Crispr rna molecules for improved ev-mediated delivery of cas12 rnp complexes

Abstract

A Cas12 RNP complex comprising: a first fusion protein comprising an extracellular vesicle (EV) polypeptide and a Cas12 protein; and a circular crRNA (CRISPR RNA) comprising: a spacer sequence, a dire

Description

TECHNICAL FIELD This invention relates to Casl2 CRISPR. RNA molecules (crRNAs) that are particularly useful for the production of extracellular vesicles (EVs) that comprise Casl2 Ribonucleoprotein (RNP) complexes and for EV-mediated delivery of Casl2 RNP complexes to target cells. The present disclosure in particular relates to Casl2 RNP complexes comprising these Casl2 crRNAs bound to a Casl2 protein, preferably wherein the Casl2 protein is fused to an EV polypeptide and to EVs comprising these Casl2 RNP complexes. BACKGROUND CRISPR / Cas systems are an invaluable tool for gene editing and have considerable potential for therapeutic application. Whilst the CRISPR / Cas9 system has been explored most widely, other CRISPR-associated proteins, such as Casl2, offer potential benefits. Casl2 has the ability to recognise a broader range of genome targets, as it has a more flexible PAM sequence than Cas9. Furthermore, Casl2 generates staggered overhang cuts in double stranded DNA, as opposed to the blunt-end cuts created by Cas9, potentially allowing for more precise gene insertions. Casl2 also offers the potential to edit multiple genetic targets within a single cell, through its ability to process multiple crRNAs from a single pre-cursor RNA molecule. The CRISPR / Casl2 system has some key differences to the CRISPR / Cas9 system. For instance, whereas the Cas9 guide RNA typically requires 2 components, the CRISPR RNA (crRNA) and the trans-activating RNA (tracrRNA) that may or may not be fused together into a single guide RNA (sgRNA), Casl2 functions with a simpler guide that only requires a single crRNA molecule. Casl2 crRNAs contain a sequence that is complementary to a target DNA sequence (known as the spacer) and the structural component required to bind Casl2 (known as the direct repeat (DR) region). Casl2, unlike Cas9, has the ability to process its own crRNA from a pre-crRNA, by first binding the DR region of a pre-crRNA and forming a stable complex, then autonomously cleaving the RNA at the 5' end of the direct repeat. This Casl2 processing activity allows for multiple mature crRNA molecules having different spacer sequences to be produced from a single pre-crRNA molecule that contains multiple spacer sequences separated by direct repeat regions. Further processing steps may be required following Casl2 cleavage to produce a mature crRNA, including the trimming of the 5' and / or 3' end of the crRNA. The delivery of CRISPR / Cas systems to target cells has posed a significant challenge and barrier to therapeutic application. Viral vectors, in particular adeno-associated viruses (AAVs), are most frequently used to deliver CRISPR / Cas to target cells, however AAVs have a number of drawbacks including safety concerns due to viral-induced immune response, vector persistency and increased risk of off-target activity. Extracellular vesicles (EVs) have the potential to provide a highly effective means of delivering RNP complexes to target cells. As compared with viral-mediated delivery approaches, EVs allow for the delivery of the gene editing machinery in a transient manner, thus offering improved safety, which is of particular importance in the treatment of paediatric diseases and disorders where the long-term expression of gene editing machinery and off-target editing is a significant concern. EV-mediated delivery is also advantageous over mRNA approaches, since delivery of an RNP complex by an EV still allows for the gene editing machinery to remain present in a target cell for long enough for gene editing to take place while avoiding the safety and tolerability issues of e.g. lipid nanoparticle-mediated mRNA delivery. Further, EV-mediated delivery of editing machinery enables reaching organs, tissues and cell types that are inaccessible to LNPs, e.g. the central nervous system. However, even though RNP complexes delivered by EVs remain in target cells for long enough for gene editing to take place, the high levels of gene editing seen with viral vectors are not normally achieved. This is because fewer copies of the editing machinery are present over a shorter timespan when delivered as an RNP as compared with when the nuclease and the gRNA are translated from a viral genome. Thus, there remains a need to increase the efficiency of gene editing achieved with EV-mediated delivery Cas RNP complexes. SUMMARY OF THE INVENTION The present inventors sought to solve the problem achieving high gene editing efficiencies whilst using EVs to deliver Casl2 RNP complexes to target cells. The present inventors recently made a surprising discovery when investigating EV-mediated delivery of Cas9 RNPs to target cells. They found that fusion of the Cas9 protein to an EV-polypeptide results in highly efficient loading of Cas9 into EVs, but that loading of the guide RNA is inefficient. Thus, low levels of gene editing were likely occurring due to only the Cas9 protein - rather than Cas9 RNP complexes - being delivered to target cells. This discovery led the present inventors to investigate whether modifying the Casl2 crRNA could improve gene editing when using EVs as a delivery vehicle for Casl2 RNP complexes to target cells. Conventional Casl2 crRNAs consist of a DR region, at the 5' end, and a spacer sequence, at the 3' end. Such Casl2 crRNAs are believed to be particularly vulnerable to exonuclease degradation, due in part to their relatively short length and lack or protective features. Hence the present inventors expressed crRNAs in EV producer cells that had been modified in a manner that they hoped may improve their stability. The present inventors hoped this would allow for improved loading of the crRNA leading to the production of EVs that display increased levels of gene editing in target cells. The present inventors tried two modified crRNAs that were hoped to display improved stability: the first crRNA included an additional direct repeat to the 3' of the spacer and the second crRNA was circular. However, neither of these modified crRNAs showed any improvement in gene editing when used for EV-mediated delivery, as compared with a conventional Casl2 crRNA. Surprisingly however, when both modifications were included in a single crRNA, a significant increase in gene editing was achieved. Hence, in a first aspect the present invention provides a Casl2 RNP complex for use with extracellular vesicles (EVs) comprising: a. a first fusion protein comprising an EV polypeptide and a Casl2 protein; and b. a circular crRNA comprising: i. a spacer sequence ii. a direct repeat region to the 5' of the spacer sequence (5' DR region); and iii. a direct repeat region to the 3' of the spacer sequence (3' DR region); wherein the Casl2 protein is bound to either the 5' DR region or the 3' DR region. In a second aspect, the present invention provides a method of producing an EV or a population of EVs comprising the following steps: a. introducing into an EV producer cell: i. a polynucleotide encoding for a circular crRNA, wherein the circular crRNA comprises: 1. a spacer sequence; 2. a direct repeat region to the 5' of the spacer sequence (5' DR region); and 3. a direct repeat region to the 3' of the spacer sequence (3' DR region); and ii. a polynucleotide encoding for a fusion protein, wherein the fusion protein comprises an EV polypeptide and a Casl2 protein; b. expressing the polynucleotide encoding for the circular crRNA and the polynucleotide encoding for the fusion protein at the same time in the EV producer cell, preferably under conditions which, in no particular order, i. the Casl2 protein binds the 5' DR region or the 3' DR region of the circular crRNA and ii. the Casl2 protein bound to circular crRNA loads into an EV. In a third aspect, the present invention provides an EV comprising the Casl2 RNP complex of the present invention. In a further aspect, the present invention provides an EV directly obtained by the method of the present invention. In a further aspect, the present invention provides a population of EVs comprising a plurality of EVs of the present invention. In a further aspect, the present invention provides a pharmaceutical composition comprising a Casl2 RNP complex of the present invention, an EV of the present invention or a population of EVs the present invention and a pharmaceutically acceptable excipient and / or carrier. In a further aspect, the present invention provides a method of delivering a Casl2 RNP complex to a target cell, comprising contacting or incubating a target cell with an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention. In a further aspect, the present invention provides an in vitro or ex vivo method of gene editing in a target cell comprising introducing into the cell an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention. In a further aspect, the present invention provides an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention for use in a method of treatment or prevention of a disease in a subject, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder. In a further aspect, the present invention provides the use of an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for treatment or prevention of a disease in a subject, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder. In a further aspect, the present invention provides a method for treating or preventing a disease in a subject comprising administering a therapeutically or prophylactically effective amount of an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder. BRIEF DESCRIPTION OF THE DRAWINGS One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows schematics of crRNAs (A) shows a conventional linear Casl2 crRNA consisting of a 5' DR region and a spacer sequence. (B) shows a linear crRNA consisting of a 5' DR region and a spacer sequence with an additional DR region to the 3' of the spacer. (C) shows a circular crRNA comprising a 5' DR region and a spacer sequence. (D) shows a circular crRNA comprising a 5' DR region and a spacer sequence that also includes an additional DR region to the 3' DR of the spacer (i.e. a crRNA of the present invention). (E) shows a pre-cursor RNA molecule for the crRNA of (D), where the sequence encoding the circular crRNA is flanked by 5' and 3' self-cleaving ribozymes. The scissors show where the ribozyme sequences are self-cleaved. Figure 2 shows the circular crRNA comprising an additional 3' DR region (i.e. a crRNA of the present invention) significantly improves gene editing in target cells with EV mediated delivery. (A)-(C) are Bar Charts showing the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Casl2 RNP complex cargo to target cells, when different crRNA designs are expressed in EV producer cells. "SL2 crRNA" is a conventional linear Casl2 crRNA as shown in Figure 1A. "SL2 crRNA DR" is a linear crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure IB. "Cir-SL2 crRNA" is a circular crRNA as shown in Figure IC. "Cir-SL2 crRNA DR" is a circular crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure ID. In (A) the target cells are dosed with 1 x 1010 EVs, in (B) the target cells are dosed with 1 x 109 EVs and in (C) the target cells are dosed with 1 x 108 EVs. Figure 3 shows the circular crRNA comprising an additional 3' DR region (i.e. a crRNA of the present invention) significantly improves gene editing in target cells with EV mediated delivery when a different EV polypeptide is used (CD63). (A) to (C) are Bar Charts showing the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Casl2 RNP complex cargo to target cells, when different crRNA designs are expressed in EV producer cells. "SL2 crRNA" is a conventional linear Casl2 crRNA as shown in Figure 1A. "SL2 crRNA DR" is a linear crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure IB. "Cir-SL2 crRNA" is a circular crRNA as shown in Figure IC. "Cir-SL2 crRNA DR" is a circular crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure ID. In (A) the target cells are dosed with 1 x 1010 EVs, in (B) the target cells are dosed with 1 x 109 EVs and in (C) the target cells are dosed with 1 x 108 EVs. Figure 4 shows the circular crRNA comprising an additional 3' DR region (i.e. a crRNA of the present invention) significantly improves gene editing in target cells with EV mediated delivery when a VSV-G scaffold is used. (A) to (C) are Bar Charts showing the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Casl2 RNP complex cargo to target cells, when different crRNA designs are expressed in EV producer cells. "SL2 crRNA" is a conventional linear Casl2 crRNA as shown in Figure 1A. "SL2 crRNA DR" is a linear crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure IB. "Cir-SL2 crRNA" is a circular crRNA as shown in Figure IC. "Cir-SL2 crRNA DR" is a circular crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure ID. In (A) the target cells are dosed with 1 x 1010 EVs, in (B) the target cells are dosed with 1 x 109 EVs and in (C) the target cells are dosed with 1 x 108 EVs. Figure 5 shows the circular crRNA comprising an additional 3' DR region (i.e. a crRNA of the present invention) significantly improves gene editing in target cells when delivery is mediated by Nanoblades, eVLPs or EVs. (A) to (C) are Bar Charts showing the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Casl2 RNP complex cargo to target cells, when different crRNA designs are expressed in EV producer cells. "Cir-SL2 crRNA" is a circular crRNA as shown in Figure IC. "Cir-SL2 crRNA DR" is a circular crRNA that includes an additional DR region to the 3' of the spacer as shown in Figure ID. In (A) the target cells are dosed with 1 x 1010 EVs, in (B) the target cells are dosed with 1 x 109 EVs and in (C) the target cells are dosed with 1 x 108 EVs. DETAILED DESCRIPTION As outlined above, the present inventors sought to solve the problem of achieving high gene editing efficiencies when using EVs to deliver Casl2 RNP complexes to target cells. Having surprisingly discovered that loading of the guide RNA may be the limiting factor in the levels of gene editing being achieved when using EVs to deliver CRSPR / Cas systems to target cells, the present inventors sought to use modified Casl2 crRNAs that were hoped to have increased stability, in an attempt to improve gene editing. Whilst two modifications aimed at preventing exonuclease degradation (i.e. the inclusion of an additional DR to the 3' of the spacer and the crRNA being circular) did not improve gene editing when used individually, the inventors were surprised to find that when these two modifications were combined in one Casl2 crRNA, gene editing is significantly increased. Without wishing to be bound by theory, the present inventors believe this could be due to this crRNA having both improved stability and an improved ability to bind Casl2. The present inventors believe the crRNA being circular may lead to improved stability, which may allow for more crRNA to be loaded into EVs, more crRNA to be delivered to target cells and / or for the crRNA to last for longer in target cells, which is of particular importance since the Casl2 RNP complex must move from the unique subcellular localisation at which it is released from the EV to the nucleus. The present inventors also believe the crRNA being symmetric may improve binding to Casl2 during EV encapsulation at a pH of around pH 6, which may allow for more functional Casl2 RNP complexes to be delivered to target cells. The crRNA comprising more than one DR may also improve loading, as the crRNA may be more likely to be bound by a Casl2 protein in the cytoplasm of an EV producer cell and, since the Casl2 protein is fused to an EV polypeptide, be pulled into a forming EV. The crRNA being circular may also be beneficial in this context since, if the Casl2 protein binds the 3' direct repeat and cleaves it prior to encapsulation in the EV, the part of the crRNA that comprises the 5' DR structure and the spacer (which has the potential form a mature crRNA) remains connected and thus is still loaded into the EV. In the EV, the 5' DR may later be bound and cleaved by a free Casl2 protein and so result in an additional functional RNP being formed. Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers or steps. Moreover, the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. crRNAs of the Present Invention Hence the present invention provides a circular Casl2 crRNA comprising: (i) a spacer sequence (ii) a direct repeat region to the 5' of the spacer sequence (referred to hereinafter as the 5' DR region) and (iii) a direct repeat region to the 3' of the spacer sequence (referred to hereinafter as the 3' DR. region). In a preferred embodiment, the spacer links the 5' DR region and the 3' DR region. In a preferred embodiment, a Casl2 protein is bound to either the 5' DR region or the 3' DR region. In an alternative preferred embodiment, a first Casl2 protein is bound to the 5' DR region and a second Casl2 protein is bound to the 3' DR region. As used herein, the term "crRNA" or "Casl2 crRNA" encompasses any RNA molecule that comprises a spacer sequence and a direct repeat region. In one embodiment, the direct repeat region is to the 5' of the spacer sequence. The term crRNA as used herein encompasses mature crRNA, intermediate crRNA and pre-crRNA. In a preferred embodiment, the crRNA is capable of binding Casl2 to form a Casl2 RNP complex. In a preferred embodiment, the RNP complex is a functional RNP complex capable of gene editing. In an alternative embodiment, the RNP complex has the potential to be a functional RNP complex capable of gene editing following further processing of the crRNA. The term "spacer" or "spacer sequence" refers to a nucleotide sequence capable of specifically binding or hybridising to a given target DNA sequence, preferably in a genomic locus of interest in a cell, preferably a target cell as described herein. In one embodiment, the spacer sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% complementary, over its entire length, to the target DNA sequence. In one embodiment the spacer sequence has a length of about 12 to 30 nucleotides, preferably 15 to 27 nucleotides, or more preferably 18 to 25 nucleotides, most preferably 18 to 22 nucleotides or 23 to 25 nucleotides. The spacer sequence comprises a seed sequence of 4 to 12 nucleotides at its 5' end, preferably having 8 to 12 nucleotides, or most preferably 8 nucleotides. The seed sequence establishes the first contact between the crRNA and the target DNA. In one embodiment, the seed sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% complementary, over its entire length, to the target DNA sequence, preferably in a genomic locus of interest in a cell. The term "direct repeat region" or "DR region" refers to a region of the Casl2 crRNA that comprises or consists of nucleotide sequence capable of forming a secondary RNA structure that a Casl2 protein is capable of binding. In a preferred embodiment, the Casl2 protein is a Casl2a protein, more preferably a Casl2a derived from Acidaminococcus sp., Lachnospiraceae bacterium or Francisella novicida. In a particularly preferred embodiment, the Casl2a is derived from Acidaminococcus sp., most preferably a Casl2a derived from Acidaminococcus BV3L6. In an alternative preferred embodiment, the Casl2 protein is a Casl2i protein. In a preferred embodiment, the direct repeat region comprises at its 3' end, or consists of, a secondary structure, sometimes referred to as a pseudoknot or a tetraloop pseudoknot, which a Casl2 protein, preferably a Casl2a protein, more preferably a Casl2a derived from Acidaminococcus sp., Lachnospiraceae bacterium or Francisella novicida, most preferably a Casl2a derived from Acidaminococcus BV3L6, is capable of binding. In a preferred embodiment, the pseudoknot consists of 18-20 nucleotides, preferably 19 nucleotides, that are found at the 3' of the direct repeat region. In a preferred embodiment of the 5' DR. region, the nucleotides forming the pseudoknot are immediately to the 5' of the spacer sequence. The pseudoknot comprises a stem loop structure. In one embodiment, the stem of the stem-loop comprises at its base a U-A base pair, most preferably formed between the base at position -2 and the base at position -15, preferably wherein the base at position -2 is an A and the base at position -15 is a U. As used herein the base at position "-1" is the most 3' base of the direct repeat region (or preferably, in the case of the 5' DR region, the base immediately to the 5' of the spacer sequence), the base at position "-2" is the base immediately to the 5' of the base at position -1, the base at position "-3" is the base immediately to the 5' of the base at position -2 etc. In an alternative embodiment, the stem of the stem-loop comprises at its base a non-canonical U-U base pair, most preferably formed between the base at position -1 and the base at position -16 and optionally the stem-loop also comprises a U-A base pair formed between the base at position -2 and the base at position -15, preferably wherein the base at position -2 is an A and the base at position -15 is a U. In a preferred embodiment, the stem of the stem-loop comprises five base pairs, preferably Watson-Crick base-pairs, that are preferably formed between the bases at position -2 to -6 and the bases at position -15 and -11 respectively, preferably with 2 or fewer mismatches, more preferably with 1 or most preferably 0 mismatches. In one embodiment, the loop of the stem-loop structure comprises 4 bases that are not paired. In a preferred embodiment, the loop comprises or consists of a nucleotide sequence, from 5' to 3', of UGUU (SEQ ID NO: 1), preferably from the base at position -10 to the base at position -7. In one embodiment, the pseudoknot further comprises either three 5'-end bases, most preferably these are bases at positions -19 to -17, or four 5'-end bases, most preferably these are bases at positions -19 to -16. In a preferred embodiment, the most 5' nucleotide, preferably the base at position -19, is an A; the base immediately to the 3' of the most 5' base, preferably the base at position -18, is an A; and / or the third base of the 5'-end bases, preferably at position -17, is a U. Optionally, the fourth base, preferably at position -16, is a U. In one embodiment, one of the "A"s at position -19 or position -18 forms, or is capable of forming, a reverse Hoogsteen A-U base pair with the most 5' U of the loop, preferably the U at base position -10. In one embodiment, the U at position -17 is reverse paired, or capable of reverse pairing, with the base at position -14, -13 and / or -12. In a preferred embodiment, the pseudoknot comprises or consists, from 5' to 3', of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 2, over its entire length. In a preferred embodiment, the nucleotide at position 1 of SEQ ID NO: 2 is an A, the nucleotide at position 4 of SEQ ID NO: 2 is a U, the nucleotide at position 10 of SEQ ID NO: 2 is a U and the nucleotide at position 19 of SEQ ID NO: 2 is a U. In a preferred embodiment, the nucleotides at position 5 to 9 of SEQ ID NO: 2 are complementary (i.e. capable of forming base pairs, preferably Watson-Crick base pairs) to the nucleotides at position 18 to 14 of SEQ ID NO: 2 respectively, preferably with 2 or fewer mismatches, preferably with 1 or most preferably 0 mismatches. Preferably, the nucleotide at position 19 of SEQ ID NO: 2 is immediately to the 5' of the most 3' nucleotide of the spacer sequence (i.e. there are no intervening nucleotides). In one embodiment, the direct repeat region is 19 to 40 nucleotides long. In some embodiments, the direct repeat region comprises additional nucleotides to the 5' of the pseudoknot as described herein. In a preferred embodiment, the 5' direct repeat region comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 3, over its entire length. In a preferred embodiment, the 3' direct repeat region of the present invention comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 3, over its entire length. In an alternative preferred embodiment, the direct repeat region comprises at its 3' end, or consists of, a stem-loop secondary structure, which a Casl2i protein is capable of binding to. In one embodiment, the direct repeat region comprises or consists of a nucleotide sequence having, over its entire length, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 36, 37, 38, 39, 40 or 41. In a one embodiment, the crRNA of the present invention comprises an AT (or AU) rich nucleotide sequence (e.g. more than 60%, 70%, 75%, 80%, 90%, 95% or 100% of the nucleotides are A or T) to the 5' of the pseudoknot within the DR. region or to the 5' of the DR region. In one embodiment, the AT (or AU) rich nucleotide sequence is to the 5' of the pseudoknot within the 5' DR region. In one embodiment, the AT (or AU) rich nucleotide sequence is to the 5' of the pseudoknot within the 3' DR region. In one embodiment, the crRNA of the present invention comprises an AT (or AU) rich nucleotide sequence to the 5' of the pseudoknot of the 5' DR region and to the 5' of the pseudoknot within the 3' DR region. Preferably the AT (or AU) rich nucleotide sequence(s) is / are immediately to the 5' of the pseudoknot, so as the most 3' nucleotide of the AT (or AU) rich nucleotide sequence is adjacent to the most 5' nucleotide of the pseudoknot. In one embodiment, the AT (or AU) rich nucleotide sequence comprises or consists of I I I I (SEQ ID NO: 30) or UUUU (SEQ ID NO: 31), TTTA (SEQ ID NO: 32) or UUUA (SEQ ID NO: 33) or preferably AAAT (SEQ ID NO: 34) or AAAU (SEQ ID NO: 35). In a one embodiment, the crRNA of the present invention comprises a GC rich nucleotide sequence (e.g. more than 60%, 70%, 75%, 80%, 90%, 95% or 100% of the nucleotides are G or C) to the 5' of the pseudoknot within the DR region or to the 5' of the DR region. In one embodiment, the GC rich nucleotide sequence is to the 5' of the pseudoknot within the 5' DR region. In one embodiment, the GC rich nucleotide sequence is to the 5' of the pseudoknot within the 3' DR region. In one embodiment, the crRNA of the present invention comprises a GC rich nucleotide sequence to the 5' of the pseudoknot of the 5' DR region and to the 5' of the pseudoknot within the 3' DR region. Preferably the GC rich nucleotide sequence(s) is / are immediately to the 5' of the pseudoknot, so as the most 3' nucleotide of the GC rich nucleotide sequence is adjacent to the most 5' nucleotide of the pseudoknot. In a preferred embodiment, the 5' DR region of the crRNA of the present invention is immediately to the 5' of the spacer sequence i.e. the most 3' nucleotide of the 5' DR region is adjacent to the most 5' nucleotide of the spacer sequence. In a preferred embodiment, the 3' DR region of the crRNA of the present invention is immediately to the 3' of the spacer sequence i.e. the most 5' nucleotide of the 3' DR region is adjacent to the most 3' nucleotide of the spacer sequence. The term "circular" when used in relation to a crRNA, refers to a crRNA that comprises a nucleotide sequence (referred to hereinafter as the "3'DR to 5'DR connecting linker") that links the 3' of crRNA and the 5' of the crRNA, thus the crRNA is a closed loop rather than having a typical linear form. Preferably, the 3'DR to 5'DR connecting linker is immediately to the 3' end of the 3' DR region so as the most 3' nucleotide of the 3' DR repeat region is adjacent to the most 5' nucleotide of the 3'DR to 5'DR connecting linker. Further preferably, the 3'DR to 5'DR connecting linker is connected immediately to the 5' end of the 5' DR region, so as the most 5' nucleotide of the 5' DR repeat region is adjacent to the most 3' nucleotide of the 3'DR to 5'DR connecting linker. In one embodiment, the 3'DR to 5'DR connecting linker comprises a stem loop. In a preferred embodiment, the stem of the stem-loop consists of 9 to 29 base pairs, preferably 14 to 25 base pairs, more preferably 17 to 21 base pairs or most preferably 19 base pairs. Preferably the base pairs are Watson-Crick base pairs. In one embodiment, the stem may comprise one, two, three or more mismatched pairs, preferably wherein the mismatched pairs are not adjacent to one another in the stem. In a preferred embodiment, the stem comprises 19 base pairs with one mismatched pair. In one embodiment, the loop of the stem-loop comprises 4 to 14 bases, preferably 6 to 12 bases, more preferably 8 to 10 bases most preferably 9 bases, which do not form base pairs. In a preferred embodiment, the stem-loop of the 3'DR to 5'DR connecting linker of the present invention comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 5 over its entire length. The 3'DR to 5'DR connecting linker may or may not further comprise additional nucleotides to the 3' and / or 5' of the stem-loop sequence. Hence, in a preferred embodiment, the circular Casl2 crRNA of the present invention comprises or consists of: (i) a 5' DR region as described herein; (ii) a spacer sequence as described herein; (iii) a 3' DR region as described herein; and (iv) a 3'DR to 5'DR connecting linker as described herein. In a preferred embodiment, the spacer links the 5' DR region and the 3' DR region. In a preferred embodiment, a Casl2 protein is bound to either the 5' DR region or the 3' DR region. In one embodiment, a first Casl2 protein is bound to the 5' DR region and a second Casl2 protein is bound to the 3' DR region. In one embodiment, the circular Casl2 crRNA of the present invention is symmetrical or substantially symmetrical. In one embodiment, the circular Casl2 crRNA of the present invention is symmetrical or substantially symmetrical in terms of its RNA secondary structures. In a particularly preferred embodiment, the circular Casl2 crRNA of the present invention comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 43 over its entire length. "N" is SEQ ID NO: 42 or SEQ ID NO: 43 may be any nucleotide, preferably any of A, U, G or C. "N[i2-30]" in SEQ ID NO: 42 denotes any nucleotide sequence of 12 to 30 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 12 to 30 nucleotides in length. "N[i8-25]" in SEQ ID NO: 43 denotes any nucleotide sequence of 18 to 25 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 18 to 25 nucleotides in length. Since SEQ ID NO: 42 and SEQ ID NO: 43 are circular Casl2 crRNAs, the most 3' nucleotide is adjacent to the most 5' nucleotide of these sequences. In one embodiment, the circular Casl2 crRNA of the present invention comprises two, three, four, five or more than five spacer sequences. Preferably, the spacer sequences are each capable of specifically binding or hybridising to different and / or distinct, target DNA sequences. In a preferred embodiment, each spacer sequence is separated from the next spacer sequence by a DR region, as described herein, and the circular Casl2 crRNA comprises a DR region to the 5' of the first spacer and to the 3' of the last spacer. Thus, in one, embodiment, the circular Casl2 crRNA of the present invention comprises or consists of: (i) a 5' (first) DR region as described herein; (ii) a first spacer sequence; (iii) a second DR region as described herein; (iv) a second spacer sequence; (v) optionally a third DR region as described herein; (vi) optionally a third spacer sequence (vii) optionally a fourth DR region as described herein; (viii) optionally a fourth spacer sequence (ix) optionally a fifth DR region as described herein (x) optionally a fifth spacer sequence; (xi) a 3' DR region as described herein; and (xii) a 3'DR to 5'DR connecting linker as described herein. Some of the DR regions, or preferably all of the DR regions, may comprise or consist of the same nucleotide sequence. In one embodiment, the circular Casl2 crRNA of the present invention is formed from a linear RNA molecule comprising 5' and 3' self-cleaving ribozyme sequences. Hence the present invention further provides a linear pre-cursor crRNA molecule (hereinafter referred to as a "pre-cursor crRNA molecule") that comprises or consists, preferably from 5' to 3': (i) a 5' self-cleaving ribozyme sequence; (ii) a 5' component of the 3'DR to 5'DR connecting linker as described herein; (iii) a polynucleotide sequence encoding a 5' DR region as described herein; (iv) a spacer sequence as described herein; (v) a polynucleotide sequence encoding a 3' DR region as described herein; (vi) a 3' component of the 3'DR to 5'DR connecting linker as described herein; and (vii) a 3' self-cleaving ribozyme sequence. In a preferred embodiment, the spacer links the 5' DR region and the 3' DR region. As used herein, the term "crRNA of the present invention" encompasses the pre-cursor crRNA molecule of the present invention. As used herein, the term "self-cleaving ribozyme sequence" or "ribozyme sequence" refers to an RNA molecule that can catalyse the cleavage of its own phosphodiester bonds in an autonomous manner i.e. without the need for other proteins or enzymes. In one embodiment, the 5' self-cleaving ribozyme sequence and 3' self-cleaving ribozyme sequence are capable of self-cleaving in a manner such that an RNA ligase, preferably RNA ligase RtcB, is capable of ligating the self-cleaved 5' end of the pre-cursor crRNA molecule of the present invention and the self-cleaved 3' end of the of the pre-cursor crRNA molecule of the present invention when they are brought into close proximity. In a preferred embodiment, the ribozyme sequences are "Twister" ribozymes, preferably as described in Roth A et al. Roth A et al. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat. Chern. Biol 10, 56-60 (2014). In a more preferred embodiment, the 5' self-cleaving ribozyme sequence of the present invention comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 7 over its entire length and / or the 3' self-cleaving ribozyme sequence of the present invention comprise or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 8 over its entire length. The 5' component of a 3'DR to 5'DR connecting linker and the 3' component of the 3'DR to 5'DR connecting linker are capable, preferably following self-cleavage of 5' and 3' ribozyme sequence, of forming a double stranded RNA duplex structure through complementary base pairing. In a preferred embodiment, the double stranded RNA duplex structure does not include the nucleotides at the self-cleaved ends or preferably does not include the nucleotides up to 2 to 15 nucleotides from the self-cleaved ends, preferably the nucleotides up to 2 to 10 nucleotides from the self-cleaved ends, more preferably nucleotides up to 2 to 7 nucleotides from the self-cleaved ends. In a particularly preferred embodiment, the 5' component of a 3'DR to 5'DR connecting linker comprises 1-5, preferably 2-4, more preferably 2-3 or most preferably 2 nucleotides at its self-cleaved 5' end that are not comprised in the RNA duplex. In a particularly preferred embodiment, the 3' component of a 3'DR to 5'DR connecting linker comprises 1-15, preferably 2-10, more preferably 5-9 or most preferably 7 nucleotides at its self-cleaved 3' end that are not comprised in the RNA duplex. In one embodiment, the double stranded RNA duplex structure comprises 9 to 29 base pairs, preferably 14 to 25 base pairs, more preferably 17 to 21 base pairs or most preferably 19 base pairs. Preferably the base pairs are Watson-Crick base pairs. The double stranded RNA duplex structure may comprise one, two, three or more mismatched pairs, preferably wherein the mismatched pairs are not adjacent to one another in the stem. In a preferred embodiment, the RNA duplex structure 19 base pairs with one mismatched pair. In one embodiment, the 5' component of the 3'DR to 5'DR connecting linker comprises 11 to 31 nucleotides, preferably 16 to 25 nucleotides, more preferably 19 to 23 nucleotides or most preferably 21 nucleotides. Preferably, the 5' component of the 3'DR to 5'DR connecting linker comprises a contiguous nucleotide sequence consisting of 9 to 29 bases, preferably 14 to 25 bases, more preferably 17 to 21 bases or most preferably 19 bases that are complementary (with preferably one mismatch) to a contiguous nucleotide sequence present in the 3' component of the 3'DR to 5'DR connecting linker. In one embodiment, the 5' component of the 3'DR to 5'DR connecting linker comprises 1 or more, preferably 1 to 10, more preferably 