Improved crispr / cas guide RNA (GRNA)

Abstract

Modified guide RNAs for EV-mediated delivery of gene editing machinery, complexes, EVs, EV producer cells and pharmaceutical compositions comprising the same, and uses thereof, for example to treat genetic diseases and disorders.

Description

[0001]Improved guide RNA (gRNA) Technical Field The present invention relates to an improved guide RNA (gRNA). The present invention also relates to uses of said gRNA in therapy. Background Art The CRISPR / Cas system, in particular the CRISPR / Cas9 system is an invaluable tool for genome modification. A Cas nuclease such as Cas9 introduces a DNA double-strand break (DSB) in a target sequence that is complementary to a 20-nt spacer sequence of its bound guide RNA (gRNA) in the presence of a protospacer adjacent motif (PAM). The gRNA can be provided as duplex of spacer containing CRISPR RNA (crRNA) and trans- activating crRNA (tracrRNA). The gRNA may optionally be a single gRNA (sgRNA), where crRNA and tracrRNA are fused by an artificial loop (Jinek et al., 2012). A gRNA consists of the target-specific spacer and a constant part typically comprised of distinct motifs including the nexus, and a first and second hairpin (Briner et al., 2014). Cellular repair of targeted DNA DSBs by error-prone end-joining pathways often lead to frameshift mutations that enable knockout studies of genes of interest and DNA donor enabled homology-directed repair (HDR) allows precise control of the inserted mutation. One widely used approach to improve gRNA targeting and therefore, genome editing efficiency is the use of chemical modifications in synthesized gRNA. These have included 5'- and 3'-end protection using phosphorothioate bonds and internal 2'-OMe residues, to improve its stability against degradation by nucleases (Hendel et al., 2015; Yin et al., 2017). The editing efficiency can be further increased by providing the cell with external non-homologous DNA and it is speculated that this non-homologous oligonucleotide enhancement (NOE) diverts cells towards error-prone instead of error-free repair pathways (Richardson et al., 2016). This effect is also achieved when using DNA as electroporation enhancer or providing a DNA donor as a repair template for HDR. Elongated crRNA / tracR hybridization as present in the bacterial CRISPR target complex, and an A-T flip that removes the TTTT transcription termination signal, improve genome editing compared to standard in-vivo transcribed sgRNA (Chen et al., 2013). These modifications are combined into Hybridization extended A-T inversion (HEAT) gRNA. Improved Cas9 activity on certain target sequences was achieved by sequence alterations, i.e. deletion and / or replacement of uridines in the linker region (34A) and in the first hairpin (U39G-U40G) of the tracrRNA (Scott et a., 2019 and PCT / US2018 / 06605). Further, genome editing efficiencies vary strongly from target to target and some targets are totally intractable to genome editing even when chemically stabilized gRNAs, HEAT gRNAs, and NOE are employed. Finally, delivery of the Cas9 / gRNA ribonucleoprotein (RNP) complex into the cystol and subsequently the nucleus of recipient cells remains challenging. In particular, the large size of Streptococcus pyogenes Cas9 (4113 nucleotides / 1371 amino acids) poses a significant challenge as this exceeds the passenger capacity of regular AAV viral vectors for DNA-based delivery of Cas9 (and AAV vectors are also unable to deliver the CRISPR / Cas9 machinery in RNP format), and it remains challenging to encapsulate such a large protein (and the corresponding RNP) in nanoparticles (Rehman et al., 2016). To date, LNP-mediated co-delivery of gRNA and mRNA encoding for Cas9 has proven to work well for editing target genes in hepatocytes preclinically and clinically, but said system is (i) currently limited to liver targets only, (ii) delivers gRNA and nuclease separately which may lead to kinetic and stoichiometric differences between the gRNA and Cas9, (iii) and LNP delivery risks triggering innate immune reactions and other tolerability issues in humans. One approach to addressing this problem is to use engineered extracellular vesicles (EVs), such as exosomes, capable of cell type-specific targeting and endosomal release, as a delivery vehicle for a bioactive cargo at a desired intracellular location, such as the cytoplasm of a cell. Extracellular vesicles (EVs), including exosomes and microvesicles among other subtypes, are nanometre sized particles released by virtually every cell type and are involved in the transfer of functionally active proteins, lipids, and nucleic acids between cells (Yáñez-Mó et al., 2015). EVs play important roles in many physiological and pathological processes related to cancer (Steinbichler et al., 2017), cardiovascular (Osteikoetxea et al., 2016), and neurodegenerative diseases (Soria et al., 2017). EVs also play a significant role in regulating intercellular communication by transferring functional proteins, metabolites, and other biological products between cells. Potential benefits of using EV-mediated delivery include being able to achieve intracellular delivery of the CRISPR / Cas complex, the ability to deliver multiple CRISPR / Cas complexes to a target cell with a single EV, the potential to achieve cell-type specific targeting, the potential to protect the CRISPR / Cas complex from degradation in the body, and the potential to prevent an adverse reaction or immune response being triggered by the CRISPR / Cas complex. In addition, there are advantages to EV-mediated delivery for gene editing specifically, as compared with existing delivery methods in the field. As compared with mRNA approaches, delivery of a CRISPR / Cas complex by an EV would allow for the gene editing machinery to remain present in a target cell for long enough for gene editing to take place. However, the expression of the gene editing machinery is still transient, thus offering strong safety advantages as compared with viral-mediated delivery approaches, such as adeno-associated virus (AAV). EV-mediated gene editing thus may allow for the treatment of paediatric diseases and disorders, where the long-term expression of gene editing machinery and off-target editing is of particular concern. However, using EVs to deliver CRISPR / Cas complexes has resulted in relatively low levels of gene editing being achieved in target cells. It is believed that these relatively low levels of gene editing may occur due to inefficient loading of CRISPR / Cas complexes (e.g. a Cas9 / gRNA RNP) into EVs, due to the size of the CRISPR / Cas protein (i.e. the RNP) or its volume to surface area ratio. Inefficient loading can reduce the number of copies of CRISPR / Cas protein (i.e. the RNP) present in the EV and therefore the editing efficiency is reduced. Hence, there is a need to increase the efficiency of gene editing when using EVs to deliver CRISPR / Cas complexes to target cells. There remains a need for engineered EVs (such as exosomes) suitable for delivery of CRISPR / Cas complexes (e.g. a Cas9 / gRNA RNPs) loaded in such a way that does not affect editing efficiency. Summary of invention It is desirable to solve the problem of using EVs to deliver CRISPR / Cas complexes, whilst achieving high gene editing efficiencies. As outlined above, it was believed that relatively low levels of editing occur when using EVs to deliver CRISPR / Cas9 complexes because of inefficient loading of the CRISPR protein, perhaps due to its relatively large size. However, the present inventors have found that, surprisingly, making modifications to the guide RNA results in much improved gene editing when EV mediated delivery of CRISPR / Cas complexes is employed. In addition, the present inventors also designed new guide RNAs with particular combinations of modifications and further previously unknown adaptations of these modifications. The resulting new guide RNAs are particularly effective at achieving high levels of gene editing when used in for EV-mediated delivery. It is also desirable to improve the genome editing efficiency of target genes by optimizing the gRNA designs to allow improved encapsulation of Cas9 / gRNA into engineered extracellular vesicles. The present inventors have overcome the problem that only Cas9 protein, not the Cas9 / gRNA RNP complex can be successfully delivered into a target cell by an engineered EV. It was further identified that gRNA did not bind to Cas9 inside of the EVs, meaning editing of a target gene could not be carried out. To address this problem, the inventors have identified a number of modified gRNAs, which may optionally be modified sgRNAs, that are able to bind to Cas9 inside of engineered EVs and achieve delivery of the large RNP complex to reporter cells. Upon gRNA transfection, the editing efficiency in reporter cells significantly increased, indicating the gRNA loading in EVs may be the limitation for high gene editing efficiency in recipient cells. It is, furthermore, desirable to improve gRNA loading into EVs by improving gRNA expression, the stability of the gRNA and the binding capacity of the gRNA to Cas9. The invention can be described as the following aspects: In one aspect the present invention provides a CRISPR / Cas system guide RNA, wherein the guide RNA comprises a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA); wherein the crRNA comprises from 5’ to 3’ a target-specific spacer sequence and a repeat region;wherein the tracrRNA comprises from 5’ to 3’ an anti-repeat region, a nexus sequence, a first hairpin loop and a second hairpin loop; wherein the repeat region of the crRNA and the anti-repeat region of the tracrRNA form a double stranded repeat / anti-repeat RNA duplex structure through complementary base pairing; wherein the double stranded repeat / anti-repeat RNA duplex structure comprises a lower stem, a bulge and an upper stem; wherein the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises fewer than four consecutive uracil nucleotides; wherein the upper stem of the double stranded repeat / anti-repeat RNA duplex has a length of at least 5 contiguous complementary base pairs wherein the first hairpin loop comprises a stem having a length of at least 5 contiguous complementary base pairs and / or wherein at least 2 of the contiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs. Preferably wherein (i) the target-specific spacer sequence is capable of hybridizing to a target sequence in a genomic locus of interest in a cell; further wherein the guide RNA is capable of effecting the manipulation of a target nucleic acid within a prokaryotic or eukaryotic cell when in complex within the cell with a CRISPR protein; and / or (ii) the guide RNA has increased stability and / or exhibits more stable binding to a CRISPR protein as compared with a guide RNA having SEQ ID NO.1. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises in 5’ to 3’ direction: the target- specific spacer sequence, the double stranded repeat / anti-repeat RNA duplex structure, the nexus sequence, the first hairpin loop and the second hairpin loop. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the lower stem of the double stranded repeat / anti-repeat RNA duplex is proximal to the spacer and the nexus; the upper stem of the double stranded repeat / anti-repeat RNA duplex is distal from the spacer and the nexus; the bulge of the double stranded repeat / anti-repeat RNA duplex is positioned between the lower stem and the upper stem; the lower stem is formed of 5 contiguous complementary base pairs; and the bulge is formed by nucleotides that are not complementary base paired. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the upper stem is formed of 9 contiguous complementary base pairs, preferably wherein 5 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the repeat region of the crRNA that forms the upper stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a repeat sequence having at least 80%, at least 90% or having 100% sequence identity to the nucleotide sequence SEQ ID NO 60; further wherein the anti-repeat region of the tracrRNA that forms the upper stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a repeat sequence having at least 80%, at least 90% or having 100% sequence identity to the nucleotide sequence SEQ ID NO 61. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the repeat region of the crRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises the nucleotide sequence UUUVA (SEQ ID NO 64); further wherein the anti-repeat region of the tracrRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises the nucleotide sequence UBAAA (SEQ ID NO 65); further wherein (i) the “V” of the UUUVA (SEQ ID NO 64) is A and the “B” of the UBAAA (SEQ ID NO 65) is U, (ii) the “V” of the UUUVA (SEQ ID NO 64) is C and the “B” of the UBAAA (SEQ ID NO 65) is G, or (iii) preferably the “V” of the UUUVA (SEQ ID NO 64) is G and the “B” of the UBAAA (SEQ ID NO 65) is C. In a further preferred embodiment, the CRISPR / Cas system guide RNA of the present invention the repeat region of the crRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 81; further wherein the anti-repeat region of the tracrRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 82, wherein: (i) the “V” of the SEQ ID NO 81 is A and the “B” of the SEQ ID NO 82 is U (ii) the “V” of the SEQ ID NO 81 is C and the “B” of the SEQ ID NO 82 is G; or (iii) preferably the “V” of the SEQ ID NO 81 is G and the “B” of the SEQ ID NO 82 is C. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the double stranded repeat / anti-repeat RNA duplex structure comprises a repeat sequence having SEQ ID NO 19; further wherein the double stranded repeat / anti-repeat RNA duplex structure comprises an anti-repeat sequence having SEQ ID NO 20; further wherein (i) the “V” of the SEQ ID NO 19 is A and the “B” of the SEQ ID NO 20 is U, (ii) the “V” of the SEQ ID NO 19 is C and the “B” of the SEQ ID NO 20 is G, or (iii) preferably, the “V” of the SEQ ID NO 19 is G and the “B” of the SEQ ID NO 20 is C. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA is a single guide RNA (sgRNA) molecule comprising a stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. Preferably wherein the stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA comprises or consists of the nucleotide sequence GAAA (SEQ ID NO 83) or AAGA (SEQ ID NO 84) In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 30; further wherein (i) the “V” of the SEQ ID NO 30 is A and the “B” is U, (ii) the “V” of the SEQ ID NO 30 is C and the “B” is G, or (iii) preferably, the “V” of the SEQ ID NO 30 is G and the “B” is C. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 21. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the first hairpin loop is a locked hairpin. Preferably, the locked hairpin comprises a super stable loop. In a further preferred embodiment, the locked hairpin has a melting temperature (Tm) of above 65 degrees. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the first hairpin loop comprises a stem having a length of contiguous 8 complementary base pairs, preferably wherein 4 of the contiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs. Preferably, the first hairpin loop comprises a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.22 or SEQ ID NO 25. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the nexus sequence comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 15 or SEQ ID NO 26. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA further comprises a terminator sequence at its 3’ end, preferably wherein the terminator sequence comprises or consists of SEQ ID NO 18. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, guide RNA (i) does not comprise any additional modifications between the second hairpin loop and the terminator sequence; or (ii) comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides, or preferably does not comprise any nucleotides, between the second hairpin loop and the terminator sequence. