizing sequence)”.

[0083] In some embodiment, the extension arm comprises or consist of a sequence that is capable of fully hybridizing to one or more donor repair template.

[0084] In some embodiment, the extension arm comprises or consist of a sequence that is capable of fully hybridizing to each extension sequence comprising in one or more donor repair template. In some embodiment, the extension arm comprises or consist of a sequence that is capable of partially hybridizing to one or more donor repair template

[0085] In some embodiment, the extension arm comprises or consist of a sequence that is capable of partially hybridizing to each extension sequence comprising in one or more donor repair template.

[0086] In some embodiments, the hybridizing sequence comprises at least 5 nucleotides, preferably at least 10 nucleotides. In some embodiments, the hybridizing sequence comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides.

[0087] In some embodiments, the hybridizing sequence comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more than 30 nucleotides, and wherein the hybridizing sequence is at least 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary with the complementary extension sequence in one or more donor repair template.

[0088] In some embodiments, the extension arm can hybridize to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 donor repair template(s) and therefore the extension arm can thus comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hybridizing sequence(s).

[0089] The guide RNA molecule of the present invention can be made by various methods known in the art including cell-based expression, in vitro transcription, and chemical synthesis. The ability to chemically synthesize relatively long RNAs (as long as 200 mers or more) using TC-RNA chemistry (see, e.g., U. S. Pat. No. 8,202,983) allows one to produce RNAs with special features that outperform those enabled by the basic four ribonucleotides (A, C, G and U). In particular, the RNA molecule of the present invention can be made with recombinant technology using a host cell system or an in vitro translation-transcription system known in the art. Details of such systems and technology can be found in e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0090] In some embodiments, the guide RNA molecule may include one or more modifications. Such modifications may include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and / or base moiety. Modified nucleotides may include 2’-O-methyl analogs, 2’-deoxy analogs, or 2’ -fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5 -bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

[0091] In some embodiments, the gRNA of the present invention may also further comprise elements, such as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3’ toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). In particular, the gRNA may contain one or more structural elements for minimizing its degradation. In particular, the gRNA of the present invention incorporates one or more stable pseudoknots at its 3’ end such as a modified prequeosinel-1 riboswitch aptamer (evopreQl) or the frameshifting pseudoknot from Moloney murine leukemia virus (MMLV)22, hereafter referred to as “mpknof ’ as described in Nelson,James W., et al. " Engineered pegRNAs improve prime editing efficiency. " Nature biotechnology 40.3 (2022): 402-410 for which the teaching is incorporated by reference. In some embodiments, the gRNA may comprise a transcriptional termination sequence at the 3’ of the molecule.

[0092] In some embodiments, the gRNA of the present invention comprises at least one locked hairpin secondary structure. As used herein, the “locked hairpin secondary structure” or “t-lock loop” in the gRNA of the present invention means that the hairpin has a high stability, particularly a high thermostability. The introduction of a highly stable hairpin into the gRNA indeed provide a nucleation site for RNA folding and thus prevent misfolding of the gRNAs regardless of the spacer sequence (Riesenberg S, Helmbrecht N, Kanis P, Maricic T, Pääbo S. Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage. Nat Commun. 2022 Jan 25;13(1):489). In particular, the term “locked hairpin secondary structure” means that the secondary structure of the first hairpin comprises a contiguous stem of base-paired nucleotide building blocks and particularly a contiguous stem of base-paired nucleotide building blocks structure connected by a loop of unpaired nucleotide building blocks. The term “base-paired” refers to base pairs formed by a purine nucleobase such as A or G to a pyrimidine nucleobase such as U or C. In particular, the term “base pair” refers to the base pairs A-U, U-A, G-C and C-G, but also includes G-U, and A-C. In some embodiments, the locked hairpin of a gRNA of the present invention has thus a secondary structure comprising a contiguous stem. The term “contiguous stem” means an uninterrupted sequence of base pairs in the stem, i.e. without any bulge formed of unpaired nucleotides. In some embodiments, the locked hairpin of a gRNA of the present invention has a secondary structure comprising a contiguous stem having a length of at least about 5 nt, e.g. about 6 nt to about 12 nt. In some embodiments, the locked hairpin has a secondary structure comprising a contiguous stem having a length of 6, 7, 8, 9 or 10 nt, more particularly of 8 nt. In some embodiments, the locked hairpin of a gRNA of the present invention has a secondary structure comprising a contiguous stem comprising at least 2 C-G base pairs, e.g. about 3 C-G base pairs to about 6 C-G base pairs. In some embodiments, the locked hairpin forms a secondary structure comprising a contiguous stem comprising 2, 3, 4 or 5 C-G base pairs, more particularly 4 C-G base pairs. In this context, the term “C-G base pair” encompasses any base pair formed by a G nucleotide and a C nucleotide irrespectively from their orientation within the stem. In some embodiments, the locked hairpin of a gRNA of the present invention forms a secondary structure comprising a contiguous stem having a melting temperature of at least about 60°C, e.g. at least about 65°C or at least about 70°C and up to about 90°C as determined by e.g. differential scanningcalorimetry and UV absorbance spectroscopy. The loop of unpaired nucleotide building blocks may have the structure Nr with N being any nucleotide e.g. selected from A, C, G, or U, and r being 3, 4, 5 or 6, particularly 4 or 5, and more particularly 4. Preferably, Nr is selected from 5’-GNRA-3’, 5’-UNCG-3’ and 5’- CUUG-3’, wherein R is a nucleotide selected from A or G, and N is any nucleotide, e.g. selected from A, C, G, or U.

[0093] In some embodiments, the gRNA of the present invention also further comprises at least two stem loop and at least one locked hairpin secondary structure located to the second loop, as previously described (Riesenberg etal., 2022).

[0094] Donor repair template:

[0095] According to the present invention, the donor repair template is used for homologous recombination-mediated repair and is particularly useful for introducing modifications within a target nucleic acid sequence.

[0096] In some embodiments, the donor repair template consists of a double-stranded DNA oligonucleotide having one single strand extension that comprises a complementary sequence for hybridization to the 3’ extension arm of gRNA (“complementary extension sequence”).

[0097] In some embodiments, the donor repair template consists of a single-strand oligodeoxynucleotide (ssODN) that comprises (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and (ii) a complementary sequence for hybridization to the 3 ’ extension arm of gRNA. In other words, the donor repair template consists of a single-strand oligodeoxynucleotide (ssODN) that comprises (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and (ii) an extension sequence comprising or consisting of a complementary sequence for hybridization to the 3’ extension arm of gRNA (“complementary extension sequence”).

[0098] In some embodiment, the complementary extension sequence is perfectly or partially complementary to the 3’ extension arm of gRNA.

[0099] In other words, in some embodiment, the donor repair templates comprise an extension sequence comprising or consisting of a sequence perfectly complementary for hybridization to the 3’ extension arm of gRNA. In some embodiment, the donor repair templates comprise anextension comprising or consisting of a sequence partially complementary for hybridization to the 3’ extension arm of gRNA.

[0100] According to the present invention, the donor repair template contains two flanking, homologous sequences on each side of the target site of the nuclease cleavage and can be oriented in the sense or antisense direction relative to the target nucleic acid molecule. The flanking sequences can comprise sequences with homology to the target sequence and / or sequences flanking the target sequence, i.e., in order to hybridize with the target nucleic acid near the target sequence and permit HDR to occur. For instance, design of donor repair templates, particularly with respect to flanking region(s) is discussed in Richardson, C., Ray, G., DeWitt, M. et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34, 339–344 (2016),' which is incorporated by reference herein in its entirety. Each flanking sequence can be at least about 10 nucleotides, e.g., at least about 10, 15, 20, 25, or 30 nucleotides.

[0101] In some embodiments, the template sequence comprises at least e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more modified nucleotides in comparison with the target nucleic acid sequence. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the template sequence of the donor repair template includes a modified nucleotide in comparison with the target nucleic acid sequence.

[0102] According to the present invention, the donor repair template comprises at one of its end (i.e. its 3’ end or 5’ end) one or more complementary extension sequence(s) that is / are capable of hybridizing to one or more hybridizing sequence(s) present at the 3 ’ extension arm of the gRNA. In some embodiments, the one or more extension sequence(s) is / are capable of perfectly hybridizing to one or more hybridizing sequence(s) present at the 3 ’ extension arm of the gRNA. In some embodiments, the one or more extension sequence(s) is / are capable of partially hybridizing to one or more hybridizing sequence(s) present at the 3 ’ extension arm of the gRNA. In other words, the one or more extension sequence(s) is / are perfectly or partially complementary to one or more hybridizing sequence(s) present at the 3’ extension arm of the gRNA.In some embodiments, the complementary sequence comprises at least 5 nucleotides, preferably at least 10 nucleotides.

[0103] In some embodiments, the complementary extension sequence comprises 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0104] In some embodiments, the complementary extension sequence comprises 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.

[0105] In some embodiments, the complementary extension sequence comprises 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, and wherein the extension sequence is at least 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary with the 3’ arm extension sequence in the gRNA.

[0106] In some embodiments, the donor repair template can be at least about 25 nucleotides in length, e.g., at least about 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, or longer. In some embodiments, the donor repair template is about 25 to about 50; about 50 to about 100; about 100 to about 150; about 150 to about 200; about 200 to about 250; about 250 to about 300; or about 25 nucleotides to about 300 nucleotides in length. In some embodiments, the donor repair template donor repair template is about 75; 80; 85; 90; 95; 100; 105; 110; 115; 120; 125; 130; 135; 140; 145; 150; 155; 160; 165; 170; 175; 180 nucleotides in length.

[0107] Combination with “cell state ” modulators:

[0108] In some embodiments, the edition is carried out in presence of one or more “cell state” modulator(s) so as to increase HDR editing.

[0109] In some embodiments, the edition is carried out in presence one or more inhibitor of the NHEJ DNA repair pathway. In some embodiments, the inhibitor of the NHEJ DNA repair pathway is a DNA-PK inhibitor. As used herein, the term " DNA-PK inhibitor" refers to an agent that inhibits DNA-PK (nuclear serine / threonine protein kinase complex composed of the catalytic subunit DNA-PKcs and a heterodimer of Ku proteins (Ku70 / Ku80)). The DNA-PK inhibitor in the present disclosure may selectively inhibit the kinase DNA-PK, or may non-selectivelyinhibit DNA-PK and inhibit also kinase(s) other than DNA-PK. The DNA-PK inhibitor in the present disclosure is not particularly limited as long as it is an agent that has the described characteristics, and preferred examples thereof can include those disclosed in WO2018 / 114999 and WO2019 / 238929. Examples of DNA-PK inhibitors which may be used according to the present disclosure are selective inhibitors of DNA-PK including M3814 / peposertib (Merck), M9831 (Merck) and AZD7648 (AstraZeneca), and non-selective inhibitors of DNA-PK including BR-101801 (Boryung Pharma), SF-2523 (SignalRx Pharmaceuticals), BR-2002 / BCN-005 (Boryung Pharma), and CC115 (Celgene).

[0110] In some embodiments, the edition is carried out in presence of one or more agents capable to increase the S / G2 / M phases of the target cells. In some embodiments, the agent is a CDK7 inhibitor. As used herein, the term " CDK7 inhibitor" refers to a compound or agent that reduces the activity of Cyclin-Dependent Kinase 7 (" CDK7"). In some embodiments, the selective CDK7 inhibitor is selected from those disclosed in WO 2015 / 058163, WO 2015 / 154022, WO 2015 / 154038, WO 2015 / 154039, WO 2015 / 058140, and WO 2014 / 063068. Selective CDK7 inhibitors include, but are not limited to, THZ1 (see, e.g., Kwiatkowski et al. (2014); Nature 511 (7511): 616-620), BS-181 (see, e.g., AN et al. (2009); Cancer Res 69(15): 6208-6215), SY-351 (Syros Pharmaceuticals).

[0111] In some embodiments, the agent is a CDC7 inhibitor. As used herein, the term " CDC7 inhibitor" refers to a compound or agent that reduces the activity of Cell division cycle 7-related protein kinase (" CDC7"). In some embodiments, the selective CDC7 inhibitor is selected from those disclosed in WO2021032170, PHA-767491 (also marketed as CAY10572 or PHA-767491 hydrochloride), TAK-931 (also known as simurosertib), PHA-848125 and XL413 (also known as BMS-863233) (see e.g. Koltun ES, et al. Discovery of XL413, a potent and selective CDC7 inhibitor. Bioorg Med Chem Lett. 2012 Jun 1;22(11):3727-31). In some embodiments, the CDC7 inhibitor is XL413.