1 to 5 or most preferably 2 additional nucleotides at its 5' end that are not capable of forming part of the double stranded RNA duplex structure. In a most preferred embodiment, the 5' component of the 3'DR to 5'DR connecting linker has nucleic acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 9 over its entire length. In one embodiment, the 3' component of the 3'DR to 5'DR connecting linker comprises 16 to 36 nucleotides, preferably 21 to 30 nucleotides, more preferably 24 to 28 nucleotides or most preferably 26 nucleotides. Preferably, the 3' component of the 3'DR. to 5'DR connecting linker comprises a contiguous nucleotide sequence consisting of 9 to 29 bases, preferably 14 to 25 bases, more preferably 17 to 21 bases or most preferably 19 bases that are complementary (with preferably one mismatch) to a contiguous nucleotide sequence present in the 5' component of the 3'DR to 5'DR connecting linker. In one embodiment, the 3' component of the 3'DR to 5'DR connecting linker comprises 1 or more, preferably 2 to 15, more preferably 5 to 9 or most preferably 7 additional nucleotides at its 3' end that are not capable of forming part of the double stranded RNA duplex structure. In a most preferred embodiment, the 3' component of the 3'DR to 5'DR connecting linker has nucleic acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 10 over its entire length. In a particularly preferred embodiment, the pre-cursor crRNA molecule of the present invention comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45 over its entire length. "N" is SEQ ID NO: 44 or SEQ ID NO: 45 may be any nucleotide, preferably any of A, U, G or C. "N[i2-30]" in SEQ ID NO: 44 denotes any nucleotide sequence of 12 to 30 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 12 to 30 nucleotides in length. "N[i8-25]" in SEQ ID NO: 45 denotes any nucleotide sequence of 18 to 25 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 18 to 25 nucleotides in length. In one embodiment, the pre-cursor crRNA molecule of the present invention comprises two, three, four, five or more than five spacer sequences. Preferably, the spacer sequences are each capable of specifically binding or hybridising to different and / or distinct, target DNA sequences. In a preferred embodiment, each spacer sequence is separated by a DR region as described herein and the pre-cursor crRNA molecule comprises a DR region to the 5' of the first spacer (i.e. the most 5' spacer) and to the 3' of the last spacer (i.e. the most 3' spacer). Thus, in one, embodiment, the circular Casl2 crRNA of the present invention comprises or consists of, from 3' to 5': (i) a 5' self-cleaving ribozyme sequence; (ii) a 5' component of the 3'DR to 5'DR connecting linker as described herein; (iii) a polynucleotide sequence encoding a 5' (first) DR region as described herein; (iv) a first spacer sequence; (v) a polynucleotide sequence encoding a second DR region as described herein; (vi) a second spacer sequence; (vii) optionally a polynucleotide sequence encoding a (third) DR region as described herein; (viii) optionally a third spacer sequence (ix) optionally a polynucleotide sequence encoding a (fourth) DR region as described herein; (x) optionally a fourth spacer sequence (xi) optionally a polynucleotide sequence encoding a (fifth) DR region as described herein (xii) optionally a fifth spacer sequence; (xiii) a polynucleotide sequence encoding a 3' DR region as described herein; (xiv) a 3' component of the 3'DR to 5'DR connecting linker as described herein; and (xv) a 3' self-cleaving ribozyme sequence Some of the DR regions or all of the DR regions may comprise or consist of the same nucleotide sequence. The present invention further provides the circular Casl2 crRNA of the present invention wherein the circular Casl2 crRNA has been cleaved by a Casl2 protein. In one embodiment, the circular Casl2 crRNA of the present invention has been cleaved by a Casl2 protein in the 5' DR region, preferably to the 5' of the pseudoknot, to produce a linearised crRNA (hereinafter referred to as the "5' DR cleaved linearised crRNA"). In one embodiment, the 5' DR cleaved linearised crRNA comprises or consists, from 5' to 3', of: (i) a Casl2 cleaved 5' DR region; (ii) a spacer sequence as described herein; (iii) a 3' DR region as described herein; and (iv) and a linker, optionally including at its 3' nucleotides of the 5' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 5' DR region. In a preferred embodiment, the spacer links the Casl2 cleaved 5' DR region and the 3' DR region. In a preferred embodiment, a Casl2 protein is, or remains, bound to the 5' DR cleaved linearised crRNA at the cleaved 5' DR region. In one embodiment, a second Casl2 protein is bound to the 3' DR region. As used herein, the term "Casl2 cleaved 5' DR. region" refers to a 5' DR region as described herein that has been cleaved by a Casl2 protein, preferably Casl2a protein or a Casl2i protein. In one embodiment, Casl2 cleaves the 5' DR region 4 nucleotides upstream (or to the 5') of the stem-loop. Thus, the Casl2 cleaved 5' DR region comprises of a pseudoknot structure at its 5' as described herein. In a preferred embodiment, the Casl2 cleaved 5' DR region of the 5' DR cleaved linearised crRNA of the present invention is immediately to the 5' of the spacer sequence i.e. the most 3' nucleotide of the 5' DR region is adjacent to the most 5' nucleotide of the spacer sequence. As such, in a preferred embodiment, the Casl2 cleaved 5' DR region consists of a pseudoknot structure as described herein. In a preferred embodiment of the 5' DR cleaved linearised crRNA of the present invention, the spacer sequence is immediately to the 5' of the 3' DR region i.e. the most 3' nucleotide of the spacer sequence is adjacent to the most 5' nucleotide of the 3' DR region. The linker of the 5' DR cleaved linearised crRNA of the present invention comprises or consists of the 3'DR to 5'DR connecting linker as described herein or, in a preferred embodiment, comprises or consists of the connecting linker as described herein connected to any nucleotides present in the 5' DR region that are upstream of the Casl2 cleavage site. In a preferred embodiment of the 5' DR cleaved linearised crRNA of the present invention, the 3' DR region is immediately to the 5' of the linker i.e. the most 3' nucleotide of the 3' DR region is adjacent to the most 5' nucleotide of the linker. In a particularly preferred embodiment, the 5' DR cleaved linearised crRNA of the present invention comprises of consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 46 or SEQ ID NO: 47 over its entire length. "N" is SEQ ID NO: 46 or SEQ ID NO: 47 may be any nucleotide, preferably any of A, U, G or C. "N[i2-30]" in SEQ ID NO: 46 denotes any nucleotide sequence of 12 to 30 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 12 to 30 nucleotides in length. "N[i8-25]" in SEQ ID NO: 47 denotes any nucleotide sequence of 18 to 25 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 18 to 25 nucleotides in length. In one embodiment, the circular Casl2 crRNA of the present invention has been cleaved by a Casl2 protein in the 3' DR region, preferably to the 5' of the pseudoknot, to produce a linearised crRNA (hereinafter referred to as the "3' DR cleaved linearised crRNA"). In one embodiment, the 3' DR cleaved linearised crRNA comprises or consists, from 5' to 3', of: (i) a Casl2 cleaved 3' DR region; (ii) a linker; (iii) a 5' DR. region as described herein; (iv) a spacer sequence as described herein; and (v) optionally, nucleotides of the 3' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 3' DR region. In a preferred embodiment, a Casl2 protein is, or remains, bound to the 3' DR cleaved linearised crRNA at the cleaved 3' DR region. In one embodiment, a second Casl2 protein is bound to the 5' DR region. As used herein, the term "Casl2 cleaved 3' DR region" refers to a 3' DR region as described herein that has been cleaved by a Casl2 protein, preferably a Casl2a protein or a Casl2i protein. In one embodiment, Casl2 cleaves the 3' DR region 4 nucleotides upstream of the stem-loop. Thus, the Casl2 cleaved 3' DR region comprises a pseudoknot structure at its 5' as described herein. In a preferred embodiment, the Casl2 cleaved 3' DR region of the 3' DR cleaved linearised crRNA of the present invention is immediately to the 5' of the linker i.e. the most 3' nucleotide of the 3' DR region is adjacent to the most 5' nucleotide of the linker. As such, in a preferred embodiment, the Casl2 cleaved 3' DR region consists of a pseudoknot structure as described herein. The linker of the 3' DR cleaved linearised crRNA of the present invention comprises or consists of the 3'DR to 5'DR connecting linker as described herein. In a preferred embodiment of the 3' DR cleaved linearised crRNA of the present invention, the linker is immediately to the 5' of the 5' DR region i.e. the most 3' nucleotide of the linker is adjacent to the most 5' nucleotide of the 5' DR region. In a preferred embodiment of the 3' DR cleaved linearised crRNA of the present invention, the 5' DR region is immediately to the 5' of the spacer sequence i.e. the most 3' nucleotide of the 5' DR region is adjacent to the most 5' nucleotide of the spacer sequence. In one embodiment, the 3' DR cleaved linearised crRNA of the present invention may include any nucleotides present in the 3' DR region that are upstream of the Casl2 cleavage site connected to the 3' of the spacer sequence. In a particularly preferred embodiment, the 3' DR cleaved linearised crRNA of the present invention comprises of consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 48 or SEQ ID NO: 49 over its entire length. "N" is SEQ ID NO: 48 or SEQ ID NO: 49 may be any nucleotide, preferably any of A, U, G or C. "N[i2-30]" in SEQ ID NO: 48 denotes any nucleotide sequence of 12 to 30 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 12 to 30 nucleotides in length. "N[i8-25]" in SEQ ID NO: 49 denotes any nucleotide sequence of 18 to 25 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 18 to 25 nucleotides in length. In one embodiment, the circular Casl2 crRNA of the present invention has been cleaved by a first Casl2 protein in the 5' DR region, preferably to the 5' of the pseudoknot, and by a second Casl2 protein in the 3' DR region, preferably to the 5' of the pseudoknot, to produce a crRNA (hereinafter referred to as the "5' DR and 3' DR cleaved crRNA") and a linearised RNA molecule (herein after referred to as the "5' DR and 3' DR cleaved linear RNA"). In one embodiment, the 5' DR and 3' DR cleaved crRNA comprises or consists, from 5' to 3', of: (i) a Casl2 cleaved 5' DR region as described herein; (ii) a spacer sequence as described herein; and (iii) optionally, nucleotides of the 3' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 3' DR region. In a preferred embodiment, a Casl2 protein is, or remains, bound to the 5' DR and 3' DR cleaved crRNA at the cleaved 5' DR region. In a further preferred embodiment, the nucleotides of the 3' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 3' DR region consists of a single Thymine. In a preferred embodiment of the 5' DR and 3' DR cleaved crRNA of the present invention, the Casl2 cleaved 5' DR region is immediately to the 5' of the spacer sequence i.e. the most 3' nucleotide of the 5' DR region is adjacent to the most 5' nucleotide of the spacer sequence. As such, in a preferred embodiment, the Casl2 cleaved 5' DR region consists of a pseudoknot structure as described herein. In a particularly preferred embodiment, the 5' DR and 3' DR cleaved crRNA of the present invention comprises of consists of a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% sequence identity to SEQ ID NO: 50 or SEQ ID NO: 51 over its entire length. "N" is SEQ ID NO: 50 or SEQ ID NO: 51 may be any nucleotide, preferably any of A, U, G or C. "N[i2-30]" in SEQ ID NO: 50 denotes any nucleotide sequence of 12 to 30 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 12 to 30 nucleotides in length. "N[i8. 25]" in SEQ ID NO: 51 denotes any nucleotide sequence of 18 to 25 nucleotides in length, preferably the nucleotide sequence is a spacer sequence as described herein of 18 to 25 nucleotides in length. In one embodiment wherein the 5' DR. and 3' DR cleaved crRNA comprises nucleotides of the 3' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 3' DR region the 5' DR and 3' DR cleaved crRNA is further processed so as it consists, from 5' to 3', of: (i) a Casl2 cleaved 5' DR region as described herein; and (ii) a spacer sequence as described herein. In a preferred embodiment, a Casl2 protein is, or remains, bound to the 5' DR and 3' DR cleaved crRNA at the cleaved 5' DR region. In one embodiment, the 5' DR and 3' DR cleaved linear RNA is a linearised RNA molecule comprising or consisting, from 5' to 3', of: (i) a Casl2 cleaved 3' DR region as described herein; and (ii) a linker as described herein, optionally including at its 3' nucleotides of the 5' DR region that are upstream (or to the 5') of where the Casl2 protein cleaves the 5' DR region. In a preferred embodiment, a Casl2 protein is, or remains, bound to the linearised RNA molecule at the cleaved 3' DR region. In one embodiment, the present invention provides a Casl2 RNP complex, comprising a Casl2 protein as described herein and a crRNA of the present invention. In one embodiment, the Casl2 RNP complex is a functional Casl2 RNP complex that is capable of gene editing in a target cell as described herein. In an alternative embodiment, the Casl2 RNP complex has the potential to be a functional RNP complex capable of gene editing in a target cell as described herein following further processing of the crRNA. In any of the embodiments described herein, the Casl2 may be Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2f, Casl2g, Casl2h, Casl2i or Casl2j. In a preferred embodiment, the Casl2 is Casl2a, Casl2c, Casl2i, Casl2j, more preferably Casl2a, Casl2i, Casl2j, or most preferably Casl2a. In a preferred embodiment, the Casl2 has RNA processing activity. In one embodiment, the Casl2 comprises a functional WED domain (also referred to as a Wedge domain). In a preferred embodiment, the Casl2 has DNA cleavage activity. In one embodiment, the Casl2 comprises a functional RuvC domain. In a preferred embodiment, the Casl2 is capable of binding to an RNA secondary structure. In a preferred embodiment, the Casl2 is capable of binding to a pseudoknot RNA structure, preferably the pseudoknot as described herein. In one embodiment, the Casl2 comprises a functional WED domain, a functional RuvC domain and a functional BH domain. Where the Casl2 is Casl2a, the Casl2a is preferably derived from Acidaminococcus sp., Lachnospiraceae bacterium or Francisella novicida, more preferably Acidaminococcus BV3L6. In a most preferred embodiment, the Casl2a comprises or consists of an amino acid sequence having sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identity to SEQ ID NO: 11, over its entire length. The present invention further provides a polynucleotide comprising or consisting of a sequence encoding for a crRNA of the present invention or preferably the pre-cursor crRNA molecule of the present invention. In one embodiment the polynucleotide is a DNA molecule. In a further aspect, the present invention provides an expression vector comprising a polynucleotide, encoding for a crRNA of the present invention, preferably the pre-cursor crRNA molecule of the present invention. In one embodiment, the crRNA of the present invention, preferably the pre-cursor crRNA molecule of the present invention, is under the control of a constitutive promoter. In a preferred embodiment, the crRNA of the present invention, preferably the pre-cursor crRNA molecule of the present invention, is under the control of a polymerase II promoter or a human U6 promoter. Cas 12 RNP complexes of the Present Invention In a preferred embodiment of any of the embodiments described herein, the Casl2 is fused to an EV polypeptide, preferably via an intein as described herein. In a preferred embodiment, the intein is capable of self-cleavage (preferably C-terminal cleavage) to release the Casl2 (and the crRNA of the present invention bound to it) from the EV polypeptide. As used herein, the term "fusion" or "fused" includes where one polypeptide is fused immediately to or into another polypeptide with no intervening amino acid residues and also includes where one polypeptide is fused to or into another polypeptide wherein further amino acid residues are present between the two polypeptide sequences, for instance the two polypeptide sequences may be fused together via an amino acid linker as described herein. As used herein, the amino acid linker may allow flexibility and enable optimal display of the Casl2 protein and, if present, the one or more additional proteins of interest described herein. Linkers also improve pharmacokinetics (PK), increase expression and improve the biological activity of the fusion polypeptides, and also to the corresponding polynucleotide constructs, and may also be used to ensure avoidance of steric hindrance and maintained functionality of the fusion polypeptides. In one embodiment, the fusion protein comprises a glycine or serine linkers which increase stability or flexibility such SEQ ID NO: 12 to 16, rigid linkers such as SEQ ID NO: 17 to 20, bending linkers (XP)n or cleavable linkers such as disulphide, protease sensitive sequences. As used herein the term "EV polypeptide" refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to an EV that is produced by a given EV producer cell as described herein or that when expressed in an EV producer cell as described herein localises or can be made to localise to an EV. In a preferred embodiment, the EV polypeptide is capable of transporting a protein to which it is fused to an EV. Preferably, the protein to which the EV polypeptide is fused is Casl2, most preferably Casl2 bound to a crRNA of the present invention. In a preferred embodiment, the EV polypeptide is of eukaryotic origin, preferably mammalian origin, more preferably human origin. In a preferred embodiment, the EV polypeptide is a microvesicle polypeptide or more preferably an exosomal polypeptide. The term "microvesicle