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, guide RNA (i) comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides or preferably does not comprise any nucleotides to the 5’ of the spacer sequence; and / or (ii) comprises fewer than 7 nucleotides, fewer than 6 nucleotides, fewer than 5 nucleotides, fewer than 4 nucleotides, fewer than 3 nucleotides, fewer than 2 nucleotides or preferably does not comprise any nucleotides between the spacer sequence and the repeat region of the crRNA. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the target-specific spacer sequence is between 18 and 52 nucleotides in length, preferably wherein the target-specific spacer sequence is between 17 and 30 nucleotides in length. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA further comprises a region between the nexus and the first hairpin loop comprising or consisting of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.41. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the second hairpin loop comprises a stem having 6 contiguous complementary base pairs, preferably wherein 5 of the contiguous complementary base pairs of the stem of the second hairpin loop are C-G or G-C base pairs. Preferably, the second hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.17. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises from 5’ to 3’ the spacer sequence, SEQ ID NO.21, SEQ ID NO.26, SEQ ID NO.16; SEQ ID NO.17 and SEQ ID NO.18. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises from 5’ to 3’, a spacer sequence and a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 27; further wherein the guide RNA comprises: (i) a G at a residue corresponding to position 5 of SEQ ID NO: 27; (ii) AAGCUG at residues corresponding to position 12 to 17 of SEQ ID NO: 27; (iii) CAGCUU at residues corresponding to position 22 and 27 of SEQ ID NO: 27; (iv) a C at a residue corresponding to position 36 of SEQ ID NO 27; and (v) GGACUUCGGUCC at residues corresponding to position 63 to 74 of SEQ ID NO 27. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA further comprises a region between the nexus and the first hairpin loop comprising or consisting of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.42. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the second hairpin loop comprises a stem having 7 contiguous complementary base pairs, preferably wherein 6 of the contiguous complementary base pairs of the stem of the second hairpin loop are C-G or G-C base pairs. Preferably, the second hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 24. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises from 5’ to 3’ the spacer sequence, SEQ ID NO.21, SEQ ID NO.26, SEQ ID NO.23; SEQ ID NO.24 and SEQ ID NO.18. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises from 5’ to 3’, a spacer sequence and a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 28; further wherein the guide RNA comprises: (i) a G at a residue corresponding to position 5 of SEQ ID NO: 28; (ii) AAGCUG at residues corresponding to position 12 to 17 of SEQ ID NO: 28; (iii) CAGCUU at residues corresponding to position 22 and 27 of SEQ ID NO: 28; (iv) a C at a residue corresponding to position 36 of SEQ ID NO 28; (v) GGACUUCGGUCC at residues corresponding to position 63 to 74 of SEQ ID NO 28; (vi) UA at residues corresponding to positions 55 and 56 of SEQ ID NO: 28; (vii) a C at a residue corresponding to position 85 of SEQ ID NO: 28; and (viii) a G at a residue corresponding to position 91 of SEQ ID NO: 28. In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA is of the +85 sgRNA architecture In a further preferred embodiment of the CRISPR / Cas system guide RNA of the present invention, the guide RNA comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In a further aspect, the present invention provides at least one nucleic acid molecule encoding a guide RNA of the present invention. In a further aspect, the present invention provides a library comprising a plurality of guide RNAs of the present invention comprising different target- specific spacer sequences and a constant gRNA backbone. In a further aspect, the present invention provides a complex comprising a Cas enzyme and a guide RNA of the present invention, preferably wherein the Cas enzyme is Cas9. In a further aspect, the present invention provides a method of preparing an EV comprising introducing into an EV-producing cell (i) a guide RNA of the present invention; or (ii) a construct encoding a guide RNA of the present invention and expressing the construct in the EV-producing cell; thereby generating EVs comprising a guide RNA of the present invention. Preferably, the construct is expressed under the control of the human U6 promoter. Further preferably, the method further comprises introducing into the EV- producing cell (i) Cas9 or (ii) a construct encoding a Cas9 and expressing the construct in the EV-producing cell, thereby generating EVs comprising a guide RNA of the present invention and a Cas9, preferably wherein the Cas9 is fused to an EV polypeptide. In a further aspect, the present invention provides an EV directly obtained by a method of the present invention. In a further aspect, the present invention provides an engineered extracellular vesicle (EV) for delivery of a bioactive cargo, the engineered EV comprising: (i) the guide RNA of the present invention or the complex one of the present invention; and optionally, (ii) an endosomal escape enhancer. In a further aspect, the present invention provides a composition comprising a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, or an EV of the present invention, and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier. In a further aspect, the present invention provides a composition comprising a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, or an EV of the present invention, for use as a medicament. In a further aspect, the present invention provides a composition comprising a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, or an EV of the present invention for the preparation of a medicament for treatment or prevention of a disease in a subject. In a further aspect, the present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a guide RNA of any one of a a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, or an EV of the present invention to a subject suffering from or susceptible to the disease. In a preferred embodiment of the uses or methods of the present invention, the subject is a mammal, preferably a human. In a further aspect, the present invention provides a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, or an EV of the present invention. Preferably, wherein the method is in vivo, in vitro or ex vivo. The present invention further provides a complex comprising a fusion protein and a guide RNA, wherein the fusion protein comprises an EV polypeptide and a CRISPR protein, wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification. The present invention further provides an EV comprising a guide RNA, wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification. The present invention further provides an EV comprising CRSIPR / Cas complex, wherein the CRISPR / Cas complex comprises a guide RNA bound to a CRSIPR protein, further wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification. The present invention further provides an EV comprising the complex of the present invention. In one embodiment of the complex or EV of the present invention the CRISPR protein is Cas12a or preferably Cas9. In one embodiment of the complex or EV of the present invention the modification changes the nucleotide sequence of the structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / or ii. a guide RNA having SEQ ID NO 1. In one embodiment of the complex or EV of the present invention the first structural component is a stem-loop or an RNA duplex. In one embodiment of the complex or EV of the present invention the modification is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component wherein the extension modification is an increase in the number of complementary base pairs present in the structural component. In one embodiment of the complex or EV of the present invention the guide RNA comprises, preferably from 5’ to 3’, a target-specific spacer sequence, a repeat / anti repeat RNA duplex, a nexus, a first hairpin and a second hairpin; wherein the repeat / anti repeat RNA duplex comprises an upper stem, a bulge and a lower stem. In one embodiment of the complex or EV of the present invention the first structural component comprising a modification is a modified upper stem, a modified first hairpin or a modified second hairpin. In one embodiment of the complex or EV of the present invention the guide RNA further comprises a second structural component, wherein the second structural component is a functional module for CRISPR protein binding, wherein the second structural component comprises a modification. I In one embodiment of the complex or EV of the present invention the modification in the second structural component changes the nucleotide sequence of the second structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / or ii. a guide RNA having SEQ ID NO 1. In one embodiment of the complex or EV of the present invention the second structural component is a stem-loop or an RNA duplex; preferably wherein i. the first structural component is an RNA duplex and the second structural component is a stem loop or ii. the first structural component is a stem loop and the second structural component is a stem loop. In one embodiment of the complex or EV of the present invention the modification in the second structural component is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component In one embodiment of the complex or EV of the present invention i. the first structural component comprising a modification is a modified upper stem and the second structural component comprising a modification is a modified first hairpin; ii. the first structural component comprising a modification is a modified upper stem and the second structural component comprising a modification is a modified second hairpin; or iii. the first structural component comprising a modification is a modified first hairpin and the second structural component comprising a modification is a modified second hairpin. In one embodiment of the complex or EV of the present invention the guide RNA further comprises a third structural component, wherein the third structural component is a functional module for CRISPR protein binding, wherein the structural component comprises a modification. In one embodiment of the complex or EV of the present invention the third structural component changes the nucleotide sequence of the third structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / or ii. a guide RNA having SEQ ID NO 1. In one embodiment of the complex or EV of the present invention the third structural component is a stem loop or an RNA duplex, preferably wherein i. the first structural component is an RNA duplex, the second structural component is a stem loop and the third structural component is a stem loop; or ii. the first structural component is a stem loop, the second structural component is a stem loop and the third structural component is a stem loop; In one embodiment of the complex or EV of the present invention the third structural component is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component. In one embodiment of the complex or EV of the present invention the first structural component comprising a modification is a modified upper stem, the second structural component comprising a modification is a modified first hairpin and the third structural component comprising a modification is a modified second hairpin. In one embodiment of the complex or EV of the present invention the modified upper stem comprises at least 5 contiguous complementary base pairs, preferably wherein the modified upper stem comprises 9 contiguous complementary base pairs wherein 5 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs. In one embodiment of the complex or EV of the present invention the modified first hairpin comprises a stem having a length of at least 5 contiguous complementary base pairs, preferably wherein the first hairpin comprises a stem having a length of 8 complementary base pairs wherein 4 of the contiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs. In one embodiment of the complex or EV of the present invention the modified second hairpin comprises a stem having 7 contiguous complementary base pairs, preferably wherein 6 of the contiguous complementary base pairs are C-G or G-C base pairs. In one embodiment of the complex or EV of the present invention the guide RNA comprises a modification in the lower stem; wherein the modified lower stem comprises fewer than four consecutive uracil nucleotides. In one embodiment of the complex or EV of the present invention i. the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides, or preferably does not comprise any nucleotides, between the second hairpin loop and the terminator sequence; ii. the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides or preferably does not comprise any nucleotides to the 5’ of the target-specific spacer sequence; and / or iii. the modified guide RNA comprises fewer than 7 nucleotides, fewer than 6 nucleotides, fewer than 5 nucleotides, fewer than 4 nucleotides, fewer than 3 nucleotides, fewer than 2 nucleotides or preferably does not comprise any nucleotides between the spacer sequence and the repeat / anti-repeat region. In one embodiment of the complex or EV of the present invention the guide RNA exhibits more stable binding to the CRISPR protein as compared with (i) a guide RNA having SEQ ID NO.1 and / or (ii) a guide RNA comprising a constant region having SEQ ID NO.40. In one embodiment of the complex or EV of the present invention the guide RNA comprises or consists, preferably from 3’ to 5’ of a target-specific spacer sequence and a constant region, wherein the cinstant region comprises a sequence having at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 63, SEQ ID NO 27 or SEQ ID NO 28. In one embodiment of the EV of the present invention, the EV is an exosome or a micovesicle. In one embodiment of the EV of the present invention, the EV is derived from a HEK cell, preferably a HEK293 cell. In one embodiment of the EV of the present invention, the EV comprises a fusogenic protein or an endosomal escape enhancer. 