[0112] In some embodiments, the edition is carried out in presence of one or more agents capable to increase the chromatin opening. In some embodiments, the agent is a HD AC inhibitor. As used herein, the term “HDAC inhibitor” refers to a compound or agent that inhibits the activity of histone deacetylases (HDACs). HDACs are enzymes that remove acetyl groups from histone proteins, leading to chromatin condensation and transcriptional repression. Inhibition of HDACs can result in chromatin opening. Examples of HDAC inhibiros include low-molecular-weight inhibitors, such as valproic acid (VP A) (Nat. Biotechnol., 26 (7): 795-797, 2008),trichostatin A, sodium butyrate (NaB or NaBut), MC 1293, and M344; nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool® (Millipore) and HuSH 29mer shRNA Constructs against HDAC1 (OriGene)); and DNA methyltransferase inhibitors (e.g., 5′ -azacytidine) (Nat. Biotechnol., 26 (7): 795-797, 2008).

[0113] In some embodiments, the edition is carried out in presence of a HDAC inhibitor and an agent capable to increase the S / G2 / M phases of the target cells.

[0114] In some embodiments, the edition is carried out in presence of a HDAC inhibitor and a CDC7 inhibitor.

[0115] In some embodiments, the edition is carried out in presence of sodium butyrate and XL413.

[0116] Uses of the methods:

[0117]

[0118] cells:

[0119] In some embodiments, the method of the present invention is used to alter a target polynucleotide sequence of interest in a cell for any purpose.

[0120] The cell can be a cell isolated from any multicellular organism, e.g., a plant cell (e.g., a rice cell, a wheat cell, a tomato cell, an Arabidopsis thaliana cell, a Zea mays cell, and the like), a cell from a multicellular protist, a cell from a multicellular fungus, an animal cell such as a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.) or a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal, etc.), a cell from a human, a cell from a healthy human, a cell from a human patient, a cell from a cancer patient, etc. In some cases, the cell with induced gene regulation can be transplanted to a subject (e.g., patient). For instance, the cell can be derived from the subject (e.g., patient) to be treated.

[0121] In some embodiments, the cell is a eukaryotic cell. Any type of eukaryotic cell may be of interest, such as a stem cell, e.g., embryonic stem cell, induced pluripotent stem cell, adult stem cell (e.g., mesenchymal stem cell, neural stem cell, hematopoietic stem cell, organ stem cell), a progenitor cell, a somatic cell (e.g., fibroblast, hepatocyte, heart cell, liver cell, pancreatic cell, muscle cell, skin cell, blood cell, neural cell, immune cell), and any other cell of the body, e.g., human body. The cells can be primary cells or eukaryotic cell cultures derived from a subject,e.g., an animal subject or a human subject, and allowed to grow in vitro for a limited number of passages. In some embodiments, the cells are disease cells or derived from a subject with a disease. For instance, the cells can be cancer or tumor cells.

[0122] In some embodiments, the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).

[0123] In some embodiments, the eukaryotic cell is a hematopoietic stem cell. As used herein, the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity to self-renew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin-CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. Ill: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life. In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.

[0124] In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells. As used herein, the term “peripheral blood cells” refer to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood.

[0125] In some embodiments, the eukaryotic cell is a bone marrow derived stem cell. As used herein the term “bone marrow-derived stem cells” refers to stem cells found in the bone marrow. Stem cells may reside in the bone marrow, either as an adherent stromal cell type that possess pluripotent capabilities, or as cells that express CD34 or CD45 cell-surface protein, which identifies hematopoietic stem cells able to differentiate into blood cells.

[0126] Gene editing:In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to generate a mutate cell, which results in a genotype that differs from its original genotype. In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element). In some embodiments, the alteration may be a homozygous alteration or a heterozygous alternation. In some embodiments, the alteration may be an insertion, deletion, or the combination thereof. As will be appreciated by those skilled in the art, an insertion / deletion in a coding region of a genomic sequence will result in a frameshift mutation or a premature stop codon. In some embodiments, the alteration may be a point mutation. In some embodiments, the alteration consists in introducing a plurality of point mutations.

[0127] In some embodiments, the method of the present invention is thus suitable for correcting or repairing a genetic mutation. As used herein, the term “genetic mutation” refers to any alteration in the DNA sequence that makes up a gene. This can include changes such as insertions, deletions, or point mutations that affect the function of the gene. Genetic mutations can lead to a variety of effects, ranging from no noticeable impact to significant changes in the organism's phenotype. For instance, the method of the present invention is suitable for correcting or modeling a mutation in the CFTR gene. As used herein, the term “CFTR gene” refers to the gene that encodes the cystic fibrosis transmembrane conductance regulator protein, which is crucial for regulating the movement of chloride and sodium ions across epithelial membranes. Mutations in this gene lead to cystic fibrosis, a condition that affects the respiratory, digestive, and reproductive systems. The CFTR gene is located on chromosome 7 and consists of 27 exons. The CFTR gene mutations were classified into six classes according to their resulting damaging effect on the protein (Elborn JS, 2016). The class I mutations contribute to the formation of proteins with incomplete length and usually involve the complete loss of its activity (e.g., W1282X, 1717-1G-> A, G542X, R553X, 2183AA> G). Mutation in the class II lead to abnormal maturation of proteins in the endoplasmic reticulum and Golgi apparatus. The effect of these mutations is premature degradation of the CFTR protein. Hence, CFTR protein does not reach the cell membrane where it should perform its function (e.g., F508del, 2184delA). The gene product having mutations of class III is properly synthesized, transported, and incorporated into the cell membrane, but has decreased activity caused by abnormal regulation of the protein. These mutations are frequently situated within one of the nucleotide bindingdomains, (e.g., G551D, R560T). Mutations of class IV cause anomalies in the structure of the transmembrane protein and thereby reduce the conduction of the chloride channel (e.g., R117H, R334W). Mutations altering the stability of the mRNA coding for the CFTR protein represent a class V of the mutations of the CFTR gene (e.g., 2789+5G-> A, A455E). Mutations in class VI lead to a decrease of the CFTR protein stability (e.g., 4326delTC, Glnl412X, 4279insA) (Harriet C., Kristin E. T., Olivier T., et al. Translating the genetics of cystic fibrosis to personalized medicine. Transl Res 2016; 168 40-49 and Elborn JS. Cystic fibrosis. Seminar.

[0128] 2016). Examples of CFTR mutations include, but are not limited to 124del23bp CFTR, CFTRdelel CFTR, Ml V CFTR, Q2X CFT, S4X CFTR, P5L CFTR, S13F CFTR, L15P CFTR, 182delT CFTR, CFTRdele2 CFTR, CFTRdele2-4 CFTR, 185+1G-> T CFTR, CFTRdele2,3 CFTR, W19X CFTR, G27R CFTR, G27X CFTR, Q30X CFTR, R31C CFTR, R31L CFTR, Q39X CFTR, A46D CFTR, 296+lG-> A CFTR, 296+lG-> T CFTR, CFTRdele3-10,14b-16 CFTR, 296+28A-+G CFTR, 296+2T-> C CFTR, 296+3insT CFTR, 297-3C-> T CFTR, 297-1G-> A CFTR, E56K CFTR, W57G CFTR, W57X CFTR, 306insA CFTR, 306delTAGA CFTR, E60X CFTR, P67L CFTR, R74W CFTR, R75X CFTR, R75Q CFTR, 365-366insT CFTR, G85E CFTR, 394delTT CFTR, L88X CFTR, G91R CFTR, CFTRdele4-7 CFTR, CFTRdele4-11 CFTR, CFTR50kbdel CFTR, 4O5+1G-> A CFTR, 405+3 A-> C CFTR, 406-2A-> G CFTR, 406-1G-+A CFTR, E92K CFTR, E92X CFTR, Q98X CFTR, Q98R CFTR, P99L CFTR, L102R CFTR, 442delA CFTR, 444delA CFTR, 457TAT-> G CFTR, D110H CFTR, D110E CFTR, R117C CFTR, R117G CFTR, R117H CFTR, R117H;5T CFTR, R117H;7T CFTR, 541delC CFTR, L138ins CFTR, H139R CFTR, 574delA CFTR, I148T CFTR, 602dell4 CFTR, Y161D CFTR, 621+1G-+T CFTR, 621+3 A-> G CFTR, L165S CFTR, R170H CFTR, 663delT CFTR, G178R CFTR, 675del4 CFTR, E193X CFTR, 711+1G-+T CFTR, 711+3A-+G CFTR, 711+5G-+A CFTR, 712-1G-+T CFTR, H199Y CFTR, V201M CFTR, P205S CFTR, L206W CFTR, W216X CFTR, Q220X CFTR, L227R CFTR, V232D CFTR, 849delG CFTR, 852del22 CFTR, CFTRdup6b-10 CFTR, M265R CFTR, 935delA CFTR, Y275X CFTR, C276X CFTR, 977insA CFTR, 991del5 CFTR, F311L CFTR, 1078delT CFTR, L320V CFTR, 1119delA CFTR, G330X CFTR, R334W CFTR, R334Q CFTR, R334L CFTR, 1138insG CFTR, I336K CFTR, T338I CFTR, S341P CFTR, 1154insTC CFTR, 1161delC CFTR, R347H CFTR, R347P CFTR, A349V CFTR, R352W CFTR, R352Q CFTR, Q359K / T360K CFTR, 1213delT CFTR, 1248+1G-+A CFTR, 1249-1G-+A CFTR, 1259insA CFTR, 1288insTA CFTR, W401X CFTR, 1341+1G-+A CFTR, 5T CFTR, 5T; TG11 CFTR, 5T; TG12 CFTR, 5T; TG13 CFTR, 7T CFTR, 9T CFTR, 1343delG CFTR, Q414X CFTR, 1429del7 CFTR, D443Y CFTR, 1461ins4 CFTR, 1471delA CFTR, L453S CFTR, A455E CFTR, 1497delGG CFTR, V456A CFTR, 1504delGCFTR, 1525-1G-> A CFTR, 1525-2A-> G CFTR, S466X CFTR, L467P CFTR, M470V CFTR, 1548delG CFTR, E474K CFTR, S489X CFTR, S492F CFTR, 1609delCA CFTR, Q493X CFTR, W496X CFTR, I502T CFTR, I507del CFTR, F508del CFTR, F508C CFTR, D513G CFTR, 1677delTA CFTR, V520F CFTR, C524X CFTR, Q525X CFTR, 1716+1G-> A CFTR, CFTRdelell CFTR, 1717-1G-> A CFTR, 1717-8G-> A CFTR, G542X CFTR, S549R CFTR, S549N CFTR, G550X CFTR, 1782delA CFTR, G551S CFTR, G551D CFTR, Q552X CFTR, R553X CFTR, 1802delC CFTR, L558S CFTR, A559T CFTR, 1811+1G-> C CFTR, R560K CFTR, R560T CFTR, 1811+1G-> A CFTR, 1811+1634A-> G or 181 l+1.6kbA-> G CFTR, 1811+1643G-> T CFTR, 1812-1G-> A CFTR, R560S CFTR, A561E CFTR, V562I CFTR, Y563N CFTR, Y563D CFTR, 1824delA CFTR, 1833delT CFTR, Y569D CFTR, P574H CFTR, F575Y CFTR, G576A CFTR, D579G CFTR, E585X CFTR, E588V CFTR, 1898+1G-> A CFTR, 1898+1G-> C CFTR, 1898+1G-> T CFTR, CFTRdelel3,14a CFTR, 1898+3A-> G CFTR, 1898+5G-> T CFTR, 1924del7 CFTR, H609R CFTR, A613T CFTR, D614G CFTR, G622D CFTR, 2055del9-> A CFTR, 2075delA CFTR, 2105-2117dell3insAGAAA CFTR, 2118del4 CFTR, R668C CFTR, 2143delT CFTR, G673X CFTR, 2183AA-> G CFTR, 2184insA CFTR, 2184delA CFTR, 2185insC CFTR, Q685X CFTR, R709X CFTR, K710X CFTR, Q715X CFTR, Q720X CFTR, 2307insA CFTR, L732X CFTR, 2347delG CFTR, 2372del8 CFTR, P750L CFTR, V754M CFTR, R764X CFTR, R785X CFTR, R792X CFTR, I807M CFTR, 2556insAT CFTR, 2585delT CFTR, 2594delGT CFTR, E822X CFTR, 2622+lG-> A CFTR, E831X CFTR, D836Y CFTR, W846X CFTR Y849X CFTR, R851X CFTR, T854T CFTR, 271 IdelT CFTR, 2721dell 1 CFTR, 2732insA CFTR, CFTRdelel4b-17b CFTR, 2752-26A-> G CFTR, W882X CFTR, 2789+2insA CFTR, 2789+5G-> A CFTR, 2790-1G-> C CFTR, Q890X CFTR, S912X CFTR, S912L CFTR, 2869insG CFTR, Y913X CFTR, 2896insAG CFTR, L927P CFTR, 2942insT CFTR, 2957delT CFTR, S945L CFTR, 2991del32 CFTR, 3007delG CFTR, 3028delA CFTR, L967S CFTR, G970R CFTR, CFTRdelel6-17b CFTR, G970D CFTR, S977F CFTR, D979V CFTR, 3120G-> A CFTR, CFTRdelel7a,17b CFTR, CFTRdelel7a-18 CFTR, 312O+1G-> ACFTR, 3121-1G-> ACFTR, 3121-2A-> GCFTR, 3121-977_3499+248del2515 CFTR, L997F CFTR, 3132delTG CFTR, A1006E CFTR, 3143del9 CFTR, 3171delC CFTR, 3171insC CFTR, Y1014C CFTR, F1016S CFTR, I1027T CFTR, Y1032C CFTR, Q1042X CFTR, 3271delGG CFTR, 3272-26A-> G CFTR, F1052V CFTR, T1053I CFTR, H1054D CFTR, G1061R CFTR, L1065P CFTR, R1066C CFTR, R1066H CFTR, G1069R CFTR, R1070W CFTR, R1070Q CFTR, 3349insT CFTR, F1074L CFTR, L1077P CFTR, W1089X CFTR, Y1092X CFTR, W1098X CFTR, W1098C CFTR, F1099L CFTR, M1101K CFTR, R1102X CFTR, E1104X CFTR, S1118F CFTR, CFTRdelel8CFTR, 3500-2A-> G CFTR, W1145X CFTR, D1152H CFTR, V1153E CFTR, 3600G-> A CFTR, CFTRdelel9 CFTR, CFTRdelel9-21 CFTR, 3600+2insT CFTR, 3600+5G-> A CFTR, R1158X CFTR, S1159P CFTR, S1159F CFTR, R1162X CFTR, R1162L CFTR, 3659delC CFTR, 3667ins4 CFTR, S1196X CFTR, 3737delA CFTR, W1204X CFTR, 3791delC CFTR, Y122X CFTR, 3821delT CFTR, I1234V CFTR, S1235R CFTR, 3849G-> A CFTR, 3849+4A-> G CFTR, 3849+5G-> A CFTR, 3849+40A-> G CFTR, 3849+10kbC-> T CFTR, 3850-1G->A CFTR, 3850-3T-> G CFTR, V1240G CFTR, G1244E CFTR, T1246I CFTR, 3876delA CFTR, 3878delG CFTR, S1251N CFTR, L1254X CFTR, S1255P CFTR, S1255X CFTR, 3905insT CFTR, D1270N CFTR, W1282X CFTR, R1283M CFTR, Q1291R CFTR, 4005+1G->A CFTR, CFTRdele21 CFTR, 4005+2T-> C CFTR, 4010del4 CFTR, 4015delA CFTR, 4016insT CFTR, 4022insT CFTR, 4040delA CFTR, N1303K CFTR, Q1313X CFTR, CFTRdele22-24 CFTR, CFTRdele22,23 CFTR, L1324P CFTR, Q1330X CFTR, L1335P CFTR, 4168delCTAAGCC CFTR, G1349D CFTR, 4209TGTT-> AA CFTR, 4218insT CFTR, E1371X CFTR, H1375P CFTR, 4259del5 CFTR, Q1382X CFTR, 4279insA CFTR, 4326delTC CFTR, Q1411X CFTR, Q1412X CFTR, 4374+1G->T CFTR, 4374+1G->A CFTR, 4382delA CFTR, 4428insGA CFTR, A1067T CFTR, E193K CFTR, K1060T CFTR, Glnl412X CFTR.