polypeptide" as used herein refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to a microvesicle that is produced by a given EV producer cell. In a preferred embodiment, the microvesicle polypeptide is capable of transporting a fusion protein into a microvesicle produced by a given EV producer cell as described herein. The term "exosomal polypeptide" as used herein refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to an exosome that is produced by a given EV producer cell. In a preferred embodiment, the exosomal polypeptide is capable of transporting a fusion protein into an exosome produced by a given EV producer cell as described herein. In a preferred embodiment, the EV polypeptide is a transmembrane EV polypeptide. In a more preferred embodiment, the EV polypeptide is a single pass transmembrane protein or a multi-pass transmembrane protein, most preferably a tetraspanin. Single pass transmembrane proteins may be advantageous, since they allow for surface and / or luminal loading to be achieved relatively easily, through engineering of the terminus that is localised to the EV surface and / or engineering of the terminus that is localised to the EV lumen. Multi-pass transmembrane proteins, such as tetraspanins, may be advantageous, since they 23 allow for further opportunities to engineering i.e. at both their termini, but also in their loops. In one embodiment the EV polypeptide is selected from the group consisting of the following non-limiting examples: CD9, CD53, CD63, CD81, CD54, CDSO, FLOT1 , FLOT2, CD49d, CD71, CD133, CD138, CD235a, AAAT, AT1B3, AT2B4, ALIX, Annexin, BASI, BASP1 , BSG, Syntenin-1 , Syntenin-2, TSP2, TSP3, Lamp2, Lamp2a, Lamp2b, TSN1 , TSN2, TSN3, TSN4, TSNS, TSN5, TSN6, TSN7, TSN8, TSN31, TSN10, TSN11 , TSN12, TSN13, TSN14, TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN4, TSN9, TSN32, TSN33, TNFR, TfRl, syndecan-1 syndecan-2, syndecan-3, syndecan-4, CD37, CD82, CD151 , CD224, CD231, CD102, NOTCH1 , NOTCH2, NOTCH3, NOTCH4, DLL1 , DLL4, JAG1 , JAG2, CD49d / ITGA4, ITGBS, ITGB6, ITGB7, CDlla, CDllb, CDllc, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, CLIC1, CLIC4, interleukin receptors, CO2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD53, CD86, CD110, CD111 , CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD 274, CD362, COL6A1 , AGRN, EGFR, FPRP, GAPDH, GLUR2, GLUR3, GP130, GPI anchor proteins, GTR1, HLAA, HLA-DM, HSPG2, ITA3, Lactadherin, L 1CAM, LAMB1, LAMC1, LIMP2, MYOF, ARRDC1 , ATP282, ATP283, ATP284, BSG, IGSF2, IGSF3, IGSF8, ITGB1 , ITGA4, ATP1A2, ATP1A3, ATP1A4, ITGA4, SLC3A2, ATP transporters, ATP1A1, ATP183, ATP281, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, a member of the myristoylated alanine rich Protein Kinase C substrate (MARCKS) protein family such as MARCKSL 1, matrix metalloproteinase-14 (MMP14), PTGFRN, BASP1, MARCKS, MARCKSL 1, PRPH2, ROM1 , SLIT2, SLC3A2, SSEA4, STX3, TCRA, TCRB, TCRD, TCRG, TFR1 , UPK1A, UPK1B, VTI1A, VTI1B, PTTG1-IP, VSVG, gag proteins such as HIV gag proteins or preferably a Murine Leukemia Virus (MLV) Gag protein or most preferably a Moloney Murine Leukaemia virus (MMLV) Gag protein, a myristoylation site preferably having the sequence is Gly-X-X-X-Ser / Thr where "X" represents any amino acid and any other EV polypeptide, and any combinations, derivatives, domains, variants, mutants, or regions thereof. Mutations, truncations, linkers or additions may be introduced into the wildtype sequence of the EV polypeptide to alter its function, for instance a preferred mutant according to the invention is a mutation of the tetraspanin CD63 which replaces the tyrosine in position 235 with alanine (denoted CD63 / Y235A). In a preferred embodiment, the EV polypeptide is selected from TSN2, TSN3, TSN4, TSN9, PTGFRN, PTTG1-IP, CD63, Lamp2b, CD81, Syntenin-1, Syntenin-2, Lamp2, Lamp2a, BASP1, MARCKS or a gag protein, preferably an MLV Gag protein or more preferably an MMLV Gag protein. In a more preferred embodiment, the EV polypeptide is a tetraspanin, preferably CD63, TSN4, TSN9 or most preferably TSN2. As used herein "TSN2", otherwise referred to as "TSPAN2", shall be understood to mean "tetraspanin-2", as well as derivatives, domains, variants, mutants, or regions thereof. The TSN2 protein, is a multi-pass transmembrane protein with four transmembrane regions. In a preferred embodiment, TSN2 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21. In a further preferred embodiment, TSN2 comprises or consists of an amino acid sequence encoded for by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22. As used herein "TSN4", otherwise referred to as "TSPAN4", shall be understood to mean "tetraspanin-4", as well as derivatives, domains, variants, mutants, or regions thereof. The TSN4 protein, is a multi-pass transmembrane protein with four transmembrane regions. As used herein "TSN9", otherwise referred to as "TSPAN9", shall be understood to mean "tetraspanin-9", as well as derivatives, domains, variants, mutants, or regions thereof. The TSN9 protein, is a multi-pass transmembrane protein with four transmembrane regions. In one embodiment, the Casl2 is fused to a Gag protein. In one embodiment, the Gag protein is a Murine Leukemia Virus (MLV) Gag protein, most preferably a Moloney Murine Leukaemia virus (MMLV) Gag protein. In a further preferred embodiment, the Casl2 protein is fused to the C-terminus of the Gag protein, or the C-terminus of the nuclear export signal(s), via an MMLV protease-cleavable linker. In a further preferred embodiment, the Casl2 protein is fused to the C-terminus of the Gag protein. In a further preferred embodiment, the Casl2 protein is fused to the C-terminus the Gag protein (preferably an MMLV Gag protein) via 1, 2 or preferably 3 nuclear export signals The present invention further provides a Casl2 RNP complex comprising a fusion protein and a crRNA of the present invention, wherein the fusion protein comprises: (i) an EV polypeptide as described herein and (ii) a Casl2 protein as described herein; wherein the Casl2 protein is bound to: a. the 5' DR region or the 3' DR region of a circular Casl2 crRNA of the present invention; b. the cleaved 5' DR region of a 5' DR cleaved linearised crRNA of the present invention; c. the cleaved 3' DR region of a 3' DR cleaved linearised crRNA of the present invention; or d. the cleaved 5' DR region of the 5' DR and 3' DR cleaved crRNA of the present invention. In a further preferred embodiment, the Casl2 is fused to a domain of the EV polypeptide that is displayed in the lumen of the EV. In the context of the present disclosure luminal loading of the cargo may have the advantage of shielding the Casl2 and crRNA cargo from an external environment and thus reduce degradation and improved stability of the cargo allowing for more efficient delivery of the cargo to target cells. In one embodiment, the Casl2 is fused to the C-terminus of a single-pass EV polypeptide. In a preferred embodiment, the Casl2 of the present invention is fused to the N-terminus or preferably the C-terminus of a tetraspanin EV polypeptide, such as CD63, TSN4, TSN9 or preferably TSN2. In a preferred embodiment of the present invention, the EV polypeptide is fused to the Casl2 protein via an intein. As used herein, the term intein refers to an in-frame intervening sequence in a protein as described by Perler (Perler, Davis et al. 1994). The term intein, as used herein, encompasses modified or mutated inteins and mini-inteins. The term intein, as used herein, includes split inteins and contiguous inteins. In a preferred embodiment, the intein is a contiguous intein. The term intein, as used herein, includes inteins that have splicing activity and inteins that have reduced, limited or no splicing activity. Such inteins having limited or preferably no splicing activity are referred to herein as "cleaving inteins". Cleaving inteins are capable of mediating the separation of a polypeptide in which they are comprised and releasing the C-terminal extein and / or the N-terminal extein. Cleaving inteins are particularly advantageous in the context of engineered EVs, as they allow for loaded cargoes to have improved bioactivity, as they are be released and can move to the subcellular location that is optimal fortheir activity. In a preferred embodiment, the intein of the present disclosure is a cleaving intein as defined herein. In a more preferred embodiment, the intein of the present invention has, or is capable of, C-terminal cleavage activity. In a most preferred embodiment, the intein of the present invention has, or is capable of, C-terminal cleavage activity, but has reduced, or preferably no, N-terminal cleavage and extein ligation activity. In any embodiment of the present invention, preferably the intein is derived from Mycobacterium tuberculosis recA. In a more preferred embodiment, the intein is the AI-CM mini-intein preferably having an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 23. In a most preferred embodiment, the intein has an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 24 or SEQ ID NO: 25. In one embodiment, the EV polypeptide of the present invention is further fused to, or the fusion protein of the Casl2 RNP complex of the present invention further comprises, one or more additional proteins of interest (POI). In one embodiment, the additional protein of interest is a targeting moiety, an Fc-binding protein, purification moity or a pharmacokinetic and / or pharmacodynamic effector moiety, most preferably a targeting moiety. The additional protein of interest may or may not be fused to the EV polypeptide via an intein as described herein. As used herein, the term "targeting moiety" refers to molecule associated with the EV that enables targeted delivery of the EV to a cell, tissue, organ, and / or compartment of interest. The targeting moiety of the present invention may be obtainable from either humans or from non-human animals. The targeting moiety may be capable of binding to a moiety present of the target cell or of a cell type present in the target tissue or organ. In a preferred embodiment, the targeting moiety targets a cell of the liver, the heart, the brain, the CNS or muscle, a neuronal cell, a hepatocyte, a card io myocyte, a cardiac smooth muscle cell, a sensory neuron, a motor neuron, an interneuron or a glia cells. Targeting can be achieved by a variety of means, for instance the use of targeting peptides. Such targeting peptides may be anywhere from a few amino acids in length to several 100s of amino acids in length, e.g. anywhere in the interval of 3-100 amino acids, 3-30 amino acids, 5-25 amino acids, e.g. 7 amino acids, 12 amino acids, 20 amino acids, etc. Targeting peptides of the present invention may also include full length proteins such as receptors, receptor ligands, etc. In one embodiment, the targeting moiety is a protein, peptide, an antibody, a VHH, a nanobody or any other derivatives of an antibody including monoclonal antibodies, single chain variable fragments (scFvs), nanobodies and other antibody domains. In one embodiment, the targeting moiety is rabies virus glycoprotein (RVG), nerve growth factor (NGF), melanotransferrin, the FC5 Peptide or Muscle Specific Peptide (MSP). As used herein, the term "Fc-binding protein" or"Fc binding polypeptide" refers to any protein, polypeptide, or sequence of amino acids which can bind or that has high affinity for an Fc domain, preferably wherein the Fc domain is comprised in a protein, most preferably wherein the protein is an additional POI as described herein. In one embodiment, the Fc polypeptide is Protein A, Protein G, Protein A / G, Z domain, ZZ domain, human FCGRI, human FCGRIIA, human FCGRIIB, human FCGRIIC, human FCGRIIIA, human, human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse, mouse FCGRn, and various combinations, derivatives, or alternatives thereof. In one embodiment, the Fc-binding protein is bound to an Fc containing protein. In a preferred embodiment, the Fc-containing protein is, or is fused to, an additional POI as described herein. As used herein, the term "Fc-containing protein" refers to any protein, polypeptide, or sequence of amino acids which comprises an Fc domain, either naturally or as a result of engineering of the protein to introduce an Fc domain. Fc stands for "fragment crystallizable" or "fragment constant", which is the name of the tail regions of antibodies. Suitable Fc domains that may be fused with a protein that natively lacking an Fc domain include the following non-limiting examples: human IGHM, human IGHA1, human IGHA2, human IGKC, human IGHG1, human IGHG2, human IGHG3, human IGHG4, human IGHD, human IGHE. In one embodiment, the Fc domain-containing protein is an antibody, an antibody derivative, a receptor such as an interleukin receptor or a ligand. Fc-binding proteins and Fc-containing protein are further described in WO 2018 / 015535 Al and WO 2018 / 015539 Al, which are incorporated by reference in their entirety. As used herein, the term "purification moiety" refers to any molecule including protein, protein domains and protein tags that can be used to purify EVs, preferably exosomes. In one embodiment, the purification moiety allows for the EVs to be purified by affinity purification. Affinity purification of EVs is described in WO 2018 / 153581 Al, WO2019 / 081474 Al and WO2019 / 238626 Al, which are incorporated by reference in their entirety. As used herein, the term "pharmacokinetic and / or pharmacodynamic effector moiety" relates to any molecule, including any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacokinetics and / or pharmacodynamics of the EV. In one embodiment, the pharmacokinetic or pharmacodynamic effector moiety is an albumin binding domain. In a preferred embodiment, the additional POI is fused or conjugated to a domain or terminus of the EV polypeptide, which is displayed on the surface of an EV, preferably an exosome. In one embodiment, the additional POI is fused or conjugated into loop 1 or loop 2 of a tetraspanin EV polypeptide or to the to the N-terminus of a single pass EV polypeptide. In a most preferred embodiment, the POI is fused or conjugated into loop 1 or loop 2 of CD63, TNS3, TSN9 or most preferably TSN2. As used herein, the term "loop 1" when used in relation to a tetraspanin refers to the domain between the first and the second transmembrane domains of the tetraspanin which is displayed on the surface of an EV. In one embodiment, loop 1 of TSN2 is defined by residues 35-54 of SEQ ID NO 21. As used herein, the term "loop 2" when used in relation to tetraspanin refers to the domain between the third and the fourth transmembrane domains of the tetraspanin, which is displayed on the surface of an EV. In one embodiment, loop 2 of TSN2 is defined by residues 112-188 of SEQ ID NO 21. As used herein the term "first transmembrane domain" when used in reference to a tetraspanin refers to the transmembrane domain most proximal to the N-terminus of the tetraspanin and the term "fourth transmembrane domain" when used in reference to a tetraspanin refers to the transmembrane domain most proximal to the C-terminus of the tetraspanin. In one embodiment, the EV polypeptide of the present invention is further fused to, or the fusion protein of the Casl2 RNP complex of the present invention further comprises, a multimerization domain, preferably a fold-on domain or a leucine zipper. In a preferred embodiment, the EV polypeptide of the present invention is further fused to, or the fusion protein of the Casl2 RNP complex of the present invention further comprises a multimerization domain together with a targeting moiety. In one embodiment of the Casl2 RNP complex of the present invention wherein the Casl2 protein is bound to 5' DR region of a circular Casl2 crRNA of the present invention, a second Casl2 protein is bound to the 3' DR region of the circular Casl2 crRNA. In one embodiment of the Casl2 RNP complex of the present invention wherein the Casl2 protein is bound to 3' DR region of a circular Casl2 crRNA of the present invention, a second Casl2 protein is bound to the 5' DR region of the circular Casl2 crRNA. In one embodiment of the Casl2 RNP complex of the present invention wherein the Casl2 protein is bound to the cleaved 5' DR region of a 5' DR cleaved linearised crRNA of the present invention, a second Casl2 protein is bound to the 3' DR region of the 5' DR cleaved linearised crRNA. In one embodiment of the Casl2 RNP complex of the present invention wherein the Casl2 protein is bound to the 3' DR region of a 3' DR cleaved linearised crRNA of the present invention, a second Casl2 protein is bound to the 5' DR region of the 3' DR cleaved linearised crRNA. In a preferred embodiment, the second Casl2 protein is fused to an EV polypeptide as described herein, preferably via an intein as described herein. In preferred embodiment, the second Casl2 protein is comprised in a second fusion protein that has the same structure as the first fusion protein as described herein i.e. wherein the first and the second fusion proteins are encoded for by the same polynucleotide sequence and / or have the same amino acid sequence. The intein may or may not have undergone self-cleavage (preferably C-terminal cleavage) to release the second Casl2 protein. The present invention further provides a Casl2 RNP complex comprising a crRNA of the present invention and a fusion protein, wherein the fusion protein comprises: (i) a Gag protein, preferably a MLV Gag protein; and (ii) a Casl2 protein as described herein, wherein the Casl2 protein is bound to: a. the 5' DR region or the 3' DR region of a circular Casl2 crRNA of the present invention; b. the cleaved 5' DR region of a 5' DR cleaved linearised crRNA of the present invention; c. the cleaved 3' DR region of a 3' DR cleaved linearised crRNA of the present invention; or d. the cleaved 5' DR region of the 5' DR and 3' DR cleaved crRNA of the present invention. In a preferred embodiment, the Casl2 protein is fused to the C-terminus of the Gag protein The present invention further provides a Casl2 RNP complex comprising a crRNA of the present invention and a fusion protein, wherein the fusion protein comprises: (i) a Gag protein, preferably an MLV Gag protein, or more preferably an MMLV Gag