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 composition comprising a complex, an EV or a population of EVs of the present invention and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier. In a further aspect, the present invention provides a complex, an EV, a population of EVs or a composition of the present invention for use in treatment or prevention of a disease in a subject. In a further aspect, the present invention provides a complex, an EV, a population of EVs or a composition of the present invention for the preparation of a medicament for treatment or prevention of a disease in a subject. In a further aspect, the present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a complex, an EV, a population of EVs or a composition of the present invention to a subject. Brief Description of Figures The disclosure will be more clearly understood by reference to the accompanying Figures, in which: Figure 1A shows a schematic illustration of the guide RNA designs according to embodiments of the present disclosure. The new “M1L” guide on the right- hand side includes an extension to the upper stem of the repeat / anti-repeat double stranded RNA duplex. As compared to the extended upper stem modification of the known “RNAFE” guide RNA, the extended upper stem of M1L has a U-A to A-U flip. Further, as compared to the to the lower stem modification of the known “RNAFE” guide RNA, the new M1L guide has the fourth U-A base pair substituted for a G-C base pair, rather than flipped. The new M1L also comprises the known “T-Lock” modification where the first hairpin is extended to form a super stable hairpin structure. Figure 1B shows a schematic illustration of the guide RNA designs according to embodiments of the present disclosure. The new “M1LR” guide shown at the bottom includes all the modifications present in M1L, but in addition also includes the substitution of “AU” to “UA” between the nexus and the first hairpin loop, as well as an additional C-G base pair in the second hairpin loop. Figure 2A-E shows a schematic illustration of the stability of different modifications according to embodiments of the present disclosure. Figure 3A shows a schematic illustration of the stability of the guide RNA design according to embodiments of the present disclosure. Figure 3B shows a schematic illustration of the stability of the guide RNA design according to embodiments of the present disclosure. Figure 4A shows a schematic illustration of the protein constructs investigated according to different embodiments of the present disclosure. Figure 4B shows a graph illustrating editing efficiencies. Figure 5A-D shows a series of graphs illustrating editing efficiencies. Figure 6A-C shows a series of graphs illustrating editing efficiencies. Figure 7A-D shows a series of graphs illustrating the improved editing efficiencies of the guide RNA designs according to the present invention. Figure 7E shows a schematic illustration of a guide comprising the known “RNAFE” modification, which includes an extension to the upper stem of the repeat / anti-repeat double stranded RNA duplex and a U-A to A-U flip in the lower stem of the repeat / anti-repeat double stranded RNA duplex. Figure 7F shows a schematic illustration of a guide comprising the known “T- Lock” modification, which includes an extension to the first hairpin to form a super-stable hairpin structure. Figure 7G on the left-hand side shows a schematic illustration of a guide comprising extension to the upper stem of the repeat / anti-repeat double stranded RNA duplex of the known “RNAFE” modification. The schematic shows that the known “RNAFE” modification also includes a U-A to A-U flip in the lower stem of the repeat / anti-repeat double stranded RNA duplex, however also shows further adaptations of this design where the U-A is instead replaced with C-G (RNAFE4C) or with G-C (RNAFE4G). On the right side, the graph shows that comparable editing efficiencies are achieved whether the U- A or replaced with C-G or G-C. Figure 8A-B shows a set of Confocal microscopy images confirming functionality of the guide RNA designs according to certain aspects of the present invention. Figure 9A-C shows a series of graphs illustrating the improved editing efficiencies of the guide RNA designs according to the present invention. Figure 10 shows a set of Confocal microscopy images confirming functionality of the guide RNA designs according to certain aspects of the present invention. Figure 11A-C shows a series of graphs illustrating the improved editing efficiencies of the guide RNA designs according to the present invention. Figure 12A-C shows a series of graphs illustrating the improved editing efficiencies of the guide RNA designs according to the present invention. Figure 13A-B shows a series of graphs illustrating the improved editing efficiencies of the guide RNA designs according to the present invention. Figure 14A-B shows graphs illustrating that editing efficiency can be improved by optimising the promoter under which the guide RNA is expressed in EV producer cells. Figure 15A-C shows graphs illustrating that EV-mediated delivery of the new “M1L” and “M1LR” designs and Cas9 also achieves efficient editing of endogenous gene targets. Figure 16A shows a graph illustrating that EV-mediated delivery of the new “M1L” and Cas9 achieves efficient gene editing when the Cas9 is loaded by fusion to a range of different EV polypeptides (Myr, a single pass EV membrane polypeptide, TSN2 and GAG). Figure 16B shows a graph confirming that EV-mediated delivery of the new “M1LR” and Cas9 achieves efficient gene editing when the Cas9 is loaded by fusion to either TSN2 or CD63. Figure 17A shows a graph illustrating that EV-mediated delivery of the new “M1LR” design and Cas9 in vivo results in highly efficient gene editing. Figure 17B shows a graph illustrating that EV-mediated delivery of the new “M1L” design and Cas9 results more efficient gene editing than delivery of the new “M1L” design and Cas9 using the gold standard transfection agent for the delivery of CRIPR / RNP complexes. Figure 18 shows a graph confirming that EV-mediated delivery of the guide RNA and Cas9 results in efficient gene editing when different endosomal escape enhancers (VSVG, CNVG or RVG) are used. Detailed Description of the Invention The present inventors sought to take advantage of the benefits of EV mediated delivery for delivering CRISPR / Cas complexes to target cells, to allow for specific gene editing. However, prior to the present invention, poor gene editing was observed when EVs were used to deliver CRISPR / Cas complexes to target cells. It was believed that this could be due to inefficient loading of the CRISPR protein into the EVs, perhaps due to its relatively large size. However, surprisingly, the present inventors found that modifying the guide RNA allows for more efficient gene editing when EVs are used to deliver CRISPR / Cas complexes to target cells. Without wishing to be bound by theory, it is believed that the modifications made to the guide RNAs increase their stability, in particular the stability of their secondary structure. It is believed that the modified guide RNAs have increased affinity for the Cas protein and that, once bound, the interaction between the Cas and the guide RNA is more stable. These guide RNA modifications are advantageous for EV-mediated delivery of CRISPR / Cas complexes, since they are believed to allow for increased loading of CRISPR / Cas complexes into EVs. The modified guides are believed to be better able to bind the Cas proteins that are being localised to the EVs (e.g. by fusion to an EV polypeptide). It is believed that the modified guide RNAs, and the CRISPR / Cas complexes they form, have a longer residence time in the cytoplasm of the producer cells and the CRISPR / Cas complexes are more stable as the multivesicular body is forming, thus more CRISPR / Cas complexes are encapsulated. Such guide RNA modifications are also believed to be of particular importance in target cells, due to the nature of EV-mediated delivery. Following uptake of EVs by target cells, the EVs are trafficked through the endolysomal system and the cargo of the EV is believed to be released at the surface of the Endoplasmic Reticulum. Hence CRISPR / Cas complexes delivered by EVs spend a longer time in the target cell before gene editing occurs, both within the EV prior to release and after release when the CRISPR / Cas complex must move from the unique subcellular localisation of release to the nucleus. Thus, the stability of the CRISPR / Cas complex is especially important in this context. Furthermore, the present inventors have identified particular guide RNA modifications that are especially effective in increasing gene editing efficiency when EV-mediated delivery of CRISPR / Cas complexes is employed. Thus, the present invention provides an EV comprising a guide RNA, wherein the guide RNA comprises a (first) structural component, wherein the (first) structural component comprises a modification as described herein. In a preferred embodiment, the structural component is a functional module for CRISPR protein binding as described herein. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. The present invention further provides an EV comprising CRSIPR / Cas complex, wherein the CRISPR / Cas complex comprises a guide RNA bound to a CRSIPR protein, further wherein the guide RNA comprises a (first) structural component, wherein the (first) structural component comprises a modification as described herein. In a preferred embodiment, the structural component is a functional module for CRISPR protein binding as described herein. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. The present invention further provides a complex, wherein the complex comprises a fusion protein and a guide RNA, wherein the fusion protein comprises an EV polypeptide and a CRISPR protein, wherein the guide RNA comprises a (first) structural component, wherein the (first) structural component comprises a modification as described herein. In a preferred embodiment, the structural component is a functional module for CRISPR protein binding as described herein. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. In a preferred embodiment, the modified guide RNA is bound to the CRISPR protein. The present invention further provides an EV comprising the complex of the present invention. The present invention further provides an EV producer cell that expresses a guide RNA, wherein the guide RNA comprises a (first) structural component, wherein the (first) structural component comprises a modification as described herein. In a preferred embodiment, the structural component is a functional module for CRISPR protein binding as described herein. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. In one embodiment, the EV producer cell further expresses a CRISPR protein, preferably Cas9 or Cas12a. In a preferred embodiment, CRISPR protein is fused to an EV polypeptide. In present invention further provides a method of preparing an EV of the present invention, or population of EVs comprising a plurality of EVs of the present invention, comprising introducing into an EV-producing cell: (i) a guide RNA; and / or (ii) a polynucleotide construct encoding a guide RNA and expressing the construct in the EV-producing cell, thereby generating EVs; further wherein the guide RNA comprises a (first) structural component, wherein the (first) structural component comprises a modification as described herein. In a preferred embodiment, the structural component is a functional module for CRISPR protein binding as described herein. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. In one embodiment, the construct is expressed under the control of a U6 promoter, preferably a human U6 promoter, most preferably a human U6 promoter having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 36. In a preferred embodiment, the method further comprises introducing into the EV-producing cell: (i) a CRISPR protein and / or (ii) a construct encoding a CRISPR protein and expressing the construct in the EV-producing cell, thereby generating EVs that further comprise a CRISPR protein, preferably wherein the CRISPR protein is bound to the guide RNA. In one embodiment, the CRISPR protein is Cas12a, or preferably Cas9. The present invention further provides an EV obtained by the method of the present invention. In one embodiment, the guide RNA of the present invention is a stabilised guide RNA as described herein. In one embodiment, the structural component of the guide RNA is a stem loop or an RNA duplex. In one embodiment, the modification is an extension modification as described herein. In one embodiment, the modification is an increase in the proportion of G-C or C-G base pairs present in the structural component. In a preferred embodiment, the modification is both an extension modification as described herein and increases in the proportion of G-C or C-G base pairs present in the structural component. In one embodiment, the modification is a stabilising modification as described herein. In a one embodiment, the guide RNA of the present invention comprises, preferably from 5’ to 3’, a target-specific spacer sequence, a repeat / anti repeat RNA duplex, a nexus, a first hairpin and / or a second hairpin; wherein the repeat / anti repeat RNA duplex comprises an upper stem, a bulge and a lower stem. In a preferred embodiment, the guide RNA of the present invention comprises, preferably from 5’ to 3’, a target-specific spacer sequence, a repeat / anti repeat RNA duplex, a nexus, a first hairpin and a second hairpin; wherein the repeat / anti repeat RNA duplex comprises an upper stem, a bulge and a lower stem. In one embodiment, the (first) structural component comprising a modification is a modified upper stem as described herein. In a preferred embodiment, the modified upper stem is a RNAFE modified upper stem as described herein, a CR3 modified upper stem as described herein or an M1L / M1LR modified upper stem as described herein. In an alternative embodiment, the (first) structural component comprising a modification is a modified first hairpin as described herein. In a further alternative embodiment, the (first) structural component comprising a modification is a modified second hairpin as described herein. In one embodiment, the guide RNA further comprises a second structural component. Preferably, the second structural component is distinct from the first structural component. In one embodiment, the second structural component comprises a modification as described herein. In a preferred embodiment, the second structural component is also a functional module for CRISPR protein binding as described herein. In one embodiment, the second structural component is a stem loop or an RNA duplex. In one embodiment, the first structural component is an RNA duplex and the second structural component is a stem loop. In one embodiment, the first structural component is a stem loop and the second structural component is a stem loop. In one embodiment, the modification in the second structural component is an extension modification as described herein. In one embodiment, the modification in the second structural component is an increase in the proportion of G-C or C-G base pairs present in the structural component. In a preferred embodiment, the modification in the second structural component is both an extension modification as described herein and increases in the proportion of G-C or C-G base pairs present in the structural component. In one embodiment, the modification is a stabilising modification as described herein. In one embodiment, the first structural component comprising a modification is a modified upper stem as described herein and the second structural component comprising a modification is a modified first hairpin as described herein. In preferred embodiment, the first structural component comprising a modification is a RNAFE modified upper stem as described herein and the second structural component comprising a modification is a modified first hairpin as described herein. In an alternative preferred embodiment, the first structural component comprising a modification is an M1L / M1LR modified upper stem as described herein and the second structural component comprising a modification is a modified first hairpin as described herein. In one embodiment, the first structural component comprising a modification is a modified upper stem as described herein and the second structural component comprising a modification is a modified second hairpin as described herein. In preferred embodiment, the first structural component comprising a modification is a CR3 modified upper stem as described herein and the second structural component comprising a modification is a modified second hairpin as described herein. In one embodiment, the first structural component comprising a modification is a modified first hairpin as described herein and the second structural component comprising a modification is a modified second hairpin as described herein. In one embodiment, the guide RNA further comprises a third structural component. Preferably, the third structural component is distinct from the first structural component and the second structural component. In one embodiment, the third structural component comprises a modification as described herein. In a preferred embodiment, the third