[0129] In some embodiments, the method of the present invention is used to generate a knock-out of a target polynucleotide sequence. The knocking out of a selected polynucleotide sequence can be useful for many applications, such as knocking out a target polynucleotide sequence of interest in the eukaryotic cell clone in vitro for research purposes; and knocking out a target polynucleotide sequence ex vivo for treating or preventing a disorder associated with increased expression of the target polynucleotide sequence. As used herein, the term "knock out" includes deleting all or a portion of the target polynucleotide sequence in a way that mutes the function of the target polynucleotide sequence.

[0130] In some embodiments, the alternation may result in a change of the target polynucleotide sequence of interest from an undesired sequence to a desired sequence. In some embodiments, the method of the present invention is used to correct any type of mutation or error in a target polynucleotide sequence of interest, including but not limited to inserting a nucleotide sequence that is missing from a target polynucleotide sequence due to a deletion, deleting a nucleotide sequence from a target polynucleotide sequence due to an insertion mutation, and replacing an incorrect nucleotide sequence with a correct nucleotide sequence.In some embodiments, the alteration results in reduced or increased expression of a target polynucleotide sequence of interest.

[0131] In some embodiments, the method can be used for increasing the fetal hemoglobin content in the eukaryotic cell.

[0132] In some embodiments, the method can be used to edit the HBG1 or HBG2 promoter and subsequently increasing the expression of y-globin. As used herein, the term “gamma globin” or “y-globin” has its general meaning in the art and refers to protein that is encoded in human by the HBG1 and HBG2 genes. The HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen and bone marrow. Two y-globin chains together with two a-globin chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) in the year following birth. The ENSEMBL IDs (i.e. the gene identifier number from the Ensembl Genome Browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565 respectively. In some embodiments, the method is suitable for introducing some mutations in the HBG1 or HBG2 promoter so that at least one transcriptional activator binding site is introduced in said promoter. In some embodiments, the method is particularly suitable for introducing a new transcriptional activator binding site for KLF 1, TAL 1 or GATA1. In some embodiments, the method herein disclosed introduces the -198T> C mutation in the HBG1 or HBG2 promoter so that the KFL1 activator can now binds to the promoter. In some embodiments, the method herein disclosed introduces the -175T> C mutation in the HBG1 or HBG2 promoter so that the TALI activator can now binds to the promoter. In some embodiments, the method herein disclosed introduces the -113A> G mutation in the HBG1 or HBG2 promoter so that the GAT Al activator can now binds to the promoter. In some embodiments, the method herein disclosed is particularly suitable for editing the -200 region in the HBG1 or HBG2 promoter so that the binding site for the LRF repressor is disrupted. In some embodiments, the method herein disclosed introduces at least one mutation selected from the group consisting of -201C>T, -200C>T, -197C>T, -196C>T, -195C>T and -194C>T in the HBG1 or HBG2 promoter so that the binding site for the LRF repressor is disrupted. In some embodiments, the gene editing herein disclosed is particularly suitable for editing the -115 region in the HBG1 or HBG2 promoter so that the binding site for the BCL11A repressor is disrupted. In some embodiments, the method herein disclosed introduces at least one mutation selected from the group consisting of -114C>T, -113C>T, -115C>T and -116C>T in the HBG1 or HBG2 promoter so that the binding site for the BCL11 A repressor is disrupted.Vectors:

[0133] In some embodiments, the different components that are suitable for implementing the method of the present invention are provided to the eukaryotic cell through expression from one or more expression vectors. For example, the nucleic acids encoding the guide RNA molecule, the nuclease and / or the donor repair template can be cloned into one or more vectors for introducing them into the cell. The vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the guide RNA molecule, the nuclease and / or the donor repair template herein disclosed. Preferably, the nucleic acids are isolated and / or purified. Thus, the present invention provides recombinant constructs or vectors having sequences encoding one or more of the guide RNA molecule(s), the nucleases described above and / or the donor repair template(s). Examples of the constructs include a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In some embodiments, the construct further includes regulatory sequences. A “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as inducible regulatory sequences. The design of the expression vector can depend on such factors as the choice of the eukaryotic cell to be transformed, transfected, or infected, the desired expression level, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with eukaryotic hosts are also described in e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). The vector can be capable of autonomous replication or integration into a host DNA. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli. Any of the procedures known in the art for introducing foreign nucleotide sequences into host cells may be used. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods forintroducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.

[0134] In some embodiments, the different components of the present invention are provided to the population of cells through the use of an RNA-encoded system. For instance, the base-editing system may be provided to the population of cells through the use of a chemically modified mRNA-encoded adenine or cytidine base editor together with modified guide RNA as described in Jiang, T., Henderson, J. M., Coote, K. et al. Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). In particular, engineered RNA-encoded nucleases system are prepared by introducing various chemical modifications to both mRNA that encoded the nuclease and guide RNA. In particular said modifications consist in uridine depleted mRNAs modified with 5-methoxyuridine: synonymous codons may be introduced to deplete uridines as much as possible without altering the coding sequence and replaced all the remaining uridines with 5-methoxyuridine. Said optimized base editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoded system. It is also possible to encapsulate the modified mRNA and guide RNA into lipid nanoparticle (LNP) for allowing lipid nanoparticle (LNP)-mediated delivery.

[0135] In some embodiments, the different components of the present invention are provided to the population of cells through the use of ribonucleoprotein (RNP) complexes. As used herein, the term “ribonucleoprotein complex,” or “ribonucleoprotein particle” refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “nucleoprotein” as provided herein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). For instance the nuclease can be pre-complexed with one or more guide RNA molecules to form a ribonucleoprotein (RNP) complex. The RNP complex can thus be introduced into the eukaryotic cell. Typically, the RNP complex is produced simply by mixing the proteins (i.e. the nuclease) and one or more guide RNA molecules in an appropriate buffer. This mixture is incubated for 10 min or more at room temperature before electroporation. Electroporation is adelivery technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane. In some embodiments, genome editing efficiency can be improved by adding a transfection enhancer oligonucleotide. RNP complex can also be delivered in cells by liposomes or other transfection systems.

[0136] In some embodiments, a plurality of successive transfections are performed for reaching a desired level of mutagenesis in the cell.

[0137] Therapeutic uses:

[0138] The methods described herein can be used for ex vivo therapy. Ex vivo therapy can comprise administering a composition (e.g., a cell) generated or modified outside of an organism to a subject (e.g., patient). In some embodiments, the composition (e.g., a cell) can be generated or modified by the methods disclosed herein. For example, ex vivo therapy can comprise administering a cell generated or modified outside of an organism to a subject (e.g., patient), wherein the primary cell has been cultured in vitro in accordance with the methods of the present invention. In some embodiments, the composition (e.g., a cell) can be derived from the subject (e.g., patient) to be treated by ex vivo therapy. In some embodiments, ex vivo therapy can include cell-based therapy, such as adoptive immunotherapy.

[0139] Thus, a further object of the present invention relates to a method of therapy in a patient in need thereof, the method comprising transplanting a therapeutically effective amount of a population of edited eukaryotic cells obtained by the methods herein disclosed.

[0140] In particular, the method is particularly suitable for treating a P-hemoglobinopathy. In some embodiments, the P-hemoglobinopathy is a sickle cell disease. In some embodiments, the P-hemoglobinopathy is a P-thalassemia.

[0141] As used herein, the term " P-hemoglobinopathy" has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the HBB gene, or mutations in, or deletions of, the promoters or enhancers of such gene that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition.As used herein, the term "sickle cell disease" has its general meaning in the art and refers to a group of autosomal recessive genetic blood disorders, which results from mutations in a globin gene and which is characterized by red blood cells that assume an abnormal, rigid, sickle shape. They are defined by the presence of pS-globin gene coding for a P-globin chain variant in which glutamic acid is substituted by valine at amino acid position 6 of the peptide: incorporation of the PS-globin in the Hb tetramers (HbS, sickle Hb) leads to Hb polymerization and to a clinical phenotype. The term includes sickle cell anemia (HbSS), sickle-hemoglobin C disease (HbSC), sickle beta-plus- thalassaemia (HbS / p+), or sickle beta-zerothalassaemia (HbS / pO).