protein; and (ii) a Casl2 protein as described herein, wherein the Casl2 protein is bound to: a. the 5' DR region or the 3' DR region of a circular Casl2 crRNA of the present invention; b. the cleaved 5' DR region of a 5' DR cleaved linearised crRNA of the present invention; c. the cleaved 3' DR region of a 3' DR cleaved linearised crRNA of the present invention; or d. the cleaved 5' DR region of the 5' DR and 3' DR cleaved crRNA of the present invention. In a preferred embodiment, the Casl2 protein is fused to the C-terminus of the Gag protein, most preferably via 1, 2 or preferably 3 nuclear export signals. Preferably the Casl2 protein is fused to the C-terminus of the Gag protein, or the C-terminus of the nuclear export signal(s), via an MMLV protease-cleavable linker. Extracellular Vesicles of the Present Invention The present invention further provides an EV comprising a crRNA of the present invention, preferably in its lumen. In a preferred embodiment, the crRNA is bound by a Casl2 protein as described herein. Preferably the Casl2 protein is fused, via an intein as described herein, to an EV polypeptide as described herein. As used herein, the term "extracellular vesicle" or "EV" refers to any type of vesicle that is obtainable from a cell in any form. Essentially, an EV may relate to any type of lipid-based structure (with vesicular morphology or with any other type of suitable morphology) that can act as a delivery or transport vehicle or that has native therapeutic or pharmacological effects. Typically, an EV comprises a lipid-based membrane that encloses an internal space (i.e., lumen). The size of EVs may vary considerably, but an EV typically comprises has a radius of below 1000 nm (preferably below 200 nm). The EVs of the present invention are genetically modified EVs. As used herein the term "modified" when used in relation to an EV can mean an EV that has been modified either using genetic or chemical approaches. As used herein the term "genetically modified" or "genetically engineered" when used in relation to an EV refers to an EV that is derived from a genetically modified cell that expresses and / or modifies the expression of proteins in the lumen, extravesicular membrane and / or displayed on the surface of the EV. Genetically modified EVs do not occur in nature. In a preferred embodiment the EV of the present invention is derived from a cell, preferably a eukaryotic cell, more preferably a mammalian cell, more preferably a human cell. In a preferred embodiment, the EV is derived from an EV producer cell as described herein. In one embodiment the EV of the present invention is a nanoblade as described herein or an eVLP as described herein. In a preferred embodiment, the EV of the present invention is a microvesicle (e.g. any vesicle shed from the plasma membrane of a cell) or more preferably an exosome (e.g. any vesicle derived from the endo-lysosomal pathway or from any other cellular pathway producing exosomes). In one embodiment, an exosome of the present invention has a radius of between 30 and 300 nm, preferably in between 40 and 250 nm, most preferably between 40 and 160 nm. The present invention further provides an EV comprising a Casl2 RNP complex of the present invention. Preferably the Casl2 RNP complex of the present invention comprises an intein as described herein between the EV polypeptide and the Casl2 protein. The present invention further provides an EV comprising a fusion protein, wherein the fusion protein comprises an EV polypeptide as described and a Casl2 protein as described herein, wherein the Casl2 protein is bound to: a. the 5' DR. of a circular Casl2 crRNA of the present invention; b. the 3' DR of a circular Casl2 crRNA of the present invention; c. the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention; d. the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention; or e. the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention Preferably the EV polypeptide is fused to the Casl2 protein via an intein as described herein. In one embodiment the EV of the present invention, the intein comprised in the fusion protein is self-cleaved (preferably at its C-terminus) to release the Casl2 protein from the EV polypeptide and preferably the intein. In a preferred embodiment, the Casl2 protein is released into the lumen of the EV. Thus, the present invention further provides an EV comprising: (i) a fusion protein comprising an EV polypeptide as described fused to an intein as described herein and (ii) a Casl2 protein as described herein, wherein the Casl2 protein is bound to a. the 5' DR of a circular Casl2 crRNA of the present invention; b. the 3' DR of a circular Casl2 crRNA of the present invention; c. the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention; d. the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention; or e. the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention. In a preferred embodiment of any of the EVs of the present invention, the EV further comprises a second Casl2 protein as described herein. In a preferred embodiment, the second Casl2 protein is comprised in a second fusion protein. In one embodiment, the second fusion protein comprises the second Casl2 protein and an EV polypeptide as described herein, preferably wherein the EV polypeptide is fused to the second Casl2 protein via an intein as described herein. In preferred embodiment, the second fusion protein that has the same structure as the first fusion protein described herein i.e. the first and the second fusion proteins are encoded for by the same polynucleotide sequence and / or have the same amino acid sequence. Thus, in one embodiment, the EV comprises: (i) a first fusion protein comprising an EV polypeptide as described herein and a Casl2 protein as described herein, wherein the EV polypeptide and the Casl2 protein are fused vis an intein and (ii) a second fusion protein comprising an EV polypeptide as described herein and a Casl2 protein as described herein, wherein the EV polypeptide and the Casl2 protein are fused via an intein further wherein: a. the Casl2 protein of the first fusion protein is bound to the 5' DR. of a circular Casl2 crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 3' DR of the circular Casl2 crRNA; b. the Casl2 protein of the first fusion protein is bound to the 3' DR of a circular Casl2 crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 5' DR of the circular Casl2 crRNA; c. the Casl2 protein of the first fusion protein is bound to the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 3' DR of the 5' DR cleaved linearised crRNA; d. the Casl2 protein of the first fusion protein is bound to 3' DR of a 5' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the cleaved 5' DR of the 5' DR cleaved linearised crRNA; e. the Casl2 protein of the first fusion protein is bound to the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 5' DR of the 3' DR cleaved linearised crRNA; f. the Casl2 protein of the first fusion protein is bound to the 5' DR of a 3' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the cleaved 3' DR of the 3' DR cleaved linearised crRNA; g. the Casl2 protein of the first fusion protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and optionally the Casl2 protein of the second fusion protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention; or h. the Casl2 protein of the second fusion protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and optionally the Casl2 protein of the first fusion protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention. In an alternative embodiment, the EV comprises: (i) a first fusion protein comprising an EV polypeptide as described herein and an intein as described herein; (ii) a first Casl2 protein as described herein; and (iii) a second fusion protein comprising an EV polypeptide as described herein and a Casl2 protein as described herein, wherein the EV polypeptide and the Casl2 protein are fused via an intein further wherein: a. the first Casl2 protein is bound to the 5' DR. of a circular Casl2 crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 3' DR of the circular Casl2 crRNA; b. the first Casl2 protein is bound to the 3' DR of a circular Casl2 crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 5' DR of the circular Casl2 crRNA; c. the first Casl2 protein is bound to the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 3' DR of the 5' DR cleaved linearised crRNA; d. the first Casl2 protein is bound to the 3' DR of a 5' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the cleaved 5' DR of the 5' DR cleaved linearised crRNA; e. the first Casl2 protein is bound to the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the 5' DR of the 3' DR cleaved linearised crRNA; f. the first Casl2 protein is bound to the 5' DR of a 3' DR cleaved linearised crRNA of the present invention and the Casl2 protein of the second fusion protein is bound to the cleaved 3' DR of the 3' DR cleaved linearised crRNA; g. the first Casl2 protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and, optionally the Casl2 protein of the second fusion protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention; or h. the Casl2 protein of the second fusion protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and, optionally the first Casl2 protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention. In one embodiment, the intein of the second fusion protein is self-cleaved (preferably at its C-terminus) to release the Casl2 protein from the EV polypeptide and preferably the intein. In a preferred embodiment, the Casl2 protein is released into the lumen of the EV. Hence, in one embodiment, the EV comprises: (i) a first fusion protein comprising an EV polypeptide as described herein and a Casl2 protein as described herein, wherein the EV polypeptide and the Casl2 protein are fused via an intein as described herein; (ii) a second fusion protein comprising an EV polypeptide as described herein and an intein as described herein; and (iii) a second Casl2 protein further wherein: a. the Casl2 protein of the first fusion protein is bound to the 5' DR. of a circular Casl2 crRNA of the present invention and the second Casl2 protein is bound to the 3' DR of the circular Casl2 crRNA; b. the Casl2 protein of the first fusion protein is bound to the 3' DR of a circular Casl2 crRNA of the present invention and the second Casl2 protein is bound to the 5' DR of the circular Casl2 crRNA; c. the Casl2 protein of the first fusion protein is bound to the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention the second Casl2 protein is bound to the 3' DR of the 5' DR cleaved linearised crRNA; d. the Casl2 protein of the first fusion protein is bound to the 3' DR of a 5' DR cleaved linearised crRNA of the present invention the second Casl2 protein is bound to the cleaved 5' DR of the 5' DR cleaved linearised crRNA; e. the Casl2 protein of the first fusion protein is bound to the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention and the second Casl2 protein is bound to the 5' DR of the 3' DR cleaved linearised crRNA; f. the Casl2 protein of the first fusion protein is bound to the 5' DR of a 3' DR cleaved linearised crRNA of the present invention and the second Casl2 protein is bound to the cleaved 3' DR of the 3' DR cleaved linearised crRNA; g. the Casl2 protein of the first fusion protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and optionally the second Casl2 protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention; or h. the second Casl2 protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and optionally the Casl2 protein of the first fusion protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention. In one embodiment, the intein of the first fusion protein is self-cleaved (preferably at its C-terminus) to release the Casl2 protein from the EV polypeptide and preferably the intein and the intein of the second fusion protein is self-cleaved (preferably at its C-terminus) to release the Casl2 protein from the EV polypeptide and preferably the intein. In a preferred embodiment, the Casl2 proteins are released into the lumen of the EV. Thus, in one embodiment, the EV comprises: (i) a first fusion protein comprising an EV polypeptide as described herein and an intein as described herein; (ii) a first Casl2 protein; (iv) a second fusion protein comprising an EV polypeptide as described herein and an intein as described herein; and (v) a second Casl2 protein further wherein: a. the first Casl2 protein is bound to the 5' DR. of a circular Casl2 crRNA of the present invention and the second Casl2 protein is bound to the 3' DR of the circular Casl2 crRNA; b. the first Casl2 protein is bound to the 3' DR of a circular Casl2 crRNA of the present invention and the second Casl2 protein is bound to the 5' DR of the circular Casl2 crRNA; c. the first Casl2 protein is bound to the cleaved 5' DR of a 5' DR cleaved linearised crRNA of the present invention the second Casl2 protein is bound to the 3' DR of the 5' DR cleaved linearised crRNA; d. the first Casl2 protein is bound to the 3' DR of a 5' DR cleaved linearised crRNA of the present invention the second Casl2 protein is bound to the cleaved 5' DR of the 5' DR cleaved linearised crRNA; e. the first Casl2 protein is bound to the cleaved 3' DR of a 3' DR cleaved linearised crRNA of the present invention and the second Casl2 protein is bound to the 5' DR of the 3' DR cleaved linearised crRNA; f. the first Casl2 protein is bound to the 5' DR of a 3' DR cleaved linearised crRNA of the present invention and the second Casl2 protein is bound to the cleaved 3' DR of the 3' DR cleaved linearised crRNA; g. the first Casl2 protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and the optionally second Casl2 protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention; or h. the second Casl2 protein is bound to the cleaved 5' DR of a 5' DR and 3' DR cleaved crRNA of the present invention and the optionally the first Casl2 protein is bound to the cleaved 3' DR of the 5' DR and 3' DR cleaved linear RNA of the present invention. In one embodiment, the EV of the present invention may comprise one, two or more than two additional fusion proteins. In one embodiment, the additional fusion protein comprises an EV polypeptide as described herein and an additional POI as described herein. In a preferred embodiment, the additional POI is a purification moiety as described herein, a targeting moiety as described herein, a pharmacokinetic and / or pharmacodynamic effector moiety as described herein and / or an Fc-binding protein as described herein. In one embodiment, the additional fusion protein comprises an EV polypeptide as described herein and two or more than two additional POIs as described herein. In a preferred embodiment, the additional fusion protein comprises a different EV polypeptide to the one comprised in the fusion protein comprising the Casl2 protein. In one embodiment, the additional POI is fused or conjugated to the EV polypeptide, such that the additional POI is displayed on the surface of the EV. In one embodiment, the additional POI is fused or conjugated to a domain or terminus of the EV polypeptide which is displayed on the surface of an EV. In one embodiment, the additional POI is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide or to the N-terminus of single-pass transmembrane EV polypeptide. In one embodiment, the additional fusion protein further comprises a multimerization domain, preferably a fold-on domain or a leucine zipper. In a preferred embodiment where the additional fusion protein comprises a targeting moiety, the additional fusion protein also comprises a multimerization domain, preferably a fold-on domain or a leucine zipper. In a preferred embodiment, the EV of the present invention further comprises a fusogen. In one embodiment, the EV of the present invention further comprises a fusogen wherein the EV polypeptide is not a fusogen. In an alternative embodiment wherein the EV of the present invention comprises an EV polypeptide that is a fusogen, the EV comprises an additional fusogen. As used herein, the term "fusogen" refers to any protein or agent that is capable of fusing lipid bilayers. In one embodiment, the fusogen is capable of merging two separate lipid bilayers into a single continuous lipid bilayer, preferably in a target cell as defined herein. In one embodiment, the lipid bilayers are cell membranes. In a preferred embodiment, the fusogen is an endosomal escape domain. The inclusion of a fusogen in the fusion protein or in the EV of the present invention may enhance bioactivity in a target cell. In one embodiment, where the EV of the present invention further comprises a fusogen, the EV comprises a further fusion protein, wherein the fusion protein comprises an EV polypeptide and the fusogen. In one embodiment, where the EV of the present invention further comprises a fusogen, the EV comprises the fusogen in one of the further fusion proteins described herein. In a preferred embodiment the fusogen is VSVG. In a preferred embodiment, the fusogen is fused to a multimerization domain, preferably a fold-on domain or a leucine zipper, most preferably a foldon domain. In one embodiment, the EV of the present invention is a nanoblade. As used herein, the term nanoblade refers to a virus-like particle (VLP) derived from a murine leukaemia virus (MLV). Nanoblades are described for instance in Mangeot et al., 2019 Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Nat. Commun. 