structural component is also a functional module for CRISPR protein binding as described herein. In one embodiment, the third structural component is a stem loop or an RNA duplex. In one embodiment, the first structural component is an RNA duplex, the second structural component is a stem loop and the third structural component is a stem loop. In one embodiment, the first structural component is a stem loop, the second structural component is a stem loop and the third structural component is a stem loop. In one embodiment, the modification in the third structural component is an extension modification as described herein. In one embodiment, the modification in the third structural component is an increase in the proportion of G-C or C-G base pairs present in the structural component. In a preferred embodiment, the modification in the third structural component is both an extension modification as described herein and increases in the proportion of G-C or C-G base pairs present in the structural component. In one embodiment, the modification is a stabilising modification as described herein. In one embodiment, the first structural component comprising a modification is a modified upper stem as described herein, the second structural component comprising a modification is a modified first hairpin as described herein and the third structural component comprising a modification is a modified second hairpin as described herein. In a preferred embodiment the modified upper stem is an M1L / M1LR modified upper stem as described herein. In one embodiment, the guide RNA further comprises a modified lower stem as described herein. In one embodiment, the guide RNA further comprises a region between the nexus and the first hairpin as described herein. In one embodiment, the a region between the nexus and the first hairpin is a modified nexus-first hairpin as described herein. In one embodiment, the guide RNA does not comprise any 3’ modifications. In one embodiment the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides, or preferably does not comprise any nucleotides, between the second hairpin loop and the terminator sequence. In one embodiment, the guide RNA does not comprise any 5’ modifications. In one embodiment, the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides or preferably does not comprise any nucleotides to the 5’, or upstream, of the spacer sequence. In one embodiment, the modified guide RNA comprises fewer than 7 nucleotides, fewer than 6 nucleotides, fewer than 5 nucleotides, fewer than 4 nucleotides, fewer than 3 nucleotides, fewer than 2 nucleotides or preferably does not comprise any nucleotides between the spacer sequence and the repeat region of the crRNA. In one embodiment the guide RNA further comprises a chemical modification (e.g. phosphorothioate bonds for 5'- and 3'-end protection and / or internal 2'- OMe residues) and / or a nucleotide substitution (e.g. a nucleotide having a locked nucleic acid (LNA) building block having a 2"-O-CH2-4" bridge). In one embodiment, the modified guide RNA includes the same combination of modifications as an “RNAFE” guide RNA as described herein, a “gRNAFE4C” guide RNA as described herein, a “gRNAFE4G” as described herein, a “gRNAT-lock” or “T-lock” guide RNA as described herein, a “T-lock T4C” guide RNA as described herein, a T-lock T4G” guide RNA as described herein, a “CR3” as described herein, an “M1L” guide RNA as described herein, an “M1LR” guide RNA as described herein, an ”M2LR” guide RNA as described herein, an “MN1LR” guide RNA as described herein or a “MN2LR” guide RNA as described herein. In a preferred embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the RNAFE upper stem as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to A-U flip as described herein. In a more preferred embodiment, the first hairpin is a non-modified hairpin as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is RNAFE as described herein. In an alternative preferred embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the RNAFE upper stem as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to C-G substitution as described herein. In a more preferred embodiment, the first hairpin is a non-modified hairpin as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is gRNAFE4C as described herein. In an alternative preferred embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the RNAFE upper stem as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to G-C substitution as described herein. In a more preferred embodiment, the first hairpin is a non-modified hairpin as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is gRNAFE4G as described herein. In an alternative preferred embodiment, the guide RNA comprises a modified first hairpin as described herein. In a preferred embodiment, the upper stem is a non-modified upper stem as described herein, the lower stem is a non- modified lower stem as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is gRNAT-lock as described herein. In a preferred embodiment, the guide RNA comprises a modified first hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to A-U flip as described herein. In a more preferred embodiment, the upper stem is a non- modified upper stem as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is T-lock T4A as described herein. In a preferred embodiment, the guide RNA comprises a modified first hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to C-G substitution as described herein. In a more preferred embodiment, the upper stem is a non-modified upper stem as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is T-lock T4C as described herein. In a preferred embodiment, the guide RNA comprises a modified first hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to G-C substitution as described herein. In a more preferred embodiment, the upper stem is a non-modified upper stem as described herein, the second hairpin is a non-modified second hairpin as described herein and the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the guide RNA is T-lock T4G as described herein. In a preferred embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the CR3 upper stem as described herein, and a modified second hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein, preferably comprising the U-A to C-G substitution as described herein. In one embodiment, the region between the nexus and the first hairpin is a modified region between the nexus and the first hairpin as described herein. In a preferred embodiment, the first hairpin is a non-modified hairpin as described herein. In one embodiment, the guide RNA is CR3 as described herein. In addition to discovering that guide RNAs having modifications allow for more efficient gene editing to be achieved when EV mediated delivery of CRISPR / Cas complexes is employed, the present inventors also designed new guide RNAs that are particularly effective in increasing gene editing efficiency in this context. These new guide RNAs comprise particular combinations of modified structural components, as well as previously unknown adaptations of these modifications. Thus, the present invention also provides guide RNAs. In one embodiment, the guide RNA is a stabilised guide RNA as described herein. In one embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the M1L / M1LR upper stem as described herein, and a modified first hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein. In a more preferred embodiment, the second hairpin is a non-modified second hairpin as described herein and / or the region between the nexus and the first hairpin is a non-modified nexus-first hairpin as described herein. In one embodiment, the modified guide RNA is M1L as described herein. In one embodiment, the modified guide RNA comprises or consists of, preferably from 5’ to 3’, the target-specific spacer sequence, SEQ ID NO.21, SEQ ID NO.26, SEQ ID NO. 41, SEQ ID NO.25, SEQ ID NO.17 and SEQ ID NO.18 In one embodiment, the modified guide RNA comprises or consists of, preferably from 5’ to 3’, a target-specific spacer sequence and a nucleotide sequence having 100% sequence identity to SEQ ID NO 27. In one embodiment, the modified guide RNA comprises or consists, preferably from 5’ to 3’, of a spacer sequence and a nucleotide sequence having 100% sequence identity to SEQ ID NO 2. In an alternative embodiment, the guide RNA comprises a modified upper stem as described herein, preferably the M1L / M1LR upper stem as described herein, a modified first hairpin as described herein and a modified second hairpin as described herein. In a preferred embodiment, the lower stem is a modified lower stem as described herein. In a more preferred embodiment, the region between the nexus and the first hairpin is a modified nexus-first hairpin as described herein. In one embodiment, the modified guide RNA is M1LR as described herein. In one embodiment, the modified guide RNA comprises or consists, preferably from 5’ to 3’, of the target-specific spacer sequence, SEQ ID NO.21, SEQ ID NO.26, SEQ ID NO.42, SEQ ID NO.25, SEQ ID NO.24 and SEQ ID NO.18. In one embodiment, the modified guide RNA comprises or consists, preferably from 5’ to 3’, of a target-specific spacer sequence and a nucleotide sequence having 100% sequence identity to SEQ ID NO 28. In one embodiment, the modified guide RNA comprises or consists, preferably from 5’ to 3’, of a target-specific spacer sequence and a nucleotide sequence having 100% sequence identity to SEQ ID NO 3. The invention further provides a nucleic acid molecule encoding a modified guide RNA of the present invention. The invention further provides a library comprising a plurality of modified guide RNAs of the present invention, preferably wherein the library comprises a plurality of guide RNAs having the same constant region but different target- specific spacer sequences. The invention further provides a complex comprising a CRISPR protein and a modified guide RNA of the present invention. Preferably wherein the CRISPR protein is a Cas enzyme, preferably Cas9. In one embodiment, the CRISPR protein forms part of a fusion protein that also comprises an EV polypeptide. The term “CRISPR / Cas system guide RNA”, “guide RNA” or “guide” refers to an RNA molecule that is capable of guiding a CRISPR protein to a specific nucleotide sequence, preferably a specific nucleotide sequence within the genome. In one embodiment, the guide RNA is capable of effecting the manipulation of a target nucleic acid within a prokaryotic or eukaryotic cell when in complex within the cell with a CRISPR protein. As used herein, the term “CRISPR / Cas complex” refers to a complex comprising or consisting of a CRISPR protein as described herein and a guide RNA as described herein. In one embodiment, the guide RNA comprises a target-specific spacer sequence as described herein. In one embodiment, the guide RNA comprises a constant region as described herein. In one embodiment, the guide RNA comprises or consists, preferably from 5’ to 3’, of a target-specific spacer sequence as defined herein and a constant region as described herein. The term “target specific spacer sequence” or “spacer” refers to a nucleotide sequence capable of specifically binding to a given target oligonucleotide sequence. In one embodiment, the target-specific spacer sequence is capable of hybridizing to a target sequence in a genomic locus of interest in a cell. In one embodiment, the target-specific spacer sequence is positioned at the 5’ end of the crRNA. In an alternative embodiment, the target-specific spacer is positioned at the 3’-end of the gRNA. In one embodiment, the target-specific spacer has a length of about 12-30 nucleotides, preferably 15-27 nucleotides, more preferably about 17-24 nucleotides. The term “constant region” when used in relation guide RNAs refers to an RNA molecule comprises or consists of all the components of the guide RNA described herein, but without the target specific spacer sequence. In one embodiment, the constant region is capable of binding to a CRISPR protein. In one embodiment, the guide RNA comprises a CRISPR RNA (crRNA) as described herein. In one embodiment the guide RNA comprises a trans- activating CRISPR RNA (tracrRNA) as described herein. In one embodiment, the guide RNA comprises or consists, preferably from 5’ to 3’, of a crRNA as described herein and a tracrRNA as described herein. In one embodiment, the crRNA comprises or consists of a target-specific spacer sequence and a repeat region. In one embodiment, the tracrRNA comprises an anti-repeat region. In a preferred embodiment, the tracrRNA comprises or consists, preferably from 5’ to 3’, of an anti-repeat region, a nexus sequence, a first hairpin loop and a second hairpin loop. The guide RNA of the present invention comprises one or more structural components. In one embodiment, a structural component has a particular secondary structure and / or a particular nucleotide sequence. In one embodiment, the secondary structure is an RNA duplex, a stem-loop or a bulge. The term “RNA duplex” as used herein refers to an RNA secondary structure of 2 or more contiguous pairs of complementary nucleotides. The term "contiguous”, when used in relation to a stem structure, means an uninterrupted sequence of base pairs in the stem, i.e. without any bulge formed of unpaired nucleotides. The term “stem-loop” refers to an RNA secondary structure of 2 or more contiguous pairs of complementary nucleotides (i.e. a stem), with a loop comprising of 3 or more unpaired nucleotides. The term “non-interrupted stem- loop structure” refers to a stem-loop where the stem does not comprise any unpaired nucleotides. The term “bulge” refers to a region within an RNA duplex or a stem where there are one or more unpaired nucleotides. In one embodiment, the structural component is a functional module for CRISPR protein binding and / or for the manipulation of the target nucleic acid. The term “functional module for CRISPR protein binding” as used herein refers to a structural component of the guide RNA that plays a role in the guide RNA forming a complex with the CRISPR protein. In one embodiment, the functional module for CRISPR protein binding is required for the guide RNA to form a complex with the CRISPR protein. Preferably, the functional module for CRISPR protein binding interacts directly with the CRISPR protein. In one embodiment, the functional module for CRISPR protein binding is a repeat / anti-repeat RNA duplex structure as described herein, a lower stem as described herein, an upper stem as described herein, the bulge of the repeat / anti-repeat RNA duplex structure as described herein, a nexus as described herein, a first hairpin loop as described herein or a second hairpin loop as described herein. The term “corresponding structural component” refers to a structural component that performs the same function. When used in relation to a functional module for CRISPR protein binding, the term refers to a structural component that performs the same function in relation to the interaction with the CRISPR protein. In one embodiment, the corresponding structural component, when used in relation to a functional module for CRISPR protein binding, refers to structural component that interacts with some of, or all of, the same residues of the CRISPR protein. In one embodiment, the structural component is a repeat / anti-repeat RNA duplex structure as described herein, a lower stem as described herein, an upper steam as described herein, a nexus as described herein, a first hairpin loop as described herein, a second hairpin loop as described herein or the region between the nexus and the first hairpin as described herein. The terms “double stranded repeat / anti-repeat RNA duplex structure”, “repeat / anti-repeat RNA duplex” or “repeat / anti-repeat RNA structure” or “repeat / anti-repeat RNA sequence” refer to a structural component of a guide RNA formed through complementary base pairing between the repeat region of the crRNA