[0142] As used herein, the term " P-thalassemia" refers to a hemoglobinopathy that results from an altered ratio of a-globin to P-like globin polypeptide chains resulting in the underproduction of normal hemoglobin tetrameric proteins and the precipitation of free, unpaired a-globin chains.

[0143] In some embodiments, the population of cell is autologous to the subject, meaning the population of cells is derived from the same subject.

[0144] In addition to its applications in ex vivo therapy, the methods described in this invention are also highly suitable for in vivo therapy, e.g. offering promising new approaches to treat monogenic diseases directly within the organism. This involves administering the therapeutic components directly into the patient's body, where precise genome editing can occur within the targeted cells. The method of the present invention can indeed allow for the specific and accurate correction of genetic mutations at their origin. This provides a powerful tool for treating various monogenic diseases. As used herein, the term “monogenic disease” refers to any disease caused by mutations in a single gene. These diseases are typically inherited and can lead to a variety of health issues depending on the gene affected. Examples of monogenic diseases include cystic fibrosis, sickle cell disease, and Duchenne muscular dystrophy. Treatment approaches for monogenic diseases often involve targeting the specific genetic mutation to correct or compensate for the defective gene function. In particular, the method of the present invention is suitable for the treatment of cystic fibrosis, which is characterized by mutations in the CFTR gene, leading to severe respiratory and digestive issues. Duchenne muscular dystrophy, resulting from mutations in the DMD gene, causing progressive muscle degeneration, can also benefit from this advanced therapeutic approach. Hemophilia, a condition where blood clotting is impaired due to mutations in clotting factor genes, is anotherexample where in vivo therapy could offer significant improvements in patient outcomes. The method of the present invention ensures that genome editing is targeted and controlled, minimizing off-target effects and enhancing the overall safety and efficacy of the treatment. This method represents a significant advancement in the field of gene therapy, providing a more precise, efficient, and safer alternative to traditional approaches. By addressing the root cause of genetic disorders at the molecular level, this in vivo therapeutic strategy has the potential to revolutionize the treatment of monogenic diseases, offering patients improved health outcomes and quality of life. Furthermore, the flexibility of this approach allows it to be tailored to individual patient needs, ensuring personalized medical care. This innovation not only expands the capabilities of gene therapy but also underscores the potential of the method of the present invention in medicinal applications, paving the way for future advancements in the treatment of genetic conditions. For in vivo applications, the components of the method, such as nucleases / nickase, guide RNA molecules (gRNA), and donor repair templates, are typically delivered by means of vectors. These vectors can include viral vectors, which are highly efficient for gene transfer into cells, the use of lipid nanoparticles (LNPs) for delivering mRNA, or virus-like-particles (VLP). These delivery systems ensure that the genetic editing components reach the target cells effectively and facilitate precise genome editing. The ability to use such vectors and delivery systems enhances the flexibility and applicability of the method, making it suitable for a wide range of therapeutic scenarios. For instance, viral vectors can be engineered to target specific cell types, increasing the precision of the treatment. On the other hand, LNPs offer a non-viral delivery method that can reduce potential immunogenicity and improve safety profiles. By leveraging these advanced delivery systems, the method can achieve high efficiency and specificity in gene editing, thereby maximizing therapeutic benefits while minimizing risks.

[0145] Kits

[0146] This invention further provides kits containing reagents for performing the above-described methods, including all component for performing the edition as described herein. To that end, one or more of the reaction components, e.g., guide RNA molecules, nucleases and / or donor repair templates for the methods disclosed herein can be supplied in the form of a kit for use. In some embodiments, the kit comprises (a) at least one nuclease or a polynucleotide encoding thereof, and (b) one or more guide RNA molecule(s) designed for guiding the nuclease(s) and (c) one or more donor repair template(s). In some embodiments, the kit can include one or moreother reaction components. In some embodiments, the kit can comprise one or more cell state modulator, such as one or more a DNA-PK inhibitors, one or more CDK7 inhibitor(s), or one or more HDAC inhibitor(s). In some embodiments, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate. Examples of additional components of the kits include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA or nucleases or verifying the target nucleic acid's status, and buffers or culture media for the reactions. The kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detection. The components used can be provided in a variety of forms. For example, the components (e.g., enzymes, RNAs, probes and / or primers) can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay. The kits of the invention can be provided at any suitable temperature. For example, for storage of kits containing protein components or complexes thereof in a liquid, it is preferred that they are provided and maintained below 0° C., preferably at or below -20° C., or otherwise in a frozen state. The kits can also include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, microparticles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like). The kits may further include instructions recorded in a tangible form for use of the components.

[0147] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0148] FIGURES:

[0149] Figure 1: Set up of Zip Editing (ZE) technology to increase HDR-editing efficiency with Cas9 nuclease in cell lines, a Representation of RNPs used in the “UNZIP" (Cas9-nuclease with free ssODN template) and in the “Zip Editing (ZE) 1” (Cas9-nuclease with ssODN import using hybridization of ssODN to the gRNA) systems, b Editing strategy to convert eGFP into BFP. c Construction of cell models and result analysis timeline, d Illustrative cytometry results of eGFP+-HEK cells, unedited, edited by UNZIP or edited by ZE1. e and f Flow cytometryquantification of the edition with Cas9 nuclease with (ZE1) or without (UNZIP) the correction template physically bound to the gRNA in eGFP+-HEK293T (n=4) and eGFP+-K562 (n=8) respectively. The proportion of cells expressing BFP (HDR-edited), eGFP (unedited) or neither of these fluorophores (imprecisely edited, with InDels) one week after transfection are reported. The mean ± SD is shown. The HDR / InDels ratio is indicated on the top of the graphs. Statistical significance is determined by 2way ANOVA. Schemas created with BioRender.com.

[0150] Figure 2: Improvement of Zip Editing (ZE) technology to increase HDR-editing efficiency with Cas9 nuclease, a Representation of RNPs used in the ZEla (ZE1 with 3-’elongated ssODN template), ZElb (ZE1 with additive t-lock loop in the gRNA), ZEID (ZE1 with additive t-lock loop in the gRNA and double ssODN template import) and ZE1L= ZEmax (ZE1 with additive t-lock loop in the gRNA and with 3-’elongated ssODN template) import systems, b Flow cytometry quantification of the HDR-edition with Cas9 nuclease with ZEla, ZElb, ZEID and ZEIL in eGFP+-K562 cells. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. Fold- changes indicate the fold-increases between the ZEla, ZElb, ZEID or ZEIL relative to ZE1. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance to ZE1 is determined by Kruskal -Wallis test, c Representation of RNPs used in the ZElLa (ZEIL with 3 -’over-elongated ssODN template) and ZEILb (ZEIL with ssODN 3 ’-hybridization to the gRNA, instead of 5’) systems, d Flow cytometry quantification of the HDR-edition with Cas9 nuclease with ZElLa and ZEILb in eGFP+-K562 cells. The proportion of cells expressing BFP (HDR-edited) one week after transfection is reported. Fold-changes indicate the fold-increases between the ZElLa or ZEILb relative to ZEIL. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance to ZEIL is determined by Kolmogorov- Smirnov test. Schemas created with BioRender.com.

[0151] Figure 3: Validation of ZEmax in cell lines and primary cells. Comparison to AZD7648. a Editing strategy to edit HBB in HEK293T cells, b Representation of the UNZIP2 system (control of ZEmax with modified gRNA but without ssODN-gRNA annealing), c Nanopore sequencing to quantify HDR-editing per base with UNZIP2 or ZEmax. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by 2way ANOVA. d Flow cytometry quantification of the edition with Cas9 nuclease with (ZEmax) or without (UNZIP) the correction template physically bound to the gRNA in eGFP+-hFFs (n=5). The proportion of cells expressing BFP (HDR-edited), eGFP (unedited) or neither of these fluorophores (imprecisely edited, with InDels) one week after transfection are reported. The mean ± SD is shown. The HDR / InDels ratios are indicatedon the top of the graphs. Statistical significance is determined by 2way ANOVA. e Flow cytometry quantification of the HDR-edition with Cas9 nuclease with UNZIP or ZEmax and with or without inhibition of NHEJ repair by AZD7648 in eGFP+-hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. The HDR / InDels ratios are indicated on the top of the graphs. Statistical significance is determined by Ordinary one-way ANOVA. f Editing strategy to edit UROS (insertion of Sad restriction site) in CD34+cells, g Capillary electrophoresis of SacI digestion products to quantify HDR-editing with UNZIP2 or ZEmax. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by unpaired t-test. Schemas created with BioRender.com.

[0152] Figure 4: Validation of ZEmax technology for CFTR editing in nasal and pulmonary basal cells. Obtention of human healthy pulmonary basal cells and editing strategy to model CFTR G542X mutation and insert Sad restriction site, b Capillary electrophoresis of SacI digestion products to quantify HDR-editing with UNZIP or ZEmax. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by paired t-test. c Obtention of human nasal and bronchial cells from a cystic fibrosis patient carrying CFTR G542X rare mutation and editing strategy to i) correct CFTR G542X mutation and ii) add other silent mutations to prevent re-cut by Cas9 nuclease, d Nanopore sequencing to quantify HDR-editing per read (total HDR, 7 mutations) with UNZIP2 or ZEmax in nasal (left panel) or bronchial (right panel) cells. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance compared to unedited cells is determined by ordinary one-way ANOVA. e Nanopore sequencing to quantify HDR-editing per base with UNZIP2 or ZEmax in nasal (left panel) or bronchial (right panel) cells. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Schemas created with BioRender.com.

[0153] Figure 5: The increased availability of the HDR template with ZEN is essential to repair SSB by HDR. a Representation of RNPs used in the “UNZIP" (Cas9-nickase with free ssODN template) and in the “Zip Editing Nickase (ZEN, Cas9-nickase with ssODN import using hybridization of 3 ’-elongated ssODN to the t-lock gRNA)” systems, b Flow cytometry quantification of the edition with Cas9 nickase with (ZEN) or without (UNZIP) the correction template physically bound to the gRNA in eGFP+-HEK293T cells, eGFP+-K562 cells, and eGFP+-hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The number of independent replicates is indicated by the squaresymbols. The mean ± SD is shown. Statistical significance is determined by unpaired t-test (eGFP+-HEK293T cells, eGFP+-hFFs) or Mann-Withney test (eGFP+-K562 cells), c Editing strategy to edit UROS in hFFs. d Representation of “UNZIP2" system (control of ZEN with modified gRNA but without ssODN-gRNA annealing), e Nanopore sequencing to quantify HDR-editing per read (total HDR, 3 mutations) with UNZIP2 or ZEN. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by unpaired t-test. f Nanopore sequencing to quantify HDR-editing per base with UNZIP2 or ZEN. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by 2way ANOVA. Schemas created with BioRender.com.

[0154] Figure 6: Iterative editing and ssODN template design improve HDR editing with ZEN. a Timeline for iterative transfections (RNPs nucleofection). b Flow cytometry quantification of the edition after 1, 2 or 3 transfection(s) with ZEN in eGFP+-hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The number of independent replicates is indicated by the square symbols. The mean ± SD is shown. Statistical significance is determined by ordinary one-way ANOVA. c Nanopore sequencing quantification of the edition after 1, 2 or 3 transfection(s) with ZEN in hFFs. Total HDR (3 mutations) rates per read are reported. The number of independent replicates is indicated by the symbols. The mean ± SD is shown. Statistical significance is determined by ordinary one-way ANOVA. d Representation of BFP-ssODN template positioning relative to the cut site used for ZEN or ZENmax. e Flow cytometry quantification of the edition with ZEN or ZENmax in eGFP+-hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The number of independent replicates is indicated by the square symbols. The mean ± SD is shown. Statistical significance is determined by unpaired t-test. f Flow cytometry quantification of the edition after 1, 2 or 3 transfection(s) with ZENmax in eGFP+-hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The number of independent replicates is indicated by the square symbols. The mean ± SD is shown. Statistical significance is determined by ordinary one-way ANOVA.