2019;10:45. In a preferred embodiment, nanoblade comprises a crRNA of the present invention bound to a Casl2 protein. In a preferred embodiment, the nanoblade comprises a crRNA of the present invention in its lumen. Preferably the Casl2 protein is fused to a gag protein, preferably a MLV gag protein. Preferably the Casl2 protein is fused to the C-terminus of the Gag protein. In one embodiment, the nanoblade may further comprise a fusogen as described herein, preferably VSV-G. In a preferred embodiment, the nanoblade may further comprise gag-pol, preferably MLV gag-pol. In a preferred embodiment, the nanoblade may further comprise the Baboon Endogenous retrovirus Riess glycoprotein. In one embodiment, the EV of the present invention is a virus-like particle (VLP) or preferably an enveloped virus-like particle (eVLP). As used herein, the term VLP refers to an assembly of viral proteins that can infect cells but lack viral genetic material. Enveloped VLPs are surrounded by a lipid bilayer. In a preferred embodiment, the VLP or eVLP comprises a crRNA of the present invention. In a preferred embodiment, the VLP or eVLP comprises the crRNA in its lumen. In a preferred embodiment, the crRNA is bound to a Casl2 protein. Preferably the Casl2 protein is fused to a gag protein, preferably a MLV gag protein. Preferably the Casl2 protein is fused to the C-terminus of the Gag protein, most preferably via 1, 2 or preferably 3 nuclear export signals. Preferably the Casl2 protein is fused to the C-terminus of the Gag protein, or the C-terminus of the nuclear export signal(s), via an MMLV protease-cleavable linker. Preferably the VLP or eVLP comprises an envelope glycoprotein, preferably the VSV-G envelope glycoprotein. In a further aspect, the present invention provides a population of EVs comprising a plurality of EVs of the present invention. As used herein the term "population of EVs" or "EV population" refers to any plurality of EVs comprising a plurality of EVs of the present invention. In one embodiment of a population of EVs of the present invention, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of all the EVs present in the population are EVs of the present invention. In a preferred embodiment of a population of EVs of the present invention, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of all the EVs present in the population comprise a crRNA of the present invention or a Casl2 RNP complex of the present invention. Methods of Loading and Producing Extracellular Vesicles of the Present Invention The present invention provides a method of loading an EV comprising the following steps: (i) introducing into an EV producer cell as described herein, a. a polynucleotide of the present invention or an expression vector of the present invention; b. a polynucleotide or expression vector encoding for a (first) fusion protein, wherein the fusion protein comprises an EV polypeptide as described herein and a Casl2 protein as described herein, preferably wherein the EV polypeptide and the Casl2 protein are fused via an intein as described herein; (ii) expressing the polynucleotide or expression vector of (i)a and (i)b, preferably at the same time, in the EV producer cell, preferably under conditions which, in no particular order: a. the Casl2 protein of the fusion protein expressed from the polynucleotide or expression vector of (i)b binds the 5' DR region or the 3'DR region of the crRNA expressed from the polynucleotide or expression vector of (i)a and b. the Casl2 protein bound to the crRNA loads into an EV. The present invention further provides a method of producing an EV or a population of EVs comprising the following steps: (i) introducing into an EV producer cell as described herein, a. a polynucleotide of the present invention or an expression vector of the present invention; b. a polynucleotide or expression vector encoding for a (first) fusion protein, wherein the fusion protein comprises an EV polypeptide as described herein and a Casl2 protein as described herein, preferably wherein the EV polypeptide and the Casl2 protein are fused via an intein as described herein; (iii) expressing the polynucleotide or expression vector of (i)a and (i)b, preferably at the same time, in the EV producer cell, preferably under conditions which, in no particular order: a. the Casl2 protein of the fusion protein expressed from the polynucleotide or expression vector of (i)b binds the 5' DR. region or the 3'DR region of the crRNA expressed from the polynucleotide or expression vector of (i)a and b. the Casl2 protein bound to the crRNA loads into an EV. In a preferred embodiment, the method thereby generates EVs comprising a crRNA of the present invention and / or a Casl2 RNP complex present invention. As used herein, the term "EV producer cell", which may also be referred to as an "EV source cell" or a "parental cell", refers to any cell that is capable of producing an EV. Generally, EVs may be derived from essentially any cell source. In a preferred embodiment, the EV producer cell is any cell that produces an EV under the conditions in which the cell is able to survive or preferably grow. In a more preferred embodiment, the EV producer cell is any cell that produces an EV under its usual, or optimum, conditions for growth. In a preferred embodiment, the EV producer cell is capable of producing a microvesicle or an exosome. In one embodiment, the EV producer cell is, or is derived from, a eukaryotic cell, preferably a mammalian cell, more preferably a human cell. In one embodiment, the EV producer cell is an ex vivo cell or preferably an in vitro cell such as a primary cell or more preferably a cellline. In a preferred embodiment, the EV producer cell-line is a HEK cell, more preferably a HEK293 cell, more preferably a Gibco™ Viral Production Cell 1.0 (VPC 1.0 cells) or Gibco™ Viral Production Cell 2.0 (VPC 2.0 cells). In a preferred embodiment, the EV producer cell is a suspension cell-line. In a preferred embodiment, the EV producer cell is grown or cultured in serum-free conditions. In a particularly preferred embodiment, the EV producer cell expresses functional RNA ligase, preferably RNA ligase RtbC. In a preferred embodiment of the methods of the present invention, the polynucleotide of the present invention or expression vector of the present invention comprise a pre-cursor crRNA molecule of the present invention. In one embodiment, following expression of the pre-cursor RNA molecule of the present invention in the EV producer cell, the 5' selfcleaving ribozyme sequence and the 3' self-cleaving ribozyme sequence undergo selfcleavage, preferably in the cytoplasm of the EV producer cell. In one embodiment, following the self-cleavage of the 5' self-cleaving ribozyme sequence and the 3' self-cleaving ribozyme sequence, the 5' component of the 3'DR to 5'DR connecting linker and the 3' component of the 3'DR to 5'DR connecting linker form a double stranded RNA duplex structure through complementary base pairing preferably in the cytoplasm of the EV producer cell. In one embodiment, following, the formation of the RNA duplex structure, an RNA ligase, preferably RNA ligase RtbC, ligates the self-cleaved 5' end of the pre-cursor RNA molecule of the present invention to the self-cleaved 3' end of the pre-cursor RNA molecule of the present invention, preferably in the cytoplasm of the EV producer cell. In a further preferred embodiment, the conditions under which the Casl2 protein binds the crRNA of the present invention and which the Casl2 protein bound to the crRNA loads into the EV are the normal conditions under which the EV producer cell is cultured. In one embodiment, the conditions under which the Casl2 protein binds the crRNA of the present invention and which the Casl2 protein bound to the crRNA loads into the EV are cell culture conditions under which the EV producer cell is able to survive or preferably grow. In one embodiment, the Casl2 protein binds to the 5' DR region or the 3' DR region of the crRNA in the cytoplasm of the EV producer cell and / or as the EV is forming. In a further preferred embodiment of the methods of the present invention, a second fusion protein is expressed in the EV producer cell. In a preferred embodiment, the second fusion protein is expressed from the same polynucleotide or expression vector encoding for the first fusion protein, thus the first and the second fusion proteins have the same amino acid sequence. In a preferred embodiment wherein the Casl2 protein of the first fusion protein binds to the 5' DR region of the crRNA of the present invention, the Casl2 protein of the second fusion protein binds to the 3' DR region. In an alternative preferred embodiment wherein the Casl2 protein of the first fusion protein binds to the 3' DR region of the crRNA of the present invention, the Casl2 protein of the second fusion protein binds to the 5' DR region. In one embodiment, the Casl2 protein of the second fusion protein binds to the 5' DR region or the 3' DR region of the crRNA in the cytoplasm of the EV producer cell and / or as the EV is forming. In one embodiment, the Casl2 protein of the second fusion protein binds to the 5' DR region or the 3' DR region of the crRNA in the lumen of the EV. In a preferred embodiment, the Casl2 protein of the first fusion protein binds in the cytoplasm of the EV producer cell and / or as the EV is forming and the Casl2 protein of the second fusion protein binds in the lumen of the EV. In an alternative preferred embodiment, the Casl2 protein of the first fusion protein and the Casl2 protein of the second fusion protein bind in the cytoplasm of the EV producer cell and / or as the EV is forming. In a preferred embodiment of the methods of the present invention wherein the Casl2 protein of a first fusion protein binds to the 5' DR region of the crRNA of the present invention and the Casl2 protein of a second fusion protein binds to the 3' DR region, the Casl2 protein of the first fusion protein cleaves the 5' DR region and / or the Casl2 protein of the second fusion protein cleaves the 3' DR. region. In a preferred embodiment of the methods of the present invention wherein the Casl2 protein of a first fusion protein binds to the 3' DR region of the crRNA of the present invention and the Casl2 protein of a second fusion protein binds to the 5' DR region, the Casl2 protein of the first fusion protein cleaves the 3' DR region and / or the Casl2 protein of the second fusion protein cleaves the 5' DR region. In one embodiment, the cleavage occurs in the cytoplasm of the EV producer cell and / or as the EV is forming. In one embodiment, the cleavage occurs in the lumen of the EV. In a preferred embodiment, the Casl2 protein of first fusion protein cleaves in the cytoplasm of the EV producer cell and / or as the EV is forming and the Casl2 protein of second fusion protein cleaves in the lumen of the EV. In an alternative preferred embodiment, both the Casl2 protein of first fusion protein and the Casl2 protein of second fusion protein cleave in the cytoplasm of the EV producer cell and / or as the EV is forming. In an alternative preferred embodiment, both the Casl2 protein of first fusion protein and the Casl2 protein of second fusion protein cleave in the lumen of the EV. In a preferred embodiment of the methods of the present invention, the methods further comprises a step of introducing into the same EV producer cell a further polynucleotide construct, preferably an expression vector, wherein the further polynucleotide construct encodes for an additional fusion protein as described herein, and expressing the further polynucleotide construct in the EV producer cell. Preferably, the additional fusion protein comprises an EV polypeptide as described herein and an additional POI as described herein. In a preferred embodiment of this method, EVs are generated that further comprise an additional POI. In a preferred embodiment, the further polynucleotide construct is expressed in the EV producer cell at the same time as the polynucleotide or expression vector of (i)a and (ii)b. In a preferred embodiment, the further polynucleotide construct comprises a different EV polypeptide to the one encoded for in (ii)b. In one embodiment, the EV polypeptide of the further polynucleotide construct is fused to the additional POI via an intein as described herein. In one embodiment, the method of producing EVs of the present invention may comprise a further step of purifying the EVs. Purification of EVs is achieved by any method including but not limited to: techniques comprising liquid chromatography (LC), high-performance liquid chromatography (HPLC), bead-eluate chromatography, ionic exchange chromatography, spin filtration, tangential flow filtration (TFF), hollow fiber filtration, centrifugation (preferably density gradient ultracentrifugation), immunoprecipitation, flow field fractionation, dialysis, microfluidic-based separation, etc., or any combination thereof. In an advantageous embodiment, the purification of the EVs is carried out using a sequential combination of filtration (preferably ultrafiltration (UF) tangential flow filtration (TFF) or hollow fibre filtration) and affinity chromatography, optionally also including size exclusion LC or bead-eluate LC. Combining purification steps normally enhances the purity of the resulting samples and, in turn leads to superior therapeutic activity. Further, as compared to UC, which is routinely employed for purifying exosomes, sequential filtrationchromatography is considerably faster and possible to scale to higher manufacturing volumes, which is a significant drawback of the current UC methodology that dominates the prior art. Another advantageous purification method is TFF, which offers scalability and purity, and which may be combined with any other type of purification technique. In a further aspect, the present invention provides an EV or a population of EVs directly obtained by the methods of the present invention. In a further aspect, the present invention provides an EV producer cell comprising a precursor crRNA molecule of the present invention, a crRNA of the present invention, a Casl2 RNP complex of the present invention or an EV the present invention or a population of EVs of the present invention In one aspect the present invention provides, the use of a pre-cursor crRNA molecule of the present invention, a crRNA of the present invention or a Casl2 RNP complex of the present invention in loading an EV. In a further preferred embodiment, the loading is luminal loading. In one aspect the present invention provides, the use of a pre-cursor crRNA molecule of the present invention, a crRNA of the present invention or a Casl2 RNP complex of the present invention in producing an EV. In a further preferred embodiment, the loading is luminal loading. As used herein, the term "EV loading" or "loading" refers to the incorporation of a cargo into the extracellular vesicle. EV loading may be exogenous or, in a preferred embodiment of the present invention, endogenous. As used herein, the term "exogenous" when used in relation to EV loading refers to the association on the cargo with EVs, or preferably the incorporation of the cargo into EVs, after they have been secreted or released by EV producer cells. As used herein, the term "endogenous" when used in relation to EV loading refers to the association on the cargo with EVs, or preferably the incorporation of the cargo into EVs, during their biogenesis in EV producer cells. In a preferred embodiment, the endogenous EV loading is active. As used herein, the term "active" when used in relation to endogenous loading refers to when loading of the cargo in the producer cell involves an EV polypeptide that is fused to the cargo or to a polypeptide that binds to the cargo (e.g. Casl2). Active loading increases EV loading as compared to passive endogenous loading, where the cargo is simply expressed in the EV producer cells. This increase in loading may include a higher proportion of the EVs produced being loaded with the cargo and / or the loaded EVs comprising a higher amount of the cargo. In a more preferred embodiment, the EV polypeptide is fused to the polypeptide that binds to the cargo via an intein as described herein, which advantageously allows for releasable loading. As used herein, the term "releasable loading" refers to the loading of an EV with a cargo by association with an EV polypeptide or EV localisation moiety, wherein the cargo is subsequently released from the EV polypeptide or EV localisation moiety. Preferably the cargo is released in the EV lumen and / or in the target cell. Compositions and methods of gene editing of the present invention In one aspect, the present invention further provides a pharmaceutical composition comprising: (i) a crRNA of the present invention, a polynucleotide or expression vector of the present invention, a Casl2 RNP complex of the present invention, an EV of the present invention ora population of EVs of the present invention; and (ii) a pharmaceutically acceptable excipient and / or carrier. The term "pharmaceutically acceptable" is used herein to refer to a material may be administered to a subject without causing any undesirable biological effects. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. In one embodiment, the pharmaceutically acceptable excipient is any substance approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopeia for use in animals, including humans. The pharmaceutical compositions of the present invention may be formulated by any known method of formulation. In one aspect, the pharmaceutical compositions of the present disclosure may be formulated as Oral formulations, including Tablet, Capsule, Sustained release, liquid; Intravenous Formulations; Parenteral Formulations; Topical Formulations; cutaneous administration including cream, ointment, gel, paste, powder; Modified release Formulations including sustained release formulation and Liquid or lyophilized formulations. In one aspect, the present invention provides a method of delivering a Casl2 RNP, preferably comprising a crRNA, to a target cell, comprising contacting or incubating a target cell with an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention. In a preferred embodiment, the target cell is exposed to the EV, population of EVs or pharmaceutical composition for at least 10 seconds, 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 5 hours, at least 12 hours, at least 24 hours or at least 48 hours. As used herein, the term "target cell" refers to a cell which an EV is capable of entering i.e. a cell that is capable of EV uptake. In one embodiment, the target cell has a genetic abnormality that is linked to a disease or a disorder. In one embodiment, the genetic abnormality is due to the expression of the gene that comprises the target DNA sequence recognised by the spacer. In one embodiment, the target cell is a cell of the liver, preferably a hepatocyte, a neuronal cell, a cell of the brain, a muscle cell, a cell of the eye, a cell of the lung, a cell of the liver, a cell of the kidneys, a cell of the heart, a cell of the stomach, a cell of the intestines, a cell of the pancreas, a red blood cell, a white blood cell including a B cell or a T cell, a cell of the lymph nodes, a cell of the bone marrow or a cell of the spleen. In a preferred embodiment, the target cell is a neuronal cell preferably a cell of the CNS or the brain, a muscle cell, a cell of the heart preferably a cardiomyocyte, a cell of the lung, a cell of the immune system, a cell of the liver preferably a hepatocyte. The target cell may be in vivo, ex vivo or in vitro. As used herein the "the target DNA sequence recognised by the spacer" refers to the sequence that is bound by the spacer sequence of the crRNA. In a preferred embodiment, the target DNA sequence recognised by the spacer is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% complementary to the spacer sequence. In a preferred embodiment, the target DNA sequence recognised by the spacer comprises a nucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% complementary to the seed sequence of the spacer sequence. In one aspect, the present invention provides the use of an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention for the delivery of a Casl2 RNP, preferably comprising a crRNA of the present invention, to a target cell as described herein. In one embodiment, the present invention provides a method of gene editing in a target cell as described herein by introducing into the cell a crRNA of the present invention, a Casl2 RNP complex of the present invention, an EV of the present invention, a population of EVs of the present invention ora pharmaceutical composition of the present invention. Preferably, wherein the method is in vivo, in vitro or ex vivo. In a preferred embodiment of any of the methods of the present invention, the gene editing is modification of a genomic locus of interest to alter gene expression in the target cell. In a preferred embodiment, the genomic locus comprises a PAM sequence on the complementary DNA strand to the strand that comprises the target DNA sequence recognised by the spacer. In a preferred embodiment, the PAM sequence comprises or consists of the following nucleotide sequence: YTN orYTTN (wherein Y may be C orT and N may be any nucleotide), preferably TTN or TTTN or more preferably TTTV (wherein V may be any of A, C or G). In a preferred embodiment, the PAM sequence is located immediately downstream (3' end) of the target DNA sequence recognised by the spacer. In one embodiment, the present invention provides a target cell as described herein comprising an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention. In one aspect, the present invention further provides the use of a crRNA of the present invention, a Casl2 RNP complex of the present invention, an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention for the manufacture of a medicament for treatment or prevention of a disease in a subject. In one aspect, the present invention further provides a crRNA of the present invention, a Casl2 RNP complex of the present invention, an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention for use in a method of treatment or prevention of a disease in a subject. In one aspect, the present invention further provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a crRNA of the present invention, a Casl2 RNP complex of the present invention, an EV of the present invention, a population of EVs of the present invention or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease. It will be clear to the skilled artisan that when describing medical and scientific uses and applications of the EVs, the present invention normally relates to a plurality of EVs, i.e. a population of EVs which may comprise thousands, millions, billions or even trillions of EVs. As can be seen from the experimental section below, EVs may be present in concentrations such as 105' 108, 1010, 1011, 1012, 1013, 1014, 1015, 1018, 1025 ,1030 EVs (often termed "particles") per unit of volume (for instance per ml or per litre), or any other number larger, smaller or anywhere in between. In the same vein, the term "population" shall be understood to encompass a plurality of entities which together constitute such a population. In other words, individual EVs when present in a plurality constitute an EV population. Thus, the present invention pertains both to individual EVs and populations comprising EVs. The dosages of EVs when applied in vivo may naturally vary considerably depending on the disease to be treated, the administration route, the activity and effects of the editing machinery, any targeting moieties present on the EVs, the pharmaceutical formulation, etc. As used herein, a "subject" refers to an animal that is the object of treatment. In a preferred embodiment the animal is a mammal, most preferably a human. In one embodiment of the uses or methods of the present invention, the disease is a genetic disorder. As used herein, the term "genetic disorder" refers to a disorder or a disease caused by one or more abnormalities in the DNA. Preferably the abnormality in the DNA is within the gene that comprises the target DNA sequence recognised by the spacer. In a preferred embodiment, the genetic disorder can be treated, or symptoms may be improved, by reducing or preventing expression of the gene comprising the target DNA sequence recognised by the spacer or by altering the sequence of the gene comprising the target DNA sequence recognised by the spacer. In a preferred embodiment, the genetic disorder is a liver disorder, a neurological disorder or a cardiac disorder. In one embodiment of the uses or methods of the present invention, the pre-cursor crRNA molecule, the crRNA, the Casl2 RNP complex, the EV, the population of EVs or the pharmaceutical composition is administered to the subject systemically or preferably vis localised administration. In a preferred embodiment, the pre-cursor crRNA molecule, the crRNA, the Casl2 RNP complex, the EV, the population of EVs or the pharmaceutical composition is locally administered into the liver, the CNS preferably the brain or the heart. EXAMPLES Example 1 - Design of the different Cas 12 crRNAs Figure 1 shows the different Casl2 crRNA designs tested by the present inventors. Figure 1A shows a linear crRNA consisting of a 5' DR region and a spacer sequence. Figure IB shows a linear crRNA consisting of a 5' DR. region, a spacer sequence and a 3' DR region. The present inventors hoped that the 3' DR region would help to protect this crRNA from exonuclease degradation. Figure IC shows a circular crRNA comprising a 5' DR region and a spacer sequence. The present inventors hoped that the crRNA being circular would help to protect it from exonuclease degradation. Figure ID shows a circular crRNA comprising a 5' DR region, a spacer sequence and a 3' DR region. This crRNA includes both the addition of a 3' DR region with being circular. Figure IE shows a pre-cursor RNA molecule for the crRNA of Figure ID. The sequence encoding the circular crRNA is flanked by 5' and 3' self-cleaving ribozymes. Upon transcription, the primary crRNA undergoes cleavage by these ribozymes. The stem-forming sequences of the 3'DR to 5'DR connecting linker hybridize and is subsequently circularized into crRNA by an endogenous RNA ligase before degradation. Example 2 - Only Cir-SL2-crRNA-DR improves gene editing To assess whether any of the crRNA designs were able to improve editing efficiency, EV producer cells (HEK293 cells) were co-transfected with a vector for the expression of: • SL2-crRNA (a crRNA as shown in Figure 1A) as encoded for by a polynucleotide having the sequence SEQ ID NO: 26; • SL2-crRNA-DR (a crRNA as shown in Figure IB) as encoded for by a polynucleotide having the sequence SEQ ID NO: 27; • cir-SL2-crRNA (a crRNA as shown in Figure IC) as encoded for by a polynucleotide having the sequence of SEQ ID NO: 28; or • cir-SL2-crRNA-DR (a crRNA as shown in Figure ID) as encoded for by a polynucleotide having the sequence of SEQ ID NO: 29 and a TSN2-Intein-Casl2a fusion protein. The intein either comprised an amino acid sequence of SEQ ID NO: 23 (referred to as "Intein" in the figures) or of SEQ ID NO: 24 (referred to as "Intein29" in the figures). The EV producer cells were also transfected with the viral fusogenic protein VSV-G-Foldon. The medium was changed to Opti-MEM 6 hours after transfection. The conditional medium was harvested 2 days after transfection and EVs were isolated by TFF followed by concentration with 10 kD spin filters. EV concentrations were determined via Zeta View and the indicated doses of isolated EVs were then incubated with HEK-SL reporter cells in 96-well plates for 72 hours. HEK-SL reporter cells stably express an "mCherry-F2A-linker-stop-GFP" contract under the control of a CMV promoter. In the absence of gene editing, the HEK-SL reporter cells express mCherry only, since the "stop" prevents GFP from being expressed. The crRNAs all include an "SL2" spacer sequence that targets the linker and when Cas9 mediated gene editing occurs, the stop is disrupted, leading to expression of GFP. GFP-positive HEK-SL reporter cells were quantified by FACS analysis. Figure 2 shows the circular crRNA comprising an additional 3' DR region (i.e. a crRNA of the present invention) significantly improves gene editing in target cells with EV mediated delivery. It is highly surprising that neither the linear crRNA comprising an additional 3' DR region (SL2-crRNA-DR) or the circular crRNA not comprising an additional 3' DR region (cir-SL2-crRNA) showed any improvement in gene editing over the conventional crRNA (SL2-crRNA), yet the circular crRNA comprising an additional 3' DR region (Cir-SL2 crRNA DR) showed a highly significant improvement. This improvement is seen at the three different doses tested and with both the different inteins tested. The present inventors went on to investigate whether the same results were obtained when different EV polypeptides were used. The experimental method described above was repeated except for a CD63-Intein-Casl2a fusion protein, rather than a TSN2-Intein-Casl2a fusion protein, was used. Figure 3 shows that again, only the circular crRNA comprising an additional 3' DR region (Cir-SL2 crRNA DR) improves gene editing as compared to the conventional crRNA (SL2-crRNA). This improvement is again seen at the three different doses tested and with both the different inteins tested. The present inventors then repeated the experimental method described above except for a single VSV-G-Foldon-Intein-Casl2a fusion protein, rather than a TSN2-Intein-Casl2a fusion protein or a CD63-Intein-Casl2a fusion protein, was used. Figure 4 shows that again, only the circular crRNA comprising an additional 3' DR region (Cir-SL2 crRNA DR) improves gene editing as compared to the conventional crRNA (SL2-crRNA) and that improvement is again seen at the three different doses tested and with both the different inteins tested. Thus Figures 3 and 4 show that any EV polypeptide may be used and the same result is observed. Example 3 - Cir-SL2-crRNA-DR improves gene editing when delivered using Nanoblades, eVLP or EVs The present inventors went on to test whether Cir-SL2-crRNA-DR improves gene editing when other high-performance LNP platforms are used. Nanoblades were produced by co-transfecting producer cells (HEK293 cells) with - a Gag polyprotein fused to Casl2a (BIC-Gag-Casl2a plasmid) - VSV-G-Foldon and - Gag-POL (MMLV) Along with either SL2-crRNA as encoded for by a polynucleotide having the sequence of SEQ ID NO: 28 or Cir-SL2-crRNA-DR as encoded for by a polynucleotide having the sequence of SEQ ID NO: 29. eVLPs were produced by co-transfecting producer cells (HEK293 cells) with - A fusion protein of MMLV-Gag-3x nuclear export signal (NES)-Casl2a - VSV-G-Foldon and - Gag-POL Along with either SL2-crRNA as encoded for by a polynucleotide having the sequence of SEQ ID NO: 28 or Cir-SL2-crRNA-DR as encoded for by a polynucleotide having the sequence of SEQ ID NO: 29. EVs were produced by co-transfecting producer cells (HEK293 cells) with - A fusion protein of TSN2-Intein29-Casl2a and - VSV-G-Foldon Along with either SL2-crRNA as encoded for by a polynucleotide having the sequence of SEQ ID NO: 28 or Cir-SL2-crRNA-DR as encoded for by a polynucleotide having the sequence of SEQ ID NO: 29. For the Nanoblades, eVLPs and EVs, the medium was changed to Opti-MEM 6 hours after transfection. The conditional medium was harvested 2 days after transfection and EVs were isolated by TFF which were concentrated by 10 kD spin filters. EV concentrations were determined via ZetaView. The indicated doses were then incubated with HEK-SL2 reporter cells in 96-well plates for 96 hours. GFP-positive cells were quantified by FACS analysis. Figure 5 shows that the circular crRNA comprising an additional 3' DR region (Cir-SL2 crRNA DR) improves gene editing when either EVs, nanoblades or eVLPs are used as a delivery vehicle. Sequence Listing SEQ ID NO SEQUENCE 1 UGUU 2 AAUUUCUACUGUUGUAGAU 3 UAAUUUCUACUGUUGUAGAU 4 Skipped sequence 5 CUGCCAUCAGUCGGCGUGGACUGUAGAACCAUGCCGACUGAUGGCAG 6 Skipped sequence 7 GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG ecu 8 AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCAC GC 9 AACCAUGCCGACUGAUGGCAG 10 CUGCCAUCAGUCGGCGUGGACUGUAG 11 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNL TDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYRN RKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVST SIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHII ASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNE LNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKF KLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSE GFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNN PEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYK DLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLY WTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDT LYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQ AANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFD YQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLN FGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTG DFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRF TGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRN SNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKE SKDLKLQNGISNQDWLAYIQELRN KRPAATKKAGQAKKKK 12 GGGGS 13 GGGGSGGGGS 14 GGGGSGGGGSGGGGS 15 GGGGGG 16 GGGGGGGG 17 EAAAK 18 EAAAKEAAAK 19 EAAAKEAAAKEAAAK 20 AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA 21 MGRFRGGLRCIKYLLLGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVG LYVLVGAGALMMAVGFFGCCGAMRESQCVLGSFFTCLLVIFAAEVTTGVFAFIGKGV AIRHVQTMYEEAYNDYLKDRGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHK NCIDEIETIISVKLQLIGIVGIGIAGLTIFGMIFSMVLCCAIRNSRDVI 22 ATGGGCCGGTTTAGGGGCGGTCTGAGATGTATCAAGTACCTCCTCCTTGGTTTCA ACCTCCTG Illi GGTTGGCCGGAAGCGCTGTGATCGCCTTCGGATTGTGGTTTAG ATTCGGAGGCGCAATCAAGGAACTGTCATCTGAGGACAAGTCACCAGAGTACTTT TACGTGGGGCTCTATGTACTCGTGGGAGCCGGGGCCCTGATGATGGCCGTGGGT TTCTTCGGGTGTTGCGGAGCAATGAGGGAAAGCCAGTGCGTGCTTGGGTCTTTCT TTACTTGTCTGCTGGTCATCTTTGCCGCCGAGGTGACTACTGGTGTCTTTGCATTC ATCGGAAAGGGCGTCGCTATCAGGCACGTGCAGACCATGTATGAAGAAGCCTATA ACGACTATCTCAAGGACCGGGGAAAGGGTAACGGCACCCTTATCACATTTCACTC TAC Illi CAGTGCTGTGGCAAAGAATCAAGTGAGCAGGTCCAACCCACTTGTCCC AAAGAACTGCTCGGCCATAAGAACTGCATCGACGAAATCGAGACTATCATCTCCG TGAAGTTGCAGCTCATAGGGATCGTAGGCATCGGTATTGCTGGTTTAACTATCTTC GGTATGATCTTCAGCATGGTACTGTGTTGTGCTATTAGAAATAGCCGAGATGTTAT A 23 ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVI GLRIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQ ALADALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHN 24 ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLRARPVVSWFDQGTRDVI GLRIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQ ALADALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVHN 25 ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLRARPVVSWFDQGTRDVI GLRIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQ ALADALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVQN 26 UAAUUUCUACUGUUGUAGAUGGAGGACAGUACUCCGGAAA 27 UAAUUUCUACUGUUGUAGAUGGAGGACAGUACUCCGGAAAUAAUUUCUACUGU UGUAGAU 28 GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCUAACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUGGAGGACAG UACUCCGGAAACUGCCAUCAGUCGGCGUGGACUGUAGAACACUGCCAAUGCCG GUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC 29 GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCUAACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUGGAGGACAG UACUCCGGAAAUAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACU GUAGAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGU CCACGC 30 Illi 31 UUUU 32 TTTA 33 UUUA 34 AAAT 35 AAAU 36 AAUUUUUGUGCCCAUCGUUGGCAC 37 GUUGGAAUGACUAAUUUUUGUGCCCACCGUUGGCAC 38 GCAACACCUAAGAAAUCCGUCUUUCAUUGACGGG 39 GUUGCAAAACCCAAGAAAUCCGUCUUUCAUUGACGG 40 AUUUUUGUGCCCAUCGUUGGCAC 41 AGAAAUCCGUCUUUCAUUGACGG 42 AACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i2- 301UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAG 43 AACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i8-251UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAG 44 GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCUAACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i2- 30]UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACAC UGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC 45 GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCG CCUAACCAUGCCGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i8- 25]UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACAC UGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC 46 AAUUUCUACUGUUGUAGAUN[i2- 30]UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACCA UGCCGACUGAUGGCAGU 47 AAUUUCUACUGUUGUAGAUN[i8- 25]UAAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACCA UGCCGACUGAUGGCAGU 48 AAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACCAUGC CGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i2-3oiU 49 AAUUUCUACUGUUGUAGAUCUGCCAUCAGUCGGCGUGGACUGUAGAACCAUGC CGACUGAUGGCAGUAAUUUCUACUGUUGUAGAUN[i8-25iU 50 AAUUUCUACUGUUGUAGAUN[i2-3oiU 51 AAUUUCUACUGUUGUAGAUN[i8-25iU