and the anti-repeat region of the tracrRNA. The double stranded repeat / anti-repeat RNA duplex structure of the present invention is distinct from the first hairpin loop as described herein and the second hairpin loop as described herein. The repeat / anti-repeat RNA duplex structure is a functional module for CRISPR protein binding as described herein. The repeat / anti- repeat RNA duplex structure of the present invention comprises at least one base pair, for example at least 2 or at least 3 base pairs that interact with a CRISPR protein. In one embodiment, the repeat / anti-repeat structure comprises at least one base pair, for example at least 2 or at least 3 base pairs wherein the 2'OH groups of both nucleotides forming the base pair interact with the CRISPR protein. In one embodiment, the repeat / anti-repeat RNA duplex structure comprises a lower stem, a bulge with a directional kink and optionally an upper stem. In certain embodiments, the bulge is replaced by a loop. In one embodiment, the lower stem of the repeat / anti-repeat RNA duplex is proximal to the spacer and the nexus, the upper stem of the repeat / anti-repeat RNA duplex is distal from the spacer and the nexus and the bulge of the repeat / anti-repeat RNA duplex is positioned between the lower stem and the upper stem. The lower stem and the upper stem of the repeat / anti-repeat RNA duplex are formed by complementary base-pairs between nucleotides from the repeat sequence of the crRNA and nucleotides from the anti-repeat sequence of the tracrRNA. The bulge of the double stranded repeat / anti-repeat RNA duplex consists of one or more unpaired nucleotides. The bulge comprises a directional kink, i.e. the stretches of unpaired nucleotides in the repeat sequence and in the anti- repeat sequence have different lengths. In one embodiment, wherein the upper stem is not a modified upper stem as described herein (referred to as a non-modified upper stem herein), the upper stem consists of 2 to 4, 3 to 4 or preferably 4 complementary base pairs. In a preferred embodiment, 1 to 3, or preferably 2, of the complementary base pairs are G-C or C-G base pairs. In a preferred embodiment, the complementary base pairs are contiguous. In a more preferred embodiment, the upper stem comprises a crRNA having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 71 and a tracrRNA having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 72. In a most preferred embodiment, the upper stem comprises a crRNA having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 73. In one embodiment, wherein the lower stem is not a modified lower stem as described herein (referred to as a non-modified lower stem herein), the lower stem consists of around 4 to 8 complementary base pairs, preferably 5 to 7 complementary base pairs or most preferably 6 complementary base pairs. In a preferred embodiment, the lower stem comprises a crRNA having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 74 and a tracrRNA having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 75. The term “nexus sequence” or “nexus” refers to a structural component of guide RNA comprising a stem-loop. In a preferred embodiment, the nexus comprises a non-interrupted stem-loop. In a preferred embodiment, the stem loop comprises a bulge. In one embodiment, the nexus sequence comprises two conserved “A” residues followed by the stem-loop having a bulge. Preferably the nexus sequence comprises a single unpaired U in the 3'-stem sequence. In one embodiment, the length of the nexus sequence is about 9 to about 12 nucleotides, preferably 11 or 12 nucleotides. In one embodiment, the nexus is capable of interacting with a Cas enzyme, preferably Cas9. In a preferred embodiment, the stem of the stem-loop consists of two contiguous complementary base pairs, preferably the two contiguous complementary base pairs are G-C or C-G base pairs. In In a preferred embodiment, the loop of the stem-loop consists or 4 to 6, or preferably 5 unpaired nucleotides. In a preferred embodiment, the nexus comprises a nucletoide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 26 or SEQ ID NO 15. The term “first hairpin” refers to a structural component of guide RNA comprising a stem-loop. In one embodiment the loop of the first hairpin comprises 3 to 6, 3 to 5, or preferably 4 unpaired nucleotides. In one embodiment, where the first hairpin is not a modified first hairpin as described herein (referred to as a non-modified first hairpin herein), the first hairpin comprises a stem that consists of 2 to 8, 2 to 7, 2 to 6, 3 to 5 or preferably 4 complementary base pairs. In a preferred embodiment, at least one, or preferably one, of the complementary base pairs are G-C or C-G base pairs. In a preferred embodiment, the complementary base pairs are contiguous. In a more preferred embodiment, the first hairpin comprises a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 80. In one embodiment, the guide RNA comprises a region between nexus and the first hairpin comprising of a nucleotide sequence, also referred to as the “nexus-first hairpin” or the “nexus-hairpin 1” herein. Preferably the nucleotide sequence consists of 2 to 10, 3 to 9, 4 to 8, 5 to 7 or most preferably 6 nucleotides. In a preferred embodiment, this nucleotide sequence does not comprise a secondary structure, in particular an RNA duplex, a stem-loop or a bulge, as described herein. In one embodiment, where the region between nexus and the first hairpin is not a modified region between nexus and the first hairpin as described herein (referred to as a non-modified nexus-first hairpin herein), the region between nexus and the first hairpin comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 41. The term “second hairpin” refers to a further structural component of guide RNA comprising a stem-loop. The second hairpin is distinct from the first hairpin as described herein. In one embodiment the loop of the second hairpin comprises 3 to 5, 3 to 4, or preferably 3 unpaired nucleotides. In one embodiment, where the second hairpin is not a modified second hairpin as described herein (referred to as a non-modified second hairpin herein), the second hairpin comprises 2 to 6, 3 to 6, 4 to 6, 5 to 6 or preferably 6 complementary base pairs. In a preferred embodiment, at least 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 5 or preferably 5 of the complementary base pairs are G-C or C-G base pairs. In a preferred embodiment, the complementary base pairs are contiguous. In a more preferred embodiment, the first hairpin comprises a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 17. In one embodiment, the guide RNA of the present invention comprises or consists, preferably from 5’ to 3’, of a target-specific spacer sequence, a double stranded repeat / anti-repeat RNA duplex structure, a nexus sequence, a first hairpin loop and a second hairpin loop. In a preferred embodiment, the guide RNA further a region between nexus and the first hairpin comprising of a nucleotide sequence as described herein. The guide RNA of the present invention may further comprise a terminator sequence at its 3’ end. The term “terminator sequence” or “terminator” refers to an RNA sequence that signals the termination of transcription to an RNA polymerase, preferably a U6 RNA polymerase or, more preferably, the human U6 RNA polymerase. Preferably wherein the terminator sequence comprises or consists of SEQ ID NO 18. The guide RNA of the present invention may consist of a single RNA molecule, e.g. a single guide RNA (sgRNA) molecule. In further embodiments, guide RNA of the present invention comprises two separate RNA molecules, e.g. crRNA molecule and a tracrRNA molecule. In still further embodiments, the guide RNA of the present invention comprises three or more separate RNA molecules, e.g. a crRNA molecule and tracrRNA molecule that is comprised of two separate RNA molecules. In a preferred embodiment, the guide RNA of the present invention a single guide RNA. The term “The single guide RNA” or “sgRNA” refers to a guide RNA comprising a stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. In one embodiment, the stretch of intervening nucleotides is 3-10, 3-8 or 3-6 nucleotides in length, preferably 3-5 nucleotides in length, most preferably 4 nucleotides in length. In one embodiment the stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA comprises or consists of the nucleotide sequence GAAA (SEQ ID NO 83) or AAGA (SEQ ID NO 84). In the single guide RNA of the present invention, the double stranded repeat / anti-repeat RNA duplex structure comprises a non- contiguous hairpin structure comprising a lower stem, a bulge with a directional kink, an upper stem and a fusion loop. The term “modified guide” or “modified guide RNA” as used herein refers to a guide RNA comprising a change in it the nucleotide sequence of its constant region, as compared with a reference constant region nucleotide sequence. In one embodiment, the reference constant region nucleotide sequence is the nucleotide sequence of the constant region of any naturally occurring guide RNA. In one embodiment, the constant region nucleotide sequence is SEQ ID NO 40. In one embodiment, the change is a substitution from one nucleobase (i.e. A, G, C or U) to a different nucleobase. In one embodiment the change is the inclusion of additional nucleotides. The term “modification” as used herein refers to the change in the nucleotide sequence of the guide RNA as compared with a naturally occurring guide RNA. In one embodiment, the modification is an extension modification as described herein, a base pair modification as described herein, or a scramble modification as described herein. In a preferred embodiment the modification is a stabilising modification. In one embodiment, a stabilising modification is a modification that increases the stability of the guide RNA such that the guide comprising the stabilising modification is a stabilised guide as described herein. The term “extension modification” refers to an increase in the number of nucleotides present in a structural component, in particular an increase in the number of complementary base pairs present in a double stranded RNA structure, such as a duplex or a stem. In one embodiment, the extension is of one base pair, two base pairs, three base pairs, four base pairs, five base pairs, six base pairs, seven base pairs or more than seven base pairs. In one embodiment, the extension modification is a modification to the upper stem as described herein, a modification to the first hairpin as described herein or a modification to the second hairpin as described herein. The term “base pair modification” refers to the substitution of one complementary base pair in a double stranded RNA, such as a duplex or a stem, for a different complementary base pair or the flipping of a base pair in a double stranded RNA, such as a duplex or a stem. In one embodiment, the base pair modification is a modification to the lower stem as described herein or a modification to an extended upper stem as described herein. The term “scramble modification” refers an alteration in the RNA sequence of the guide RNA. In one, embodiment, the scramble modification involves changing the order of the nucleotides present in a sub-section or region of the RNA sequence. In one embodiment, scramble modification is a modification to the region between nexus and the first hairpin as described herein. In one embodiment, the modification is in a structural component of the guide RNA as described herein. In one embodiment the modification is the upper stem, the lower stem, the first hairpin modification, the second hairpin or the modification in the region between the nexus and the first hairpin. In one embodiment, the modification in the upper stem is a modification as compared with an upper stem having a reference upper stem nucleotide sequence. In one embodiment the reference upper stem nucleotide sequence is the nucleotide sequence of the upper stem of any naturally occurring guide RNA. In one embodiment, the upper stem nucleotide reference sequence has a crRNA sequence of SEQ ID NO 71 and a tracRNA sequence of SEQ ID NO 72. In one embodiment, the upper stem nucleotide reference sequence has the sequence of SEQ ID NO 73. In one embodiment, the modification in the lower stem is a modification as compared with the lower stem having a reference lower stem nucleotide sequence. In one embodiment the reference lower stem nucleotide sequence is the nucleotide sequence of the lower stem of any naturally occurring guide RNA. In one embodiment, the lower stem nucleotide reference sequence has a crRNA sequence of SEQ ID NO 74 and a tracRNA sequence of SEQ ID NO 75. In one embodiment, the lower stem reference sequence has the sequence of SEQ ID NO 76. In one embodiment, the modification in the region between the nexus and the first hairpin is a modification as compared with a region between the nexus and the first hairpin having a reference nexus-first hairpin nucleotide sequence. In one embodiment the reference nexus-first hairpin nucleotide sequence is the nucleotide sequence of the region between the nexus and the first hairpin of any naturally occurring guide RNA. In one embodiment, the nexus-first hairpin nucleotide reference sequence has the sequence of SEQ ID NO 41. In one embodiment, the modification in the first hairpin is a modification as compared with the first hairpin of a reference first hairpin nucleotide sequence. In one embodiment the reference first hairpin nucleotide sequence is a nucleotide sequence of the first hairpin having any naturally occurring guide RNA. In one embodiment, the first hairpin nucleotide reference sequence has the sequence of SEQ ID NO 80. In one embodiment, the modification in the second hairpin is a modification as compared with the second hairpin of a reference second hairpin nucleotide sequence. In one embodiment the reference second hairpin nucleotide sequence is a nucleotide sequence of the second hairpin having any naturally occurring guide RNA. In one embodiment, the second hairpin nucleotide reference sequence has the sequence of SEQ ID NO 17. The term “modified upper stem” refers to an upper stem as described herein that comprises a modification as described herein. In one embodiment, the upper stem comprises an extension modification. In one embodiment, the modified upper stem consists of 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 complementary base pairs. In a preferred embodiment, the upper stem consists of 5 to 12, 5 to 10, 5 to 9, 6 to 9 or 7 to 9 complementary base pairs. In a most preferred embodiment, the modified upper stem consists of 9 complementary base pairs. In one embodiment, at least 2, at least 3, at least 4 or at least 5 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment 3 to 7, 4 to 6 or most preferably 5 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment, the complementary base pairs of the upper stem are contiguous. In a most preferred embodiment, the upper stem consists of 9 contiguous complementary base pairs, preferably wherein 5 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs. In one embodiment, the modified upper stem is an “RNAFE upper stem” as described herein, “a CR3” upper stem as described herein or an “M1L / M1LR” upper stem as described herein. In one embodiment, the RNAFE upper stem comprises or consists of an upper stem having a repeat region of the crRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 54 and the tracrRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 55. In one embodiment, wherein the guide RNA is an sgRNA, the upper stem consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 56. In one embodiment, the CR3 upper stem comprises or consists of an upper stem having a repeat region of the crRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 57 and the tracrRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 58. In one embodiment, wherein the guide RNA is an sgRNA, the upper