[0155] Figure 7: Modulation of cell cycle or chromatin opening can be associated with ZEN to increase HDR-editing. a Flow cytometry quantification of the HDR-edition with Cas9 nickase (ZENmax) with or without the synchronisation of eGFP+-hFF s in G0 / G1 phase with palbociclib or in S phase with XL413. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. Fold-changes indicate the fold-increases between the conditionswith or without drug. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significances to ZENmax without drug are determined by Kolmogorov- Smirnov test, b Flow cytometry quantification of the HDR-edition with Cas9 nickase (ZENmax) with or without NaBut exposure. The proportion of cells expressing BFP (HDR-edited) one week after transfection is reported. Fold change indicates the fold increase between the condition with NaBut and the condition without. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by Kolmogorov- Smirnov test, c Nanopore sequencing to quantify UROS HDR-editing per read with ZEN with or without NaBut in hFFs. Fold-changes indicate the fold-increases between the conditions with or without NaBut. The mean ± SD is shown. The number of independent replicates is indicated by the square symbols. Statistical significance is determined by Kolmogorov- Smirnov test.

[0156] Figure 8: Mechanistic behind ZIP efficiency, a. Results of gRNA and ssODN alignment for ZIPmax_V3 and ZIPmax_V4. Alignment performed with Clustal Omega. Stars indicate complementary nucleotides between the two sequences, b. Flow cytometry quantification of HDR edition with Cas9 nuclease with UNZIP (n=6), ZIPmax (=ZEmax) (n=6), ZIPmax_V3 (n=6), ZIPmax_V4 (n=6), UNZIP 2 (n=3) or UNZIP 3 (n=6) in eGFP + -hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. Fold changes indicate fold increases between ZIPmax, ZIPmax_V3, ZIPmax_V4, UNZIP 2 or UNZIP_3 relative to UNZIP. The mean ± SD is shown. Statistical significance to UNZIP determined by Kruskal -Wallis test. c. Flow cytometry quantification of HDR edition with Cas9 nuclease with UNZIP (n=6), ZIPmax (n=6), ZIPmax_V3 (n=6), ZIPmax_V4 (n=6), UNZIP 2 (n=3) or UNZIP 3 (n=6) in eGFP + -hFFs. The proportions of cells expressing BFP (HDR-edited) one week after transfection are reported. The mean ± SD is shown. Statistical significance to UNZIP determined by Ordinary one-way ANOVA.

[0157] EXAMPLE:

[0158] Introduction:

[0159] The CRISPR-Cas9 system induces DNA double-strand breaks (DSBs) at targeted sites to activate mainly two competitive DNA repair pathways: homology-directed repair (HDR) which allows precise editing with a homologous DNA template, and non-conservative NHEJ / MMEJ pathways which connect two ends of the broken DNA and are often accompanied by random insertions and deletions (InDels). The relatively low efficiency of HDR compared to NHEJ / MMEJ has been a major bottleneck in achieving precise gene editing at desired loci.Because of higher efficacy, clinical genome editing protocols have been restricted to imperfect but efficient NHEJ-based approaches. To obtain a higher precise genome editing rate, authors proposed i) to modulate repair pathways, by pharmacological NHEJ / MMEJ inhibition (Bischoff et al., 2020; Selvaraj et al., 2024; Riesenberg et al., 2023; Wimberger et al., 2023; Schimmel et al., 2023), by HDR activation through expression of HDR actors (Jin et al., 2024; Park et al., 2024) or Cas9-fusion proteins to enhance HDR pathway at the targeted locus (Charpentier et al., 2018; Ma et al., 2020; Jayavaradhan et al., 2019; Carusillo et al., 2023) or ii) to enhance HDR by S / G2 synchronization (Lin et al., 2014) or iii) to use DSB-less CRISPR tools (Komor et al., 2016; Anzalone etal., 2019; Ferreira da Silva etal., 2024). While promising, these approaches could alter the normal behavior of the target cells, raising substantial safety concerns when explored in a therapeutic context (Wang et al., 2024) and HDR remains suboptimal. Studies also revealed that exogenous DNA template presence at the DNA cut is critical for HDR. For example, fluorescently labeled donor DNA can be used to enrich cells that are likely to be edited via HDR (Cullot et al., 2019; Lee et al., 2017). Here, we describe the development of new efficient CRISPR-Cas9 tools, named ZIP-editors, that carry a ssODN template annealed to a modified gRNA allowing RNP stability. This specific heteroduplex gRNA-ssODN is very easy to design. It does not require any specialized and / or expensive chemical modifications nor modified Cas9. The simplicity of using unmodified Cas9, ssODN and easily-available modified gRNA allows its easy transferability to all laboratories. Importantly, it is versatile, allowing increased HDR for many targets, in cell lines and primary cells, with nuclease and unlocking HDR using safer single nickase.

[0160] Material & Methods:

[0161] Ethical statement

[0162] Our research complies with all relevant ethical regulations. Human CD34+HSPCs were isolated from cord blood of healthy donors from Bagatelle Hospital, according to the hospital’s ethical institutional review board (Maison de Sante Protestante de Bordeaux, Talence, France) and with mother’s informed consent.

[0163] Cystic fibrotic patient was recruited at the pediatric cystic fibrosis resource and skills center (CPP 23.00854.000207). Nasal and bronchial cells were obtained respectively by nasal or bronchial superficial ciliary brushing according to the hospital’s ethical institutional review board and with parent’s informed consent.Non-cystic fibrotic subjects (CPP 23.00854.000207) were recruited after surgical resection at the University Hospital of Bordeaux. Bronchial specimens from all subjects were obtained by either fibroscopic bronchoscopy or lobectomy in macroscopically normal areas, as previously described (Trian etal., 2015). All subjects gave their written informed consent to participate in the study after the nature of the procedure had been fully explained.

[0164] Cell culture

[0165] Human embryonic kidney (HEK) cell line immortalized with large T antigen HEK293T (ATCC®) was maintained in Dulbecco’s modified Eagle’s medium (DMEM), low glucose (1 g. L-1), L-Glutamine (1 g. L-1) and pyruvate (Gibco® by Lifetechnologies™) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100μg / mL streptomycin (all from Eurobio).

[0166] K-562 cell line (ATCC®) was maintained in Roswell Park Memorial Institute (RPMI) Medium 1640, L-Glutamine, 25 mM HEPES (Gibco®) supplemented with 10% fetal bovine serum, GlutaMAX (Gibco®), 100 U / mL penicillin, and 100 pg / mL streptomycin.

[0167] hTERT immortalized human foreskin fibroblasts (hFFs, (ATCC®) were maintained in DMEM, high glucose (4.5 g. L-1), L-Glutamine (1 g. L-1) and pyruvate (Gibco® by LifetechnologiesTM) supplemented with 10% fetal bovine serum, 1% essential amino acids (Gibco®), 100 U / mL penicillin, 100μg / mL streptomycin (Eurobio™), 10 pg / mL ciprofloxacin (Biogaran™) and 0.5 pg / mL amphotericin B (Sigma®).

[0168] Human CD34+stem and progenitor cells (HSPCs) were isolated from the cord blood of healthy donors. Briefly, mononuclear cells were isolated by Ficoll gradients. hCD34+cells were purified according to the manufacturer’s instructions (Human CD34-Positive Selection kit II from Stem Cell Technologies) and purity was analyzed by flow cytometry using phycoerythrin-conjugated anti-CD34 antibody (Biolegend) Cryopreserved hCD34+cells were thawed and cultured in expansion medium consisting in StemSpan SFEM (Stem Cell Technologies) supplemented with Flt3-L (100 ng / mL), SCF (100 ng / mL), human TPO (100 ng / mL), vitamin C (0.35 mg / mL), ciprofloxacin (10 pg / mL) and 100 U / mL penicillin, and 100 pg / mL streptomycin (Eurobio).Nasal and bronchial epithelial cell culture were established from nasal or bronchial brushings, as previously described (Trian et al., 2015). Nasal and basal epithelial cells were cultured using PneumaCult™-Ex Plus Medium (StemCell).

[0169] All cell types were cultured at 37 °C, 5% CO2 in a humidified chamber.

[0170] Construction of eGFP+cell models

[0171] HEK293T cells, K562 cells and hFFs were transduced with a lentivirus containing the gene encoding eGFP (enhanced green fluorescent protein) and a puromycin resistance gene, under the dependence of the ubiquitous strong constitutive promoter MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted). This lentivirus was produced and titrated by the Vect'UB vectorology platform of Bordeaux University. HEK293T cells, K562 cells and hFFs were respectively transduced at a multiplicity of infection (MOI) of 0.1, 1 and 2 to have a low copy number of the integrated vector. 96h after transduction, a selection of transduced cells was performed by adding puromycin (2 pg / mL for 48h, Gibco) to their culture medium. Hereafter these cells will be referred to as eGFP+-HEK293T cells, eGFP+-K562 cells and eGFP+-hFFs.

[0172] RNP transfection and gene editing tools

[0173] The crRNAs were designed using CHOP-CHOP software (chopchop.cbu.uib.no). Then the tracrRNA part (eventually modified for one of the loop and in length) was added to reconstitute the gRNA. gRNA and ssODN template for HDR correction were ordered from Integrated DNA Technologies. Their sequences are given respectively in the Tables 1 and 2.

[0174] Table 1:

[0175] sgRNA Sequence (5’ to 3’)

[0176] sgRNA eGFP CTCGTGACCACCCTGACCTAGTTTTAGAGCTAGAAATAGCA (UNZIP) AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTT

[0177] sgRNA eGFP CTCGTGACCACCCTGACCTAGTTTTAGAGCTAGAAATAGCA (ZE1, ZElb) AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGCTCAGTCT

[0178] sgRNA eGFP CTCGTGACCACCCTGACCTAGTTTTAGAGCTAGAAATAGCA (ZEla, ZElD, AGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTC

[0179]

[0180] ZElLa, ZEILb, CAAGTGGCACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGC ZEmax, ZEN, TCAGTCTGCTGCC

[0181] ZENmax)

[0182] sgRNA HBB GTAACGGCAGACTTCTCCTCGTTTTAGAGCTAGAAATAGCA (UNZIP2, AGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTC ZEmax) CAAGTGGCACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGC TCAGTCTGCTGCC

[0183] sgRNA UROS GGAAGCAGCAGAGTTATGTTGTTTTAGAGCTAGAAATAGCA (UNZIP2, AGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTC ZEmax, ZEN) CAAGTGGCACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGC TCAGTCTGCTGCC

[0184] sgRNA GACAATATAGTTCTTGGAGAGTTTTAGAGCTAGAAATAGCA CFTR_G542X_m AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG odeling (UNZIP) CACCGAGTCGGTGCTTTT

[0185] sgRNA GACAATATAGTTCTTGGAGAGTTTTAGAGCTAGAAATAGCA CFTR_G542X_m AGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTC odeling (ZEmax) CAAGTGGCACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGC TCATTCTGCCATCAAAGCGTGCTCA

[0186] sgRNA GACAATATAGTTCTTTGAGAGTTTTAGAGCTAGAAATAGCA CFTR_G542X_c AGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTC orrection CAAGTGGCACCGAGTCGGTGCTTTTCTGCCATCAAAGCGTGC (UNZIP2, TCAGTCTGCTGCC

[0187] ZEmax)

[0188]

[0189] Table 2:

[0190] HDR template Sequence (5’ to 3’)

[0191] BFP (UNZIP) AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAG CCACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGA BFP (ZE1, ZE la) TGAGCACGCTTTGATGGCAGAAGCTGCCCGTGCCCTGGCCC ACCCTCGTGACCACCCTGAGCCACGGCGTGCAGTGCTTCGC CCGCTACCCCGACCACAT

[0192]

[0193] BFP (ZEID 1 / 2) GGCAGCAGACTGAGCAAGCTGCCCGTGCCCTGGCCCACCC TCGTGACCACCCTGAGCCACGGCGTGCAGTGCTTCGCCCGC TACCCCGACCACAT BFP (ZEID 2 / 2) ACGCTTTGATGGCAGAAGCTGCCCGTGCCCTGGCCCACCCT CGTGACCACCCTGAGCCACGGCGTGCAGTGCTTCGCCCGCT ACCCCGACCACAT BFP (ZElb, GGCAGCAGACTGAGCACGCTTTGATGGCAGAAGCTGCCCG ZEmax, ZEN) TGCCCTGGCCCACCCTCGTGACCACCCTGAGCCACGGCGTG CAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACG ACTTCTTCAAGTCCGCCATGCCCGAAGGC BFP (ZElLa) TGAGCACGCTTTGATGGCAGAAGCTGCCCGTGCCCTGGCCC ACCCTCGTGACCACCCTGAGCCACGGCGTGCAGTGCTTCGC CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGT CCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCACCTACAAGAC BFP (ZEILb) AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAG CCACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGA AGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTG AGCACGCTTTGATGGCAG BFP (ZENmax) GGCAGCAGACTGAGCACGCTTTGATGGCAGCACCTACGGC AAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC CGTGCCCTGGCCCACCCTCGTGACCACCCTGAGCCACGGCG TGCAGTGCTTCGCCCGCTACCCCGACCA HBB (UNZIP2) TGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCAT GGTGCATCTGACTCCCGAGGAAAAATCCGCAGTCACTGCC CTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAG HBB (ZEmax) GGCAGCAGACTGAGCACGCTTTGATGGCAGTGACACAACT GTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTG ACTCCCGAGGAAAAATCCGCAGTCACTGCCCTGTGGGGCA AGGTGAACGTGGATGAAGTTGGTGGTGAG UROS (UNZIP2) CTGAAGATTACGGGGGACTCATTTTTACCAGCCCCAGAGCA GTGGAAGCAGCAGAGCTCTGTTTAGAGCAAAACAATAAAA CTGAAGGTGAGGGTGGGTCTGCTGTGGATTCCACTGGAC