Claims

1. A Casl2 RNP complex for use with extracellular vesicles (EVs) comprising:a. a first fusion protein comprising an EV polypeptide and a Casl2 protein; andb. a circular crRNA comprising:i. a spacer sequenceii. a direct repeat region to the 5' of the spacer sequence (5' DR region); andiii. a direct repeat region to the 3' of the spacer sequence (3' DR region); wherein the Casl2 protein is bound to either the 5' DR region or the 3' DR region.

2. The Casl2 RNP complex of claim 1, wherein the 5' DR region and the 3' DR region comprise a pseudoknot structure, preferably wherein the pseudoknot structure comprises or consists of a nucleotide sequence having 90%, 91%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 over its entire length.

3. The Casl2 RNP complex of claim 1 or claim 2, wherein the 3' end of the 3' DR repeat region is connected to the 5' end of the 5' DR repeat region via a nucleotide linker comprising a stem loop structure, preferably comprising a stem of 14 to 25 base pairs, more preferably 17 to 21 base pairs or most preferably 19 base pairs and a loop of 4 to 14 bases, more preferably 7 to 11 bases or most preferably 9 bases.

4. The Casl2 RNP complex of any one of the preceding claims, wherein the Casl2 protein is Casl2a, preferably derived from Acidaminococcus BV3L6.

5. The Casl2 RNP complex of any one of the preceding claims, wherein the Casl2 protein is fused to a domain of the EV polypeptide that is displayed in the EV lumen.

6. The Casl2 RNP complex of any one of the preceding claims, wherein the Casl2 protein is fused to the EV polypeptide via an intein.

7. The Casl2 RNP complex of any one of the preceding claims, wherein:a. the Casl2 protein of the first fusion protein is bound to the 5' DR region and a second Casl2 protein is bound to the 3' DR region; orb. the Casl2 protein of the first fusion protein is bound to the 3' DR region and a second Casl2 protein is bound to the 5' DR region.

8. The Casl2 RNP complex of any one of claims 1 to 6, further comprising a second fusion protein comprising an EV polypeptide and a Casl2 protein, wherein:a. the Casl2 protein of the first fusion protein is bound to the 5' DR. region and the Casl2 protein of the second fusion protein is bound to the 3' DR region; orb. the Casl2 protein of the first fusion protein is bound to the 3' DR region and the Casl2 protein of the second fusion protein is bound to the 5' DR region.

9. The Casl2 RNP complex of claim 7 or claim 8, wherein the second Casl2 protein or the Casl2 protein of the second fusion protein is a Casl2a, preferably derived from Acidaminococcus BV3L6.

10. The Casl2 RNP complex of claim 8 or claim 9, wherein the first and the second fusion proteins have the same amino acid sequence.

11. The Casl2 RNP complex of any one of the preceding claims, wherein the Casl2 protein of the first fusion protein is bound to the 5' DR region and the 5' DR region is cleaved by the Casl2 protein, preferably to the 5' of the pseudoknot structure.

12. The Casl2 RNP complex of any one of the preceding claims, wherein the Casl2 protein of the first fusion protein is bound to the 3' DR region and the 3' DR region is cleaved by the Casl2 protein, preferably to the 5' of the pseudoknot structure.

13. The Casl2 RNP complex of any one of claims 7 to 10, wherein the 5' DR region is cleaved, preferably to the 5' of the pseudoknot structure, and the 3' DR region is cleaved, preferably to the 5' of the pseudoknot structure.

14. A method of producing an EV or a population of EVs comprising the following steps:a. introducing into an EV producer cell:i. a polynucleotide encoding for a circular crRNA, wherein the circular crRNA comprises:

1. a spacer sequence;2. a direct repeat region to the 5' of the spacer sequence (5' DR region); and3. a direct repeat region to the 3' of the spacer sequence (3' DR region);andii. a polynucleotide encoding for a fusion protein, wherein the fusion protein comprises an EV polypeptide and a Casl2 protein;b. expressing the polynucleotide encoding for the circular crRNA and the polynucleotide encoding for the fusion protein at the same time in the EV producer cell, preferably under conditions which, in no particular order,i. the Casl2 protein binds the 5' DR. region or the 3' DR region of the circular crRNA andii. the Casl2 protein bound to circular crRNA loads into an EV.

15. The method of claim 14, wherein the method produces an EV or a population of EVs comprising a Casl2 RNP complex as defined in any one of claims 1 to 13.

16. The method of claim 14 or claim 15 wherein the polynucleotide encoding for the circular crRNA comprises or consists of, from 5' to 3':a. a 5' self-cleaving ribozyme sequence;b. a 5' connecting linker;c. a sequence encoding the 5' DR region;d. a spacer sequence;e. a sequence encoding the 3' DR region;f. a 3' connecting linker; andg. a 3' self-cleaving ribozyme sequence.wherein, following the self-cleavage of the ribozyme sequences, the 5' connecting linker and the 3' connecting linker are capable of forming a double stranded RNA duplex structure through complementary base pairing, wherein the 5' connecting linker comprises 1-5, preferably 2-4, more preferably 2-3 or most preferably 2 nucleotides at its self-cleaved 5' end that are not comprised in the RNA duplex and the 3' component of a 3'DR to 5'DR connecting linker comprises 1-15, preferably 2-10, more preferably 5-9 or most preferably 7 nucleotides at its self-cleaved 3' end that are not comprised in the RNA duplex.

17. The method of any one of claims 14 to 16, wherein the method further comprises a further step of purifying the EVs produced.

18. An EV comprising the Casl2 RNP complex of any one of claims 1 to 13.

19. An EV directly obtained by the method of any one of claims 14 to 1720. The EV of claim 18 or 19, wherein the EV is an exosome or microvesicle.

21. The EV of any one of claims 18 to 20, wherein the Casl2 protein of the first fusion protein is fused to the EV polypeptide of the first fusion protein via an intein, wherein the intein is self-cleaved, preferably at its C-terminus, to release the Casl2 protein of the first fusion protein from the EV polypeptide of the first fusion protein.

22. The EV of any one of claims 18 to 21, wherein the EV comprises the Casl2 RNP complex of claims 8 to 13, wherein the intein of the second fusion protein is self-cleaved,preferably at its C-terminus, to release the Casl2 protein of the second fusion protein from the EV polypeptide of the second fusion protein.

23. A population of EVs comprising a plurality of the EVs of any one of claims 18 to 22.

24. A pharmaceutical composition comprising a Casl2 RNP complex of claims 1 to 13, an EV of claims 18 to 22 or a population of EVs of claim 23 and a pharmaceutically acceptable excipient and / or carrier.

25. A method of delivering a Casl2 RNP complex to a target cell, comprising contacting or incubating a target cell with an EV of claims 18 to 22, a population of EVs of claim 23 or a pharmaceutical composition of claim 24.

26. An in vitro or ex vivo method of gene editing in a target cell comprising introducing into the cell an EV of claims 18 to 22, a population of EVs of claim 23 or a pharmaceutical composition of claim 24.

27. An EV of claims 18 to 22, a population of EVs of claim 23 or a pharmaceutical composition of claim 24 for use in a method of treatment or prevention of a disease in a subject, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder.

28. Use of an EV of claims 18 to 22, a population of EVs of claim 23 or a pharmaceutical composition of claim 24 in the manufacture of a medicament for treatment or prevention of a disease in a subject, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder.

29. A method for treating or preventing a disease in a subject comprising administering a therapeutically or prophylactically effective amount of an EV of claims 18 to 22, a population of EVs of claim 23 or a pharmaceutical composition of claim 24 to a subject suffering from or susceptible to the disease, preferably wherein the disease is a genetic disorder, most preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder.s

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