stem consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 59. In one embodiment, the M1L / M1LR upper stem comprises or consists of an upper stem having a repeat region of the crRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 60 and the tracrRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 61. In one embodiment, wherein the guide RNA is an sgRNA, the upper stem consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 62. The term “modified lower stem” refers to a lower stem as described herein that comprises a modification as described herein. In one embodiment, the lower stem comprises a base pair modification. In one embodiment, the modified lower stem comprises fewer than four consecutive uracil nucleotides. In one embodiment, the lower stem crRNA repeat region comprises the nucleotide sequence UUUVA (SEQ ID NO 64) and the lower stem tracrRNA anti-repeat region comprises the nucleotide sequence UBAAA (SEQ ID NO 65) further wherein: (i) the “V” of the UUUVA (SEQ ID NO 64) is A and the “B” of the UBAAA (SEQ ID NO 65) is U (referred to herein as “U-A to A-U flip” or “U-A flip”), (ii) the “V” of the UUUVA (SEQ ID NO 64) is C and the “B” of the UBAAA (SEQ ID NO 65) is G (referred to herein as “G-C substitution”), or (iii) the “V” of the UUUVA (SEQ ID NO 64) is G and the “B” of the UBAAA (SEQ ID NO 65) is C (referred to herein as “C-G substitution”). In one embodiment, the modified lower stem comprises of a crRNA repeat region comprising or consisting of the nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 81 and a tracrRNA anti-repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 82, wherein: (i) the “V” of the SEQ ID NO 81 is A and the “B” of the SEQ ID NO 82 is U (ii) the “V” of the SEQ ID NO 81 is C and the “B” of the SEQ ID NO 82 is G; or (iii) preferably the “V” of the SEQ ID NO 81 is G and the “B” of the SEQ ID NO 82 is C. In one embodiment, wherein the guide RNA comprises both a RNAFE upper stem modification as described herein and a lower stem modification as described herein, the repeat / anti-repeat RNA duplex structure comprises a repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 49 and an anti-repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 50, further wherein: (i) the “V” of the SEQ ID NO 49 is A and the “B” of the SEQ ID NO 50 is U, (ii) the “V” of the SEQ ID NO 49 is C and the “B” of the SEQ ID NO 50 is G, or (iii) preferably, the “V” of the SEQ ID NO 49 is G and the “B” of the SEQ ID NO 50 is C. In one embodiment, wherein the guide RNA comprises both a RNAFE upper stem modification as described herein and a lower stem modification as described herein, further wherein the guide RNA is a single guide RNA, the repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 51, further wherein: (i) the “V” of the SEQ ID NO 51 is A and the “B” is U, (ii) the “V” of the SEQ ID NO 51 is C and the “B” is G, or (iii) preferably, the “V” of the SEQ ID NO 51 is G and the “B” is C. In one embodiment, wherein the guide RNA comprises both a CR3 upper stem modification as described herein and a lower stem modification as described herein, the repeat / anti-repeat RNA duplex structure comprises a repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 66 and an anti-repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 67, further wherein: (i) the “V” of the SEQ ID NO 66 is A and the “B” of the SEQ ID NO 67 is U, (ii) the “V” of the SEQ ID NO 66 is C and the “B” of the SEQ ID NO 67 is G, or (iii) preferably, the “V” of the SEQ ID NO 66 is G and the “B” of the SEQ ID NO 67 is C. In one embodiment, wherein the guide RNA comprises both a CR3 upper stem modification as described herein and a lower stem modification as described herein, further wherein the guide RNA is a single guide RNA, the repeat / anti- repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 68, further wherein: (i) the “V” of the SEQ ID NO 68 is A and the “B” is U, (ii) the “V” of the SEQ ID NO 68 is C and the “B” is G, or (iii) preferably, the “V” of the SEQ ID NO 68 is G and the “B” is C. In one embodiment, wherein the guide RNA comprises both a M1L / M1LR upper stem modification as described herein and a lower stem modification as described herein, the repeat / anti-repeat RNA duplex structure comprises a repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 69 and an anti-repeat region comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 70, further wherein: (i) the “V” of the SEQ ID NO 69 is A and the “B” of the SEQ ID NO 70 is U, (ii) the “V” of the SEQ ID NO 69 is C and the “B” of the SEQ ID NO 70 is G, or (iii) preferably, the “V” of the SEQ ID NO 69 is G and the “B” of the SEQ ID NO 70 is C. In one embodiment, wherein the guide RNA comprises both a M1L / M1LR upper stem modification as described herein and a lower stem modification as described herein, further wherein the guide RNA is a single guide RNA, the repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 30, further wherein: (i) the “V” of the SEQ ID NO 30 is A and the “B” is U, (ii) the “V” of the SEQ ID NO 30 is C and the “B” is G, or (iii) preferably, the “V” of the SEQ ID NO 30 is G and the “B” is C. The term “modified region between nexus and the first hairpin”, “modified nexus-hairpin 1 region”, “modified nexus-first hairpin region” refers to the nucleotide sequence between the nexus and the first hairpin as described herein that comprises a modification as described herein. In one embodiment, the nucleotide sequence between the nexus and the first hairpin comprises a scramble modification as described herein. In one embodiment the scramble modification is the reordering of two consecutive nucleotides, preferably an A and a U. In a preferred embodiment, the modified region between nexus and the first hairpin comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 41. The term “modified first hairpin” or “modified hairpin 1” refers to a first hairpin as described herein that comprises a modification as described herein. In one embodiment, the first hairpin comprises an extension modification. In one embodiment, the modified first hairpin comprises a locked hairpin, preferably wherein the locked hairpin comprises a super stable loop and / or a melting temperature (Tm) of above 65 degrees. In one embodiment, the modified first hairpin comprises a stem consisting of 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 complementary base pairs. In a preferred embodiment, the first hairpin comprises a stem consisting of 5 to 12, 5 to 10, 6 to 10 or 7 to 9 complementary base pairs. In a most preferred embodiment, the first hairpin comprises a stem consisting of 8 complementary base pairs. In one embodiment, at least 2, at least 3 or at least 4 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment 2 to 6, 3 to 5 or most preferably 4 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment, the complementary base pairs of the first hairpin are contiguous. In a most preferred embodiment, the first hairpin comprises a stem consisting of 7 contiguous complementary base pairs, preferably wherein 4 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs. In a still further preferred embodiment, the modified first hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.22 or SEQ ID NO.25. In one embodiment, wherein the guide RNA comprises both a modified region between nexus and the first hairpin as described herein and a modified first hairpin as described herein, the region of the guide RNA between the nexus and hairpin 2 comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.23 or SEQ ID NO.16. The term “modified second hairpin” or “modified hairpin 2” refers to a second hairpin as described herein that comprises a modification as described herein. In one embodiment, the second hairpin comprises an extension modification. In one embodiment, the modified second hairpin comprises a stem consisting of 7, 8, 9, 10, 11, 12, 13,14 or more than 14 complementary base pairs. In a preferred embodiment, the second hairpin comprises a stem consisting of 7 to 14, 7 to 10, 7 to 9 or 7 to 8 complementary base pairs. In a most preferred embodiment, the second hairpin comprises a stem consisting of 7 complementary base pairs. In one embodiment, at least 3, at least 4, at least 5 or at least 6 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment 4 to 8, 5 to 7 or most preferably 6 of the complementary base pairs are C-G or G-C base pairs. In a preferred embodiment, the complementary base pairs of the second hairpin are contiguous. In a most preferred embodiment, the second hairpin comprises a stem consisting of 7 contiguous complementary base pairs, preferably wherein 6 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs. In a still further preferred embodiment, the modified first hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.24. In one embodiment, the guide RNA of the present invention is a stabilised guide RNA. In one embodiment, a stabilised guide RNA has increased stability. In one embodiment, a stabilised guide RNA has increased stability as compared with a reference guide RNA, as described herein. In one embodiment, the increased stability is in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is an EV producer cell and / or a target cell. In a more preferred embodiment, the EV producer cell is a HEK cell, most preferably a HEK293 cell. In a more preferred embodiment, the cell is a target cell is a neuronal cell or a cardiomyocyte. The stability, or relative stability, of the stabilised guide RNA may be measured in accordance with any method known in the art. In one embodiment, the reference guide RNA comprises a reference constant region nucleotide sequence as defined herein. In a preferred embodiment, the reference guide RNA consists, from 5’ to 3’, of a target-specific spacer sequence and a reference constant region nucleotide sequence as defined herein. In a most preferred embodiment, the reference guide RNA comprises the same target -specific spacer sequence as the guide RNA of the invention. In one embodiment a stabilised guide RNA has a more stable secondary structure, in particular the modified structural component(s) have a more stable secondary structure. In one embodiment, a stabilised guide RNA has a more stable secondary structure as compared with a reference guide RNA, as described herein. In one embodiment, the modified structural component(s) of the stabilised guide RNA have a more stable secondary structure as compared with the same structural component(s) in a reference guide RNA, as described herein. In one embodiment the stabilised guide RNA has a more stable secondary structure in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is an EV producer cell and / or a target cell. In a more preferred embodiment, the EV producer cell is a HEK cell, most preferably a HEK293 cell. In a more preferred embodiment, the cell is a target cell is a neuronal cell or a cardiomyocyte. The stability, or relative stability, of the stabilised guide RNA’s secondary structure may be measured in accordance with any method known in the art. In one embodiment a stabilised guide RNA has an increased affinity for a CRISPR protein. In one embodiment a stabilised guide RNA has an increased affinity for a CRISPR protein as compared with a reference guide RNA, as described herein. In one embodiment, the CRISPR protein is a Cas12a, or preferably a Cas9.In one embodiment the stabilised guide RNA has a higher affinity for the CRISPR protein in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is an EV producer cell and / or a target cell. In a more preferred embodiment, the EV producer cell is a HEK cell, most preferably a HEK293 cell. In a more preferred embodiment, the cell is a target cell is a neuronal cell or a cardiomyocyte. The affinity, or relative affinity, of the stabilised guide RNA for the CRISPR protein may be measured in accordance with any method known in the art. In one embodiment a stabilised guide RNA has an exhibits more stable binding to a CRISPR protein. In one embodiment a stabilised guide RNA exhibits more stable binding to a CRISPR protein, as compared with a reference guide RNA, as described herein. In one embodiment, the CRISPR protein is a Cas12a, or preferably a Cas9.In one embodiment the stabilised guide RNA exhibits more stable binding to the CRISPR protein in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is an EV producer cell and / or a target cell. In a more preferred embodiment, the EV producer cell is a HEK cell, most preferably a HEK293 cell. In a more preferred embodiment, the cell is a target cell is a neuronal cell or a cardiomyocyte. The stability, or relative stability, of the binding of the stabilised guide RNA to the CRISPR protein may be measured in accordance with any method known in the art. The term “RNAFE” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 43. RNAFE comprises the modified RNAFE upper stem as described herein and a modified lower stem as described herein, wherein the modified lower stem comprises the U-A to A-U flip. The term “gRNAFE4C” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 44. gRNAFE4C comprises the modified RNAFE upper stem as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to C-G. The term “gRNAFE4G” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 45. gRNAFE4G comprises the modified RNAFE upper stem as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to G-C. The term “gRNAT-lock” or “gRNA T-lock” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 46. gRNAT- lock comprises the first hairpin modification as described herein. The term “T-lock T4A” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 85. T-lock T4A comprises the first hairpin modification as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a from U-A to A-U flip. The term “T-lock T4C” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 47. T-lock T4C comprises the first hairpin modification as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to C-G. The term “T-lock T4G” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 48. T-lock T4G comprises the first hairpin modification as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to G-C. The term “CR3” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO 63. CR3 comprises the CR3 upper stem modification as described herein, nexus-first hairpin modification as described herein and the first hairpin as described herein and the second hairpin modification as described herein. The term “M1L” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO. 27. M1L comprises the M1L / M1LR upper stem modification as described herein, the first hairpin modification as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to G-C. The term “M1LR” refers to a modified guide RNA consisting of, from 5’ to 3’, of the spacer sequence and SEQ ID NO. 28. M1L comprises the M1L / M1LR upper stem modification as described herein, the first hairpin modification as described herein, the nexus-first hairpin modification as described herein, the second hairpin modification as described herein and the lower stem modification as described herein, wherein the lower stem modification comprises a substitution from U-A to G-C. In one embodiment, the guide RNA of the present invention is more efficiently loaded into an EV when expressed in an EV producer cell. In one embodiment, the modified guide RNA is more efficiently loaded as compared with a reference guide RNA, as described herein. In one embodiment, the EV producer cell is a mammalian cell, preferably a HEK cell, most preferably a HEK293 cell. In a preferred embodiment, the EV producer cell also expresses a CRISPR protein, preferably Cas12a or more preferably Cas9. Most preferably, the CRISPR protein forms part of a fusion protein that also comprises an EV polypeptide, most preferably the EV polypeptide is TSN2. In one embodiment, the modified guide RNA is loaded as part of a CRISPR / Cas complex with the CRISPR protein. The loading, or relative loading, of the modified guide RNA