[0194]

[0195] UROS (ZEmax, GGCAGCAGACTGAGCACGCTTTGATGGCAGCTGAAGATTA ZEN) CGGGGGACTCATTTTTACCAGCCCCAGAGCAGTGGAAGCA GCAGAGCTCTGTTTAGAGCAAAACAATAAAACTGAAGGTG AGGGTGGGTCTGCTGTGGATTCCACTGGAC CFTR_G542X_mo ACATCTCCAAGTTTGCAGAGAAAGACAATATAGTTCTTTAG deling (UNZIP) GAGCTCGGAATCACACTGAGTGGAGGTCAACGAGCAAGA CFTR_G542X_mo TGAGCACGCTTTGATGGCAGaattttctatttttggtaatagGACATCTCC deling (ZEmax) AAGTTTGCAGAGAAAGACAATATAGTTTAGGAGCTCGGAA TCACACTGAGTGGAGGTCAACGAGCAAGAATTTCTTTAGC AAGgtgaat

[0196] CFTR_G542X_cor ttctatttttggtaatagGACATCTCCAAGTTTGCAGAGAAAGATAACA rection (UNZIP2) TCGTCCTCGGAGAGGGTGGAATCACACTGAGTGGAGGTCA ACGAGCAAGAATTTCTTTAGCAAGgtgaata CFTR_G542X_cor GGCAGCAGACTGAGCACGCTTTGATGGCAGttctatttttggtaatagG rection (ZEmax) ACATCTCCAAGTTTGCAGAGAAAGATAACATCGTCCTCGG AGAGGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGA ATTTCTTTAGCAAGgtgaata

[0197]

[0198] Eventually, annealing between sgRNA and ssODN template(s) to form sgRNA-template hybrids (ZE1, ZEla, ZElb, ZEIL, ZElLa and ZEILb, ZEN and ZENmax, ZIPmax_V3, ZIPmax_V4) or sgRNA-templates hybrid (ZEID), were performed by mixing the components either in equimolar proportion (1: 1) for ZE1, ZEla, ZElb, ZEIL, ZElLa and ZEILb ZEN and ZENmax systems, or with a double template (1:2) for the ZEID. The components were then heated for 5 minutes in a dry bath at 95 °C before allowing them to cool to room temperature.

[0199] The different components of the CRISPR-Cas9 system (Alt-R® S.p. HiFi Cas9 Nuclease V3 (abbreviated hereafter as Cas9) or Alt-R S.p. Cas9 D10A Nickase V3 protein (abbreviated hereafter as nickase) both from Integrated DNA Technologies, with either split sgRNA and ssODN template or sgRNA-template(s) hybrids) were combined to form a RNP. They were mixed and then incubated for 10-20 minutes at room temperature. For this, 10 pg (for HEK293T cells, K562 cells and hFFs) or 17 pg (for other cells) of nuclease or nickase Cas9 and 3.9 pM (for HEK293T cells, K562 cells and hFFs) or 6.5 pM (for other cells) of sgRNA, ssODN template or sgRNA-template(s) hybrids for ZEs were mixed. Finally, 3.9 pM of Alt-R Cas9Electroporation Enhancer solution (Integrated DNA Technologies) was added to the RNP complex to improve electroporation efficiency.

[0200] Cells were transfected by electroporation using the Nucleofector 4D AMAXA electroporation system (Lonza®). In brief, 5.104to 3.105depending on the cell type were resuspended in SF Cell Line 4D-Nucleofector® (HEK293T or K562 cells) or P3 Primary Cell Line 4D-Nucleofector® (hFFs, CD34+HSPCs, pulmonary basal, nasal and bronchial cells) and added to the RNP complex. Then cells were nucleofected using DG-150, FF-120, CZ-167, DO-100 or DC- 100 programs respectively.

[0201] Cells were then cultured as described above for a week before the editing analysis.

[0202] Edition analysis

[0203] HDR and imprecise editing (InDels) analysis by flow cytometry

[0204] For the eGFP target, precise editing allows eGFP to BFP switch. At least 6 days after editing and before analysis by flow cytometry, cells were recovered in a PBS (phosphate buffered saline, Gibco) - EDTA (ethylenediaminetetraacetic acid, 2 mM, Sigma) solution. Cells were analyzed for fluorescence using the BD FACSCanto II analyzer (BD Biosciences) of the cytometry platform of the University of Bordeaux (UB'FACSility). Excitation wavelengths of 488 and 405 nm were used to detect eGFP and BFP fluorescence in the FITC (Fluorescein-5-isothiocyanate) (530 / 30 filter) and Pacific Blue (450 / 50 filter) channels respectively. The data presented are from the analysis of at least 10,000 events. The percentages of eGFP+ / BFP-, eGFP- / BFP+, and eGFP- / BFP-correspond to cells unedited, or edited by HDR or with InDels respectively. The percentages of BFP+or eGFPTBFP' cells shown in the graphs were calculated by subtracting the percentages of BFP+or eGFPTBFP' cells, respectively, from a sample of cells of the corresponding lineage not transfected with RNP when these were non-zero. Then, the percentage of BFP+, eGFP- / BFP-or eGFP+ / BFP-cells were normalized to recover a total of 100%.

[0205] HDR analysis by RFLP

[0206] If a restriction site is added in ssODN template sequence, HDR rate can be measured by RFLP (restriction fragment length polymorphism). At least 5 days after editing genomic DNA was extracted from cell pellets using Nucleospin® Tissue (Macherey-Nagel, Duren, Germany) according to the manufacturer’s protocol. The genomic region flanking the edited site was amplified by PCR (HotStarTaq Plus DNA polymerase, Qiagen®, Venlo, Netherlands) with adequate primers (Table 3). PCR products were purified with Nucleospin® Gel and PCRClean-up (Macherey -Nagel) and digested with SacI restriction enzyme (New England Biolabs) for at least 1 h at 37 °C. Then, digestion products were loaded into the Agilent® 2200 TapeStation (Santa Clara) capillary electrophoresis using D1000 ScreenTape and D1000 reagents according to the manufacturer’s protocol. Quality control of enzymatic digestion efficiency is included in each assay.

[0207] Table 3:

[0208] PCR primers Sequence (5’ to 3’)

[0209] HBB_424pb_F CTGATGGTATGGGGCCAAGAG

[0210] HBB_424pb_R GTCTCCACATGCCCAGTTTCT

[0211] UROS_405pb_F tagttccaggcacatagtaagcac

[0212] UROS_405pb_R TCCCAAGGCAGAGTCTGTGA

[0213] UROS lkb F CTCTAATCCCAGGCTGCGTC

[0214] UROS lkb R GCCTCCGCTCATCAGTGTAA

[0215] CFTR_415pb_F CAGCAATGTTGTTTTTGACCAACT

[0216] CFTR_415pb_R ACCCACTAGCCATAAAACCCC

[0217] CFTR lkb F AGGTCGTGAGAATGAGGTGC

[0218] CFTR lkb R TGGAGTGGCAGGGTCTATGA

[0219]

[0220] HDR analysis by Nanopore sequencing

[0221] At least 5 days after editing genomic DNA was extracted from cell pellets using Nucleospin® Tissue (Macherey-Nagel, Duren, Germany) according to the manufacturer’s protocol. The genomic region flanking the edited site was amplified by High-Fidelity PCR (Phusion High-Fidelity DNA Polymerase, New England Biolabs) with adequate primers (Table 3). Sequencing library was prepared using Rapid Barcoding Kit 96 V14 (SQK-RBK114.96, Oxford Nanopore Technologies) according to the manufacturer’ s protocol. Sequencing was then performed using a MinlON MklB device and a Flow Cell R10.4.1 (FLO-MIN114) both from Oxford Nanopore Technologies to reach at least 2000 reads per sample. After sequencing, super-accurate basecalling was performed using MinKNOW (v24.02.6) and fastq were aligned on humanreference genome GrCH38 (GCA_0000001405.15) using Epi2Me (v5.1.9) and wf-alignement pipeline (v1.1.2). Then, BAM were analyzed using two homemade bioinformatic pipelines in Python (v3.8) using pysam (v0.16). Briefly, one searches specific sequences in the BAM corresponding to unedited, HDR-editing sequences or eventually predominant Indels seen with IGV (Integrative Genomics Viewer) visualization to determine the HDR rate per read whereas the other one calculates the proportion of each nucleotide for each selected position to determine the HDR rate per base returning these data in xlsx format (pandas v1.1.3).

[0222] Pharmacological drugs

[0223] Cell cycle synchronization

[0224] To evaluate the impact of synchronization on the HDR editing efficiency, cells were synchronized in G0 / G1 phase by incubation with palbociclib, also named PF-00080665 or PD 0332991 (1 μM, Sigma Aldrich) 24 h before and 24 h after RNP transfection. To synchronize cells in the S phase, they were incubated with XL413 (20 μM, Selleckchem) for 24 h before RNP transfection and 24-48h after RNP transfection.

[0225] Cell cycle analyses were performed to check synchronization efficiency (not shown). Briefly, cells either synchronized or not were harvested, fixed with 4 % paraformaldehyde (Thermoscientific) and permeabilized with Triton 0.5 % (Sigma- Aldrich). Cells were then incubated overnight with RNAse (100 μg / mL, MoBiTec) and then for 15 minutes with propidium iodide (4 μg / mL, BioLegend). The samples were examined on a BD Biociences Accuri C6 Plus flow cytometer and the data were analyzed with BD CSampler™ software (BD Biosciences).

[0226] HD AC inhibition

[0227] To evaluate the impact of HD AC inhibition, cells were incubated 24h before and 48h after edition with sodium butyrate (NaBut, 1 mM, Sigma).

[0228] NHEJ inhibition

[0229] To modulate the NHEJ pathway, we incubated the cells 24h before and 48h after edition with AZD7648 (1 μM, MedChemExpress).

[0230] Statistical analysis and reproducibility

[0231] The experimental data presented are from the analysis of several independent experiments. No statistical method was used to predetermine sample size. No data were excluded from theanalyses. The experiments were not randomized. Statistical significance was inferred when necessary and formatted using GraphPad Prism 10.3.1 software. To confirm significance, p-values were calculated when analyzing at least three independent experiments. If not indicated, the results are not significant. The details (number of independent replicates, statistical test used) are indicated in the legends of the figures.

[0232] Results:

[0233] ZIP editing strategy to improve precise genome editing (PGE) using nuclease Cas9 To increase the effective concentration of the donor template at the DSB and thus the HDR-editing efficiency, we reasoned a strategy to hybridize the ssODN donor template to the gRNA. First, we added a 5 ’-extension on the 80nt ssODN template and a 3 ’-extension on the gRNA for annealing. The RNP complex formed is named ZIP editing 1 (ZE1) and the control RNP with regular free ssODN and gRNA is named UNZIP (Figure 1a). For the quantification of CRISPR-Cas9-mediated genome editing, we used a rapid flow cytometry-mediated assay based on eGFP to BFP conversion (Figure 1b / c) (Glaser et al., 2016). In case of HDR-editing, a substitution of a histidine in place of a tyrosine at position 66 in the chromophore of eGFP shifts its emission toward the blue spectrum (Heim et al., 1994) (Figure 1b). Three cell types, HEK293T cells, K562 cells and human foreskin fibroblast (hFFs), were stably transduced at low multiplicity of infection with a lentiviral construct containing eGFP / puromycin resistance cassette (to obtain less than 10% of eGFP+cells). Then, after selection, we obtained cells with one copy of eGFP by cell. Reliable simultaneous quantification of HDR- and imprecise-editing was obtained by flow cytometry one week after editing as BFP fluorescence appearance or eGFP fluorescence loss respectively (Figure 1c). Interestingly, PGE (HDR-editing) efficiency was significantly 2-fold increased with ZE1 compared to UNZIP system nucleofection in eGFP+-HEK293T cells (14.9% ± 0.7 vs 8% ± 1 respectively, Figure 1d, e) and confirmed in eGFP+-K562 cell line (10.2% ± 3.8 vs 5.2% ± 2.6 respectively, Figure 1f). These data, obtained in two cell lines, demonstrated that ZIP Editing system increases PGE using CRISPR-Cas9 nuclease, with a 2-fold increase of HDR / Indels ratio.

[0234] ZIP editing improvements

[0235] We hypothesized that ssODN template stability and quantity was critical for PGE. For that, we designed new generations of ZIP editing systems. First, we elongated the ssODN template to limit its degradation kinetic. Elongated hybridized ssODN (ZE1a, 120nt versus 80nt in ZE1, Figure 2a) moderately improved PGE (+26.5%, ZE1b) in eGFP+-K562cells. Because thiselongation could affect the gRNA stability and Cas9 interaction, we added a t-lock element in the second loop of the gRNA, as previously described (Riesenberg etal., 2022), to reinforce its stability. ZE1L (120nt + t-lock) induces a higher HDR-editing rate compared to UNZIP (3-fold increase), to ZE1 (1.7-fold increase, Figure 2b) and even to ZE1a (1.3-fold increase). Importantly, at this target, the t-lock element alone did not modify editing efficiency, confirming that higher ssODN concentration at the DNA break is crucial for PGE (ZE1b, Figure 2b). We tested to further elongate the ssODN template (170 nt, ZElLa, Figure 2c) or to flip the template with a 3 ’-extension for hybridization to the gRNA (ZEILb, Figure 2c).

[0236] Neither of these constructs improve HDR-editing compared to ZE1L (Figure 2d). To explore whether two tethered templates improve HDR-editing rate, we designed ZE1D (Figure 2a) in which ssODN are each hybridized by a distinct sequence to the gRNA with a t-lock element. ZEID significantly increased HDR-editing rate in eGFP+-K562 cell line compared to UNZIP (2.7-fold increase), to ZE1 (+ 37%, Figure 2b) but was less efficient than ZE1L. Altogether, ssODN elongation (120nt) associated with a t-lock loop in the gRNA (ZE1L) were the best improvements. This system was used for the following experiments and named ZEmax.

[0237] To check whether the increase in HDR editing with ZEmax was due to the initial complementary extension sequences chosen, we randomly created a new pair of complementary sequences with the same nucleotides and compared this ZEmax_V2 to ZEmax. We did not observe any difference in terms of HDR-editing suggesting that the sgRNA / ssODN complementary extensions, no matter their sequences, are important in ZIP CRISPR (data not shown). We decided to keep the initial sequence for the following experiments.

[0238] Encouraged by these observations, we tested ZEmax to edit HBB endogenous locus (Figure 3a). Because the t-lock element in gRNA can improve HDR-editing by itself in some targets (Riesenberg et al., 2022), we decided to use the same gRNA (with t-lock stabilization and 3’-extension) as control in UNZIP2 (Figure 3b). Only the templates, with or without the 5’-extension to anneal (or not) to the gRNA, change between UNZIP2 and ZEmax. NGS analysis by Nanopore sequencing revealed that the seven nucleotides inserted in the template were edited with an important increase (2 -fold) of HDR-editing rate using ZEmax (around 7 to 13.5%, Figure 3c). This result showed that ZEmax allows editing in a large window from -15 to + 13 bases around the cut site. Then, we evaluated our innovative tool in eGFP+-hFFs, known to be difficult to be precisely edited. Compared to the UNZIP control, ZEmax induced a tremendous editing improvement with up to 12-fold regarding HDR-editing and 16-fold for the HDR / InDels ratio (Figure 3d). Another way to increase the HDR / InDels ratio is to mitigate theDNA repair pathways. To date, the most potent molecule to inhibit NHEJ pathway is AZD7648, a potent inhibitor of DNA PKcs (Selvaraj et al., 2024). We decided to challenge the ZEmax system with this compound. Using eGFP+-hFFs, we found that ZEmax allows a 2-fold higher precise editing efficacy compared to UNZIP with 1 μM AZD7648 exposure 11.1% ± 1.9 vs 5.3% ± 1.7 respectively, (Figure 3e). Importantly, a complementary additive effect was observed with simultaneous use of ZEmax and AZD7648 leading to 16.5 ± 3.7% of HDR-edited cells. Due to recent data showing alarming genotoxicity of AZD7648 (Cullot et al., 2024), we decided to use only ZEmax without AZD7648 for the following experiments. In order to check the ZIP Editing system versatility in clinically-relevant cells, we evaluated it in cord blood stem and progenitor CD34+cells (HSPCs), by targeting the exon 4 of UROS, an enzyme of heme biosynthesis (Figure 3f). In these primary cells, PGE was measured by Restriction Fragment Length Polymorphism (RFLP). Digestion required the perfect editing of 2 nucleotides to create a SacI restriction site. HDR rate was more than 2-fold increased with ZEmax compared to the UNZIP2 system (16.5% ± 5.1 vs 7.3% ± 4.6 respectively, Figure 3g). Taken together, these data showed the high efficacy and versatility of this very easy-to-design ZEmax tool in cell lines and primary cells to improve HDR-editing efficiency.

[0239] CFTR gene editing in human pulmonary basal and nasal cells

[0240] First, to evaluate our ZIP editors in these human primary cells, we harvested “healthy” pulmonary basal cells to model class I nonsense CFTR p. GlyG542Ter, usually named G542X responsible for cystic fibrosis (NM_000492.4(CFTR):c.1624G>T, Figure 4a). Cells were edited with UNZIP or ZEmax by nucleofection. The template contains i) non coding TAG codon to replace GGA codon (Gly) and ii) insertion of a Sad restriction site that allows HDR-editing analysis by RFLP and mutes the PAM to avoid Cas9 recut. The analysis revealed a 3-fold higher percentage of HDR-edited alleles with the c.1624G>T (G542X) mutation and the other 4 nucleotides responsible for Sad cut with ZEmax compared to UNZIP, reaching up to 22% (Figure 4b). We then decided to correct this nonsense mutation, not eligible for actual pharmacological treatments (Fang et al., 2022). We obtained nasal and bronchial cells from a cystic fibrosis patient with compound heterozygous G542X mutation. For editing, we used a mutation-specific gRNA targeting the G542X-mutated allele and a correcting template coding GGA (Gly) instead of TGA (nonsense) in position p.542. We added silent mutations to avoid Cas9 recut and analyzed editing by Nanopore NGS sequencing (Figure 4c). Whereas the UNZIP2 system was inefficient to edit these nasal or bronchial cells, ZEmax allowed moderate complete (7 mutations) HDR-editing rates (Figure 4d left and right panels, around 2%).Regarding specifically the c.1624T>G correction, cells were initially 50% C.1624G. We obtained around 56 or 57% of C.1624G after editing, meaning 6 or 7% of genomic edition in bronchial and nasal cells respectively (Figure 4e). This result, higher than for other substitutions, suggests an additive pathway to correct C.1624T, in accordance with previous reports of gene conversion with the endogenous allele in case of heterozygosity (Javidi et al., 2020; Mianné et al., 2022).

[0241] ZIP editing strategy coupled with Cas9 nickase to obtain PGE.

[0242] Cas9 nuclease is known to induce DSB-dependent ON-target genotoxicity (Boutin etal., 2022). We thus applied the ZIP editing strategy to a simple DSB-free Cas9 (D10A nickase), not known to allow HDR-editing using RNP transfection. To edit cells without DSB, we applied the best ZIP Editing system (ZEmax, with the t-lock element in gRNA and a 5’-extended (120nt) ssODN template) to Cas9D10Anickase. This new system is named ZEN (ZEmax with Nickase) (Figure 5a). As expected, UNZIP nickase with free BFP-ssODN template did not edit or edit at very low levels eGFP+-HEK293T cells, eGFP+-K562 cells and eGFP+-hFFs (Figure 5b), confirming previous publications showing that it is very difficult to edit the genome using a unique nick (Vriend and Krawczyk, 2016; David and maizels, 2014). Importantly, ZEN induced a 3-fold increase of PGE (BFP+cells) in eGFP+-HEK293T cell line (Figure 5b, left) and unlocked PGE in eGFP+-K562 cell line (Fig. 5b middle) and in primary eGFP+-hFFs (35-fold increase, Figure 5b right). To confirm these results, we moved to the edition of an endogenous locus (UROS) in hFFs with a template containing 3 substitutions (Figure 5c). To be sure that the difference between control and ZEN is really due to the import system and not to other modifications (t-lock or 3 ’-extension in the gRNA), we designed a new control with Cas9D10Anickase with a gRNA containing the t-lock element and the 3 ’-extension, but with a free 120nt template (without 5’-extension, UNZIP2, Figure 5d). Nanopore sequencing revealed a 5 -fold improvement of perfect UROS HDR-editing (3 concomitant edited bases, Figure 5e), confirmed by individual base analysis (Fig. 5f), without any detection of InDels.

[0243] Unlike nuclease, the absence of InDels using nickase is an opportunity to re-edit the unedited cells using the same gRNA to enrich the HDR-edited population (Figure 6a). We made 3 iterative RNP transfections with ZEN in eGFP+-hFF and obtained an interesting additive effect leading to more than 3% of BFP+cells (Figure 6b). We confirmed this additive effect targeting UROS in hFF (Figure 6c).Then, to take advantage of our easy-to-design ZE tool, we evaluated the impact of ssODN design, and compared 2 asymmetric templates with either the larger homology arm on the 3’-(ZEN template) or on the 5 ’-extremity (ZENmax template), relatively to the cutting site (Figure 6d). Interestingly, we observed a 2-fold increase of HDR-editing in eGFP+-hFF with ZENmax compared to ZEN (Figure 6e). Thus, a very versatile tool such as ZE could be a great help to test and optimize the template designs. With ZENmax in eGFP+-hFF, we confirm that iterative transfections lead to an additive effect, and allow to reach more than 5% of HDR-edited cells (Figure 6f).

[0244] ZEN strategy can be combined with “cell state” modulation to increase nick-based HDR-editing

[0245] To further improve HDR-editing efficiency and benefit from the high versatility of the ZEN tool, we investigated various pathways known to modulate genome editing. It is well known that HDR-editing using Cas9 nuclease depends on S / G2 phase but the impact of cell cycle on nick-mediated HDR-editing has been less studied (Zhang et al., 2021). We observed higher HDR-editing rates in high proliferative HEK293T cells than in less proliferative hFFs (Figure 5b), suggesting that editing could be preferentially done during S / G2 / M-phases. To decipher the role of cell cycle on HDR-based nick-repair, and S / G2 / M-phases in particular, we modulated hFFs cell cycle. We used palbociclib, a CDK4 / 6 inhibitor, to reduce the S / G2 / M eGFP+-hFFs proportions during editing. We observed a 2-fold decrease in HDR-editing rates (Figure 7a). In contrast, using XL413, a CDK7 inhibitor during editing to increase S / G2 / M eGFP+-hFFs proportions, HDR-editing efficiency was improved by almost 2-fold (+85.4%, Figure 7a). Taken together, these data suggested that HDR-editing with nickase is S / G2 / M dependent.

[0246] Chromatin access is also known to be important for CRISPR tools efficiency (Björnson et al., 2023; Liu et al., 2022). We modulated chromatin opening using sodium butyrate (NaBut), an HD AC inhibitor in hFFs. We observed an increase (around 2-fold) in HDR-editing rates in the presence of NaBut both for eGFP (Figure 7b) and UROS (Figure 7c). Therefore, thanks to ZEN that reaches detectable HDR edition rates in several targets and cell types, we were able to better understand how it works, to demonstrate the implication of several parameters in nick-mediated HDR-editing and to propose drugs to increase its efficiency.Mechanism behind ZIP efficiency

[0247] To determine if the increase in HDR editing efficiency observed using the ZIP (ZEN) strategy was really due to an improved proximity between the ssODN template and the gRNA, we performed a FRET experiment using a Cy3-ssODN (containing the hybridization sequence) and a Cy5-probe annealed to the gRNA (containing or not the hybridization sequence in the ZIP and UNZIP_3 conditions respectively) (data not shown). We analyzed FRET signals (Cy5 emission after Cy3 excitation) of UNZIP_3 or ZIP gRNA / ssODN configurations either directly by spectrofluorometry or after cell transfection by confocal microscopy (data not shown). Using spectrofluorometry, we observed an increase by 2.4-fold in FRET signal (p= 0.1) with the ZIP compared to UNZIP_3 gRNA / ssODN configuration (data not shown). This means that the proximity between ssODN and gRNA seems to be effectively increased when they contain complementary extensions. We confirmed this amplification of the FRET signal by confocal microscopy, seven hours after cell transfection with the ZIP compared to the UNZIP_3 gRNA / ssODN configuration. Indeed, the number of FRET -positive pixels for 100 cells is increased when gRNA and ssODN contain complementary extensions using the same doses as for editing experiments (data not shown). These data confirm gRNA-ssODN proximity into cells after 37°C culture. To test this proximity at low concentration, we used a lower dose (⅓ of the usual dose) and confirmed a rise in the number of FRET-positive pixels for 100 cells under the ZIP gRNA / ssODN configuration (data not shown). Surprisingly, by confocal microscopy analysis, we observed at usual and low doses, while using the same amount of components, that the Cy signals are higher in the ZIP gRNA / ssODN configuration (data not shown), in favor of a better transfection efficiency. When normalizing on Cy3 / Cy5 transfection efficiency, FRET-positive pixels are still increased in the ZIP gRNA / ssODN configuration (data not shown). Taken together, these FRET results suggest that ZIP strategy allows the formation of gRNA-ssODN complexes in the cells but we can not exclude that at least a part of the increase in HDR editing observed using the ZIP strategy could be due to a better transfection efficiency perhaps through an increase in charges. Considering that whatever the length of the hybridization sequence (15, 20 or 30nt) we get the same HDR editing efficiency, we then challenged hybridization’s role in ZIP’s efficiency. To try to distinguish increase in lengths (and associated charges) and annealing effects, we first try to design non-complementary extended gRNAs and ssODNs (scramble sequences but with possible partial hybridization, indicated by stars in Figure 8a). We get the same efficiency as with ZIPmax where extensions are fully complementary (Figure 8b / c). In parallel, we tested annealing between 5nt-extensions (slight increase in charges) in a ZIPmax_hyb5 configuration. This new construction is asefficient as previously studied ZIPmax_30nt (data not shown), and its efficiency is not reduced when temperature increases (data not shown) unlike such a short annealing is not supposed to remain annealed at room and even more at 37°C temperatures. Altogether, these results suggest that only a small degree of complementarity between gRNA and ssODN extensions is sufficient and that a large increase in charges is not necessary. However, it is important to note that efficiency was drastically reduced when only gRNAs or only ssODN templates were extended, indicating that both extensions are necessary (Figure 8b / c) to achieve ZIP efficiency.

[0248] REFERENCES:

[0249] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0250] Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785): 149-157. doi: 10.1038 / s41586-019-1711- 4. Bischoff N et al. Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion? Cells. 2020;9:1318. doi: 10.3390 / cells9051318.

[0251] - Boutin J, et al. ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. CRISPR J. 2022 Feb;5(l): 19-30. doi: 10.1089 / crispr.2021.0120.

[0252] - Björnson Y, Huang CY, Rollins JL, Castañeda G, Kaur N, Yamamoto E, Johnston JM.

[0253] The effect of histone deacetylase inhibitors on the efficiency of the CRISPR / Cas9 system. Biochem Biophys Rep. 2023 Jul 16;35: 101513. doi: 10.1016 / j.bbrep.2023.101513.

[0254] Carusillo A et al. A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells. Nucleic Acids Res. 2023;51:4660- 4673. doi: 10.1093 / nar / gkad255.

[0255] Charpentier M et al. CtIP fusion to Cas9 enhances transgene integration by homology- dependent repair. Nat Commun. 2018;9:l 133. doi: 10.1038 / s41467-018-03475-7. Cullot G et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun. 2019; 10: 1136. doi: 10.1038 / s41467-019-09006-2.

[0256] Cullot G, et al. Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations. Nat Biotechnol. 2024 Nov 27. doi: 10.1038 / s41587-024-02488-6.- Davis L, Maizels N. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair. Proc Natl Acad Sci U S A. 2014 Mar 11;111(10):E924-32. doi: 10.1073 / pnas.1400236111.

[0257] - Fang X, et al. Pharmacological Responses of the G542X-CFTR to CFTR Modulators.

[0258] Front Mol Biosci. 2022 Jun 24;9:921680. doi: 10.3389 / fmolb.2022.921680.

[0259] - Ferreira da Silva J, et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Nat Biotechnol. 2024 Jul 22:10.1038 / s41587-024-02324-x. doi: 10.1038 / s41587-024-02324-x.

[0260] Glaser A, et al. GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR / Cas9-mediated Genome Editing. Mol. Ther. Nucleic Acids. 2016;5:e334. doi: 10.1038 / mtna.2016.48.

[0261] - Heim R, et al. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A. 1994;91:12501-4. doi: 10.1073 / pnas.91.26.12501.

[0262] Javidi-Parsijani P, et al. CRISPR / Cas9 increases mitotic gene conversion in human cells. Gene Ther 2020;27:281-296. DOI: 10.1038 / s41434-020-0126-z.

[0263] Jayavaradhan R, et al. CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. Nat Commun. 2019;10:2866. doi: 10.1038 / s41467-019-10735-7.

[0264] Jin YY, et al. Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor. Nat Commun. 2024;15:6843. doi: 10.1038 / s41467-024-50788- x.

[0265] - Komor AC, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 19;533(7603):420-4. doi: 10.1038 / naturel7946.

[0266] - Lee K, et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife. 2017;6:e25312. doi: 10.7554 / eLife.25312.

[0267] - Lin S, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery. Elife. 2014;3:e04766. doi: 10.7554 / eLife.04766. - Liu N, et al. HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing. Mol Ther Nucleic Acids. 2022 Jun 2;29:36-46. doi: 10.1016 / j.omtn.2022.05.036. Erratum in: Mol Ther Nucleic Acids. 2022 Oct 06;30:173. doi: 10.1016 / j.omtn.2022.09.021.- Ma L, et al. MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits. Nat Commun. 2020; 11:6082. doi: 10.1038 / s41467- 020-19842-2.

[0268] - Mianne J, et al. CRISPR / Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles. BMC Biol. 2022 Jan 7;20(l):8. doi: 10.1186 / s12915-021-01214-x.

[0269] - Park SJ, et al. Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation. Mol Ther Nucleic Acids.

[0270] 2024;35: 102274. doi: 10.1016 / j.omtn.2024.102274.

[0271] - Riesenberg S, et al. Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage. Nat Commun. 2022; 13:489. doi: 10.1038 / s41467- 022- 28137-7.

[0272] - Riesenberg S, et al. Efficient high-precision homology-directed repair-dependent genome editing by HDRobust. Nat Methods. 2023;20:1388-1399. doi: 10.1038 / s41592- 023-01949-1.

[0273] Schimmel J, et al. Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Rep. 2023;42:112019. doi: 10.1016 / j.celrep.2023.112019.

[0274] Selvaraj S, et al. MH. High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nat Biotechnol. 2024;42:731-744. doi: 10.1038 / s41587-023-01888-4.

[0275] Trian T, et al. House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway. Am J Re spir Crit Care Med. 2015 Mar l;191(5):538-46. doi: 10.1164 / rccm.201409-15820C.

[0276] Vriend LE, Krawczyk PM. Nick-initiated homologous recombination: Protecting the genome, one strand at a time. DNA Repair (Amst). 2017 Feb;50:l-13. doi: 10.1016 / j.dnarep.2016.12.005.

[0277] - Wang J, et al. DNA-PKcs suppresses illegitimate chromosome rearrangements. Nucleic Acids Res. 2024;52:5048-5066. doi: 10.1093 / nar / gkae140.

[0278] - Wimberger S, et al. Simultaneous inhibition of DNA-PK and Pol0 improves integration efficiency and precision of genome editing. Nat Commun. 2023; 14:4761. doi: 10.1038 / s41467-023-40344-4.- Zhang Y, et al. Pathways and signatures of mutagenesis at targeted DNA nicks. PLoS Genet. 2021 Apr 15;17(4):e1009329. doi: 10.1371 / journal.pgen.1009329.

Claims

CLAIMS:

1. A method of altering a target sequence of a nucleic acid molecule comprising the step consisting of contacting the target nucleic acid sequence with (a) a nuclease (b) a guide RNA (gRNA); and (c) one or more donor repair template(s) wherein:- the gRNA comprises in a 5’ to 3’ orientation:(i) a CRISPR RNA (crRNA) complementary to a portion of the target nucleic acid molecule;(ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and,(iii) a 3’ extension arm that is capable of hybridizing to one or more donor repair template(s), and,- the donor repair template comprises:(i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and(ii) an extension comprising a complementary sequence (“complementary extension sequence”) for hybridization to the 3’ extension arm of gRNA.

2. The method according to claim 1 wherein the nuclease is a CRISPR / Cas nuclease, more particularly a Cas9 nuclease, even more particularly a Cas9 nuclease that comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

3. The method according to claim 1 wherein the nuclease is a nickase, more particularly a Cas9 nickase, even more particularly a nickase that comprises the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO:3.

4. The method according to any one of claims 1 to 3 wherein the extension arm is located at the 3’ end of the guide RNA, and comprises a hybridizing sequence that is capable of hybridizing to one or more donor repair template(s).

5. The method according to claim 4 wherein the extension arm can hybridize to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 donor repair template(s) and therefore the extension arm can thus comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hybridizing sequence(s).

6. The method according to any one of claims 1 to 5 wherein the gRNA further comprises at least one locked hairpin secondary structure.

7. The method according to any one of claims 1 to 6 wherein the donor repair template consists of a double-stranded DNA oligonucleotide having one single strand extension that comprises a complementary extension sequence for hybridization to the 3’ extension arm of gRNA.

8. The method according to any one of claims 1 to 6 wherein the donor repair template consists of a single-strand oligodeoxynucleotide (ssODN) that comprises (i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and (ii) an extension sequence comprising a complementary sequence (“complementary extension sequence”) for hybridization to the 3’ extension arm of gRNA.

9. The method according to any one of claims 1 to 8, wherein the complementary extension sequence is perfectly or partially complementary to the 3’ extension arm of gRNA.

10. The method according to claim 1, wherein the complementary extension sequence comprises 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and wherein the extension sequence is at least 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary with the 3’ arm extension sequence in the gRNA.

11. The method according to any one of claims 1 to 10 wherein the donor repair template donor repair template is about 75; 80; 85; 90; 95; 100; 105; 110; 115; 120; 125; 130; 135; 140; 145; 150; 155; 160; 165; 170; 175; 180 nucleotides in length.

12. The method according to any one of claims 1 to 10 wherein the edition is carried out in presence of one or more agents selected from the group consisting of a DNA-PK inhibitors, CDK7 inhibitors, CDC7 inhibitors and HDAC inhibitors.

13. The method according to any one of claims 1 to 12 for editing a eukaryotic cell selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).

14. The method according to any one of claims 1 to 13 for correcting or repairing a genetic mutation.

15. The method according to any one of claims 1 to 14 for use in therapy.

16. A kit that comprises (a) at least one nuclease or a polynucleotide encoding thereof, and (b) one or more guide RNA molecule(s) designed for guiding the nuclease(s) and (c) one or more donor repair template(s), wherein:- the gRNA comprises in a 5’ to 3’ orientation:(i) a CRISPR RNA (crRNA) complementary to a portion of the target nucleic acid molecule;(ii) a trans-activating crRNA (tracrRNA) that links the crRNA to the nuclease and,(iii) a 3’ extension arm that is capable of hybridizing to one or more donor repair template(s), and,- the donor repair template comprises:(i) a template sequence with one or more nucleotide changes flanked by segments of homologous sequence to the target nucleic acid sequence and(ii) an extension sequence comprising a complementary sequence (“complementary extension sequence”) for hybridization to the 3’ extension arm of gRNA.

17. The kit according to claim 16 that comprise one or more agents selected from the group consisting of a DNA-PK inhibitors, CDK7 inhibitors, CDC7 inhibitors and HDAC inhibitors.

Images