and / or a CRISPR / Cas complex comprising the modified guide RNA, may be measured in accordance with any method known in the art. In one embodiment, the guide RNA of the invention exhibits improved gene editing when delivered to a target cell by an EV. In one embodiment, the modified guide RNA exhibits improved gene editing as compared with a reference guide RNA, as described herein. In one embodiment the modified guide RNA exhibits the improved gene editing in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is a target cell, preferably the target cell is a neuronal cell or a cardiomyocyte. Gene editing, or relative gene editing, may be measured in accordance with any method known in the art. In one embodiment, the guide RNA of the invention exhibits improved or comparable recognition of a target DNA sequence (i.e. the protospacer). In one embodiment, the guide RNA exhibits improved or comparable recognition of a target DNA sequence, as compared with a reference guide RNA, as described herein. In one embodiment the modified guide RNA exhibits improved or comparable recognition of a target DNA sequence in a cell, preferably a mammalian cell. In a preferred embodiment, the cell is a target cell, preferably the target cell is a neuronal cell or a cardiomyocyte. The recognition, or relative recognition, of a target DNA sequence may be measured in accordance with any method known in the art. In one embodiment, the EV of the present invention is an exosome or a micovesicle. In a preferred embodiment, the EV is derived from a mammalian cell preferably a HEK cell, most preferably a HEK293 cell. In a further preferred embodiment, the EV comprises a fusogenic protein or an endosomal escape enhancer. In one embodiment, the EV producer cell of the present invention further expresses a fusogenic protein or an endosomal escape enhancer. The fusogenic protein or an endosomal escape enhancer may or may not be fused to an EV polypeptide. In a preferred embodiment, the EV producer cell is a mammalian cell, preferably a HEK cell, most preferably a HEK293 cell. In one embodiment, the fusion protein of the present invention comprises an intein positioned between the EV polypeptide and the CRISPR protein. In a preferred embodiment, the CRISPR protein of the present invention is Cas12a, or more preferably Cas9. In one embodiment, the CRISPR protein of the present invention is fused (preferably via an intein) to a domain of the EV polypeptide that is localised to the lumen of the EV. In a preferred embodiment, the EV polypeptide of the present invention is TSN2. The invention further provides a population of EVs comprising a plurality of EVs of the present invention. The invention further provides the use of a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, an EV of the present invention or a population of EVs of the present invention for CRISPR-mediated oligonucleotide binding and / or editing, particularly for genome editing. In one embodiment, the use is in vitro or ex vivo. The invention further provides a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, an EV of the present invention or a population of EVs of the present invention. The invention further provides composition comprising a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, an EV of the present invention or a population of EVs of the present invention and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier. In one embodiment, the composition is for use as a medicament. In one embodiment, the composition is for use is the treatment or prevention of a disease in a subject. In one embodiment, the composition is for the preparation of a medicament for treatment or prevention of a disease in a subject. The invention further provides method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a guide RNA of the present invention, a nucleic acid molecule of the present invention, a library of the present invention, a complex of the present invention, an EV of the present invention or a population of EVs of the present invention to a subject. In a preferred embodiment, the disease or disorder is a genetic disease or disorder. In a further preferred embodiment, the disease or disorder is a paediatric disease or disorder and / or a disease or disorder wherein symptoms are exhibited during childhood. In a further preferred embodiment, the disease or disorder is a heart or cardiovascular disease or disorder In a further preferred embodiment, the disease or disorder is a neurological disease or disorder, such as ALS or Angelman’s syndrome. In a particularly preferred embodiment, the disease or disorder is Angelman’s syndrome. In a preferred embodiment, the subject to be treated is a human child. In one embodiment, the child is under the age of 21 years old, under the age of 18 years old, under the age of 16 years old or under the age of 14 years old. In a preferred embodiment, the child is pre-pubescent. Further detailed description The present inventors have provided a means to solve the problem of low genome editing in a target gene by providing modified gRNAs with increased efficacy of CRISPR-mediated oligonucleotide binding and / or editing. In particular, the inventors have engineered gRNAs with combinations of modified hairpin structures and additional base pair modifications that results in increased editing efficiency of a target. The present inventors also provide optimized gRNA designs that allow improved encapsulation of Cas9 / gRNA into engineered extracellular vesicles (EVs). The modified gRNA designs are more stable and so are less susceptible to degradation. This means that more gRNA comes into contact with Cas and so the probability of binding is increased, which in turn improves the loading of the gRNA and Cas9 / gRNA complex into an EV. Further, by allowing more Cas to associate with the more stable gRNA, therefore improving the ratio of Cas to Cas-and-gRNA, more is likely to be loaded into the EV due to size limitations. Finally, having more stable gRNA that exists without degradation for longer, means more is likely to bind to Cas and be delivered as an RNP. These approaches to improve stability and therefore loading into an EV, therefore result in better editing efficiency of a target because more Cas9 / gRNA complex is delivered to a target. Finally, some DNA sequences are refractory to CRISPR / Cas9 cleavage, which is at least in part due to gRNA misfolding caused by unstable gRNA secondary structures, which in turn leads to lack of association with Cas9. Improving the stability of the gRNA will therefore allow sequences to be targeted that may have previously proved difficult or been impossible to target and also allow gRNA and Cas9 to associate. Both of these advantages will in turn improve loading of the modified gRNA / Cas9 RNP complex into an EV and as a result, improve the editing efficiency of the RNP complex. Also provided is a way to overcome the problem that only Cas9 protein, not the Cas9 / gRNA RNP complex is successfully delivered into a target cell by an engineered EV. It is, furthermore, desirable to improve gRNA loading into EVs by improving gRNA expression, the stability of the gRNA and the binding capacity of the gRNA to Cas9. The inventors have also found that the encapsulation of Cas9 / gRNA complex into engineered EVs can be improved as a result of increased binding of the modified gRNA with Cas9 during the biogenesis of EVs. To address these problems, the inventors have identified a number of modified gRNA that are able to bind to Cas9 inside of engineered EVs and achieve delivery of the large RNP complex to reporter cells. Upon gRNA transfection the editing efficiency in reporter cells significantly increased, indicating the gRNA loading in EVs may be the limitation for high gene editing efficiency in recipient cells. The disclosure will be more clearly understood from the following description of some embodiments thereof, given by way of example only, and with reference to the Figures where appropriate. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted as prior art to the claimed disclosure. In the case of conflict, the present specification, including definitions, will control. In a further aspect of the invention, there is provided a CRISPR / Cas system guide RNA (gRNA), optionally a chimeric single guide RNA molecule (sgRNA), comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and capable of effecting the manipulation of a target nucleic acid within a prokaryotic or eukaryotic cell when in complex within the cell with a CRISPR protein, wherein the guide RNA or sgRNA comprises the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; and a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”. As discussed in the background section, a gRNA may be a duplex of a crRNA and a tracrRNA. Thus, the gRNA of the first aspect may comprise a crRNA and a tracrRNA. Alternatively, the gRNA may be a single molecule wherein these sequences are fused. Such gRNAs are referred to as sgRNAs. As such, in a preferred embodiment, the gRNA is an sgRNA. The gRNA modifications are described with reference to a backbone sequence (SEQ ID NO: 1). This backbone represents the well-known +85 sgRNA architecture. As the skilled person would appreciate, the invention is not limited to this particular sequence. For instance, the recited modifications may be applied to other +85 sgRNA sequences comprising further modifications, such as further insertions, substitutions, or deletions. In addition, the modifications may be applied to other gRNA architectures, and so the invention is not limited to the +85 architecture. Examples of other architectures include the following sgRNAs: +48; +54; +67; and + 85 (e.g. from Hsu et al., 2013). The skilled person would be able to identify the residue of these sgRNAs that corresponds to a particular position of SEQ ID NO: 1 by techniques known in the art, for instance alignment of the relevant feature. In a particular embodiment, the gRNA has the +85 architecture. The gRNA may include the sequence of SEQ ID NO: 1 as a backbone sequence to which the modifications are applied. The gRNA may be of the sequence according to SEQ ID NO: 1, apart from the modifications. In an example, the gRNA comprises only the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; and a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”. The gRNA may be an sgRNA. An exemplary sgRNA with said modifications is referred to as M1L herein. These modifications provide a gRNA with an extended stem loop and with a “G-C” base pair within the stem loop. The replacement of positions 73 to 76 results in hairpin 1 being a superstable hairpin structure. The stem loop and hairpin 1 are discussed further herein. In some embodiments, the gRNA may include one or more or all of the following modifications: an insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; an insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1; “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; an insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The gRNA may comprise: “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The “UA” modification is present within hairpin 1 of the gRNA. The insertion of the “C” and “G” provides an additional “C-G” base pair in hairpin 2. Hairpin 1 and hairpin 2 are discussed further herein. Thus, in some embodiments, the gRNA may include the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The gRNA may be an sgRNA. In an example, the gRNA comprises only these modifications. An exemplary sgRNA with said modifications is referred to as M1LR herein. The gRNA may comprise: an insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1 and an insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1. The gRNA may comprise: An insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1, an insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1, and an insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1. The insertion of “CUUCG” provides an extension to hairpin 2. In some embodiments, the gRNA may include the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; an insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; an insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1; “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The gRNA may be an sgRNA. In an example, the gRNA comprises only these modifications. An exemplary sgRNA with said modifications is referred to as MN1LR herein. In some embodiments, the gRNA may include the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; an insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The gRNA may be an sgRNA. In an example, the gRNA comprises only these modifications. An exemplary sgRNA with said modifications is referred to as M2LR herein. In some embodiments, the gRNA may include the following modifications: a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; a “C” at a residue corresponding to position 46 of SEQ ID NO 1; an insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; an insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1; “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”; an insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; an insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1; and an insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The gRNA may be an sgRNA. In an example, the gRNA comprises only these modifications. An exemplary sgRNA with said modifications is referred to as MN2LR herein. The gRNA of the invention may have a sequence according to SEQ ID NO: 1 and comprises at least any set of modifications disclosed herein. The gRNA of the invention may have a sequence according to SEQ ID NO: 1 and comprises any modifications or sets of modifications disclosed herein, and also comprises one or more insertions, substitutions, or deletions. The gRNA of the invention may have a sequence according to SEQ ID NO: 1 and comprises any modifications or sets of modifications disclosed herein, and also further comprises one to five, one to three, one or two, or one insertions, substitutions, or deletions. The gRNA of the invention may have a sequence according to SEQ ID NO: 1 and comprises only the modifications or sets of modifications disclosed herein. The gRNA of the present disclosure comprises a spacer sequence, i.e. a sequence capable of specifically binding to a given target oligonucleotide sequence. In certain aspects, the target-specific spacer sequence is positioned at the 5'-end of the gRNA. Typically, the target-specific spacer sequence has a length of about 15-27 nt, particularly about 17-24 nt. In some examples, the spacer is at least 20 nucleotides in length. In particular embodiments, the spacer is 20 nucleotides in length. In some embodiments, the spacer sequence comprises the sequence of SEQ ID NO: 29 (GGACAGUACUCCGCUCGAGU). The gRNA of the present disclosure may comprise an extended stem loop. Optionally the extended stem loop is according to SEQ ID NO: 14 (GUUUGAGAGCUAUGCUGGAAACAGCAUAGCAAGUUCAAAU). The gRNA of the present disclosure may comprise a nexus sequence, optionally wherein the nexus is according to SEQ ID NO 15 (AAGGCUAGUCC). A nexus sequence is a sequence capable of forming a secondary structure and interacting with a Cas enzyme. Typically, the nexus sequence comprises two conserved A residues followed by a stem-loop structure having a bulge, particularly a single unpaired U in the 3'-stem sequence, particularly a nexus sequence. The length of the nexus sequence is typically about 9 to about 12 nt. A CRISPR / Cas9-gRNA typically comprises a hairpin sequence comprising a first hairpin and a second hairpin. The term "first hairpin" relates to the most 5'-positioned hairpin in the hairpin sequence. The hairpin sequence including the first hairpin is distinct from a repeat / anti- repeat secondary structure of the gRNA, which typically comprises a lower stem, a bulge with a directional kink and an upper stem. Further, the hairpin sequence including the first hairpin is distinct from the nexus as outlined above. The gRNA of the present disclosure may comprise a first hairpin, optionally wherein the hairpin is according to SEQ ID NO 16. The first hairpin is a sequence, which forms a non-interrupted stem-loop structure. In a standard gRNA, the first hairpin typically forms a contiguous secondary structure of paired nucleotide building blocks, i.e. a stem, with a length of 4 nt and a loop consisting of 4 nt. At its 3'-end, a CRISPR / Cas9-gRNA typically comprises a stretch of U nucleotides, e.g. about 2 to about 10 U nucleotides. In a particular example, the gRNA has a sequence of six U nucleotides at the 3’ end (not including any residues of hairpin 2). This may be referred to as a “terminator”. In a further aspect, the gRNA comprises a locked hairpin. A locked hairpin secondary structure does not interfere with Oligonucleotide binding and / or editing. The term "locked hairpin " in any gRNA of the invention means that the hairpin has a high stability, particularly a high thermostability. In certain aspects, the locked hairpin of a gRNA of the present invention has a secondary structure comprising a contiguous stem. The term "contiguous stem" means an uninterrupted sequence of base pairs in the stem, i.e. without any bulge formed of unpaired nucleotides. In certain embodiments, the locked hairpin of a gRNA of the present invention has a secondary structure comprising a contiguous stem having a length of at least about 5 nt, e.g. about 6 nt to about 12 nt. In particular embodiments, the locked hairpin has a secondary structure comprising a contiguous stem having a length of 6, 7, 8, 9 or 10 nt, more particularly of 8 nt. In certain aspects, the locked hairpin of a gRNA of the present invention has a secondary structure comprising a contiguous stem comprising at least 2 C-G base pairs, e.g. about 3 C-G base pairs to about 6 C-G base pairs. In particular aspects, the locked hairpin forms a secondary structure comprising a contiguous stem comprising 2, 3, 4 or 5 C-G base pairs, more particularly 4 C- G base pairs. In this context, the term "C-G base pair" encompasses any base pair formed by a G nucleotide and a C nucleotide irrespectively from their orientation within the stem. Further, the term "locked hairpin secondary structure" in any gRNA of invention means that the hairpin has a high stability, particularly a high thermostability e.g. compared to an unlocked hairpin of a standard gRNA. The term "locked secondary structure" further means that the secondary structure of the first hairpin comprises a contiguous stem of base-paired nucleotide building blocks and particularly a contiguous stem of base-paired nucleotide building blocks structure connected by a loop of unpaired nucleotide building blocks. In particular aspects, the hairpin having a locked secondary structure does not comprise a base pair where the 2'OH groups of both nucleotides forming the base pair interact with an Oligonucleotide binding and / or editing enzyme. In this context, it is to be understood that an unlocked hairpin of a standard CRISPR / Cas9-gRNA, forms a stem-loop structure having a contiguous stem with a length of 4 nt and a loop of 4 nt. Further, the locked hairpin secondary structure of the invention is different from a CRISPR repeat / anti-repeat structure. In certain embodiments, a repeat / anti- repeat structure of a gRNA comprises a lower stem, optionally a bulge with a directional kink and optionally an upper stem. Further, a repeat / anti-repeat structure of a gRNA comprises at least one base pair, e.g.2 or 3 base pairs that interact with an Oligonucleotide binding and / or editing enzyme wherein the repeat / anti-repeat structure comprises at least one base pair, e.g.2 or 3 base pairs where the 2'OH groups of both nucleotides forming the base pair interact with an Oligonucleotide binding and / or editing enzyme. In contrast, a locked hairpin secondary structure as defined herein typically has an uninterrupted stem. The term "CRISPR repeat / hairpin" as used above relates to a CRISPR hairpin structure present in a single RNA molecule. The term "CRISPR repeat / anti- repeat" as used above refers to a CRISPR hairpin structure present in two separate RNA molecules forming the gRNA, e.g. a crRNA molecule and a tracrRNA molecule. The term "hairpin(s)" as used above refers to a non- CRISPR repeat or repeat / anti-repeat structure. A hairpin having a locked secondary structure according to the present invention may be an extension of an already present non-CRISPR hairpin or a newly introduced hairpin, e.g.5' or 3' to a "CRISPR repeat / hairpin" or "CRISPR repeat / anti-repeat" structure. The guide RNA or optionally sgRNA of the invention, further comprises a scaffold that comprises a second hairpin, optionally wherein the second hairpin is according to SEQ ID NO 17. In some aspects, the gRNA may comprise a second hairpin that isn’t a locked hairpin. The term "hairpin" as used above relates to a CRISPR hairpin structure present in a single RNA molecule. In some aspects, the gRNA may comprise a second hairpin which comprises an insertion of C and G base pair in the stem. The guide RNA may further comprise a terminator sequence, optionally wherein the terminator sequence is according to SEQ ID NO 18 (UUUUUU). As discussed herein, in some examples there is provided an sgRNA which is one of the following formats: +48; +54; +67; or + 85 (e.g. from Hsu et al., 2013). These numbers refer to the length of the entire sgRNA. It will be appreciated that the guides are target-specific, so can be swapped in. As such, where we refer to +85 or another of the numbers provided above, it will be understood that this includes a guide sequence, not necessarily the specific guide sequence used in the present Examples or the literature. Other target-specific changes are also envisaged. In some embodiments, the sgRNA is of the +85 sgRNA architecture. The modifications in respect of the +85 architecture may comprise as follows: 1. A replacement of U with G corresponding to position 5 of +85 backbone 2. An insertion of AGCUG between residues corresponding to position 12 and 13 of +85 backbone 3. An insertion of CAGCU between residues corresponding to position 16 and 17 of +85 backbone 4. A replacement of A with C corresponding to position 26 of +85 backbone 5. A replacement of GAAA with GGACUUCGGUCC between residues corresponding to position 52 and 57 of +85 backbone Thus, in an example, the gRNA may be of the following sequence (referred to herein as M1L) SEQ ID NO:2: NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUU AGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUCGGUCC AAGUGGCACCGAGUCGGUGCUUUUUU In some embodiments, the modifications in respect of the +85 architecture may comprise as follows: 1. A replacement of U with G corresponding to position 5 of +85 backbone 2. An insertion of AGCUG between residues corresponding to position 12 and 13 of +85 backbone 3. An insertion of CAGCU between residues corresponding to position 16 and 17 of +85 backbone 4. A replacement of A with C corresponding to position 26 of +85 backbone 5. A replacement of AU to UA between residues corresponding to position 44 and 47 of +85 backbone 6. A replacement of GAAA with GGACUUCGGUCC between residues corresponding to position 52 and 57 of +85 backbone 7. An insertion of C between residues corresponding to position 66 and 68 of +85 backbone 8. An insertion of G between residues corresponding to position 71 and 73 of +85 backbone Thus, in an example, the gRNA may be of the following sequence (referred to herein as M1LR): NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUU AGCAAGUUCAAAUAAGGCUAGUCCGUUUACAACUUGGACUUCGGUCC AAGUGGCACCCGAGUCGGGUGCUUUUUU SEQ ID NO: 3 In some embodiments, the modifications in respect of the +85 architecture may comprise as follows: 1. A replacement of U with G corresponding to position 5 of +85 backbone 2. An insertion of AGCUG between residues corresponding to position 12 and 13 of +85 backbone 3. An insertion of CAGCU between residues corresponding to position 16 and 17 of +85 backbone 4. A replacement of A with C corresponding to position 26 of +85 backbone 5. An insertion of C between residues corresponding to position 33 and 34 of +85 backbone 6. An insertion of G between residues corresponding to position 43 and 44 of +85 backbone 7. A replacement of AU to UA between residues corresponding to position 44 and 47 of +85 backbone 8. A replacement of GAAA with GGACUUCGGUCC between residues corresponding to position 52 and 57 of +85 backbone 9. An insertion of C between residues corresponding to position 66 and 68 of +85 backbone 10. An insertion of G between residues corresponding to position 71 and 73 of +85 backbone Thus, in an example, the gRNA may be of the following sequence (referred to herein as MN1LR): NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUU AGCAAGUUCAAAUAAGCGCUAGUCCGUGUUACAACUUGGACUUCGGU CCAAGUGGCACCcGAGUCGGGUGCUUUUUU SEQ ID NO: 5 In some embodiments, the modifications in respect of the +85 architecture may comprise as follows: 1. A replacement of U with G corresponding to position 5 of +85 backbone 2. An insertion of AGCUG between residues corresponding to position 12 and 13 of +85 backbone 3. An insertion of CAGCU between residues corresponding to position 16 and 17 of +85 backbone 4. A replacement of A with C corresponding to position 26 of +85 backbone 5. A replacement of AU to UA between residues corresponding to position 44 and 47 of +85 backbone 6. A replacement of GAAA with GGACUUCGGUCC between residues corresponding to position 52 and 57 of +85 backbone 7. An insertion of C between residues corresponding to position 66 and 68 of +85 backbone 8. A replacement of G with CUUCGG between residues corresponding to position 69 of +85 backbone 9. An insertion of G between residues corresponding to position 71 and 73 of +85 backbone Thus, in an example, the gRNA may be of the following sequence (referred to herein as M2LR): NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUU AGCAAGUUCAAAUAAGGCUAGUCCGUUUACAACUUGGACUUCGGUCC AAGUGGCACCcGACUUCGGUCGGGUGCUUUUUU SEQ ID NO: 6 In some embodiments, the modifications in respect of the +85 architecture may comprise as follows: 1. A replacement of U with G corresponding to position 5 of +85 backbone 2. An insertion of AGCUG between residues corresponding to position 12 and 13 of +85 backbone 3. An insertion of CAGCU between residues corresponding to position 16 and 17 of +85 backbone 4. A replacement of A with C corresponding to position 26 of +85 backbone 5. An insertion of C between residues corresponding to position 33 and 34 of +85 backbone 6. An insertion of G between residues corresponding to position 43 and 44 of +85 backbone 7. A replacement of AU to UA between residues corresponding to position 44 and 47 of +85 backbone 8. A replacement of GAAA with GGACUUCGGUCC between residues corresponding to position 52 and 57 of +85 backbone 9. An insertion of C between residues corresponding to position 66 and 68 of +85 backbone 10. A replacement of G with CUUCGG between residues corresponding to position 69 of +85 backbone 11. An insertion of G between residues corresponding to position 71 and 73 of +85 backbone Thus, in an example, the gRNA may be of the following sequence (referred to herein as MN2LR): NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUU AGCAAGUUCAAAUAAGCGCUAGUCCGUGUUACAACUUGGACUUCGGU CCAAGUGGCACCCGACUUCGGUCGGGUGCUUUUUU SEQ ID NO: 7 The gRNA or sgRNA of the invention is particularly suitable for CRISPR / Cas- mediated oligonucleotide binding and / or editing, wherein oligonucleotide binding and / or editing is mediated by a complex comprising a gRNA of the invention and a Cas enzyme, which may be selected from any suitable Cas enzyme. The gRNAs of the present disclosure are capable of forming a complex with a Cas enzyme. Preferably, the Cas enzyme is Cas9. “Cas enzyme” as used herein includes a catalytically fully active Cas nuclease capable of creating a double-strand break in a DNA template, a catalytically partially active Cas nickase capable of creating a single-strand break in a double stranded DNA template, e.g. Cas9 D10A or Cas9 H840A, and a catalytically inactive Cas enzyme, e.g. dCas9. Further, the term “Cas enzyme” includes split-fusion versions of the above enzymes, e.g. split-fusion versions of Cas9 or Cas9 D10A. In certain aspects, the Cas enzyme is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, and Cas14 including any recombinant variant thereof and particularly from Cas9 including any recombinant variant thereof. The Cas9 enzyme may be a Streptococcus, e.g. a S. pyogenes or Lactobacillus Cas9 enzyme as described by Briner et al. (2014), the content of which is herein incorporated by reference, including any recombinant variant thereof. In particular, the term "Cas enzyme" includes optimized Cas enzymes, e.g. Cas enzymes resulting in less off-target events such as R691A Cas9-Hifi and / or having a higher catalytic efficacy such as K918N SpyCECas9. The optimized guides provide herein may be used with a range of CRISPR enzymes including wildtype (wt) nucleases, as well as nickases and deadCas9s. The term "CRISPR-mediated oligonucleotide binding and / or editing" includes binding and optionally cleaving of any DNA or RNA template, particularly a double-stranded DNA template or a single-stranded DNA template or a single- stranded RNA template, e.g. an oligonucleotide, a plasmid, a chromosome in vitro and in vivo. For example, the term includes activation and / or repression, e.g. Cas9 gene activation and / or repression, genome editing, epigenome editing and / or prime editing. In certain examples, the locked hairpin of a gRNA or sgRNA of the present invention further comprises a super stable loop. The super stable loop which prevents misfolding of the gRNA, and optionally comprises at least one modified nucleotide building block. A "nucleic acid" or “nucleotide” can refer to a polynucleotide and includes poly- ribonucleotides and poly-deoxyribonucleotides. Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, e.g., cytosine (C), thymine (T) and uracil (U), and adenine (A) and guanine (G), respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) and G. Michael Blackburn, Michael J. Gait, David Loakes and David M. Williams, Nucleic Acids in Chemistry and Biology 3rd edition, (RSC publishing 2006), which are herein incorporated in their entirety for all purposes). Indeed, the present invention contemplates any deoxyribonucleotide or ribonucleotide component, and any chemical variants thereof. The polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. An "oligonucleotide" or "polynucleotide" can mean a nucleic acid ranging from at least 2, at least 8, at least 15 or at least 25 nucleotides in length, but may be up to 50, 100, 1000, 5000, 10000, 15000, or 20000 nucleotides long or a compound that specifically hybridises to a polynucleotide. Polynucleotides include sequences of DNA or RNA or mimetics thereof, which may be isolated from natural sources, recombinantly produced or artificially synthesised. The invention also encompasses situations in which there is a non-traditional base pairing, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and postulated to exist in a triple helix. "Polynucleotide" and "oligonucleotide" are used interchangeably herein. It will be understood that when a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), this also includes the corresponding RNA sequence (e.g., A, U, G, C) in which "U" replaces "T". The term "A" as used herein particularly denotes a nucleotide building block comprising an adenine nucleobase, a ribose a phosphate group or internucleosidic linkage. In some embodiments, the term specifies an unmodified nucleotide building block. In some embodiments, the term also includes a modified nucleotide building block. The term "G" as used herein particularly denotes a nucleotide building block comprising a guanine nucleobase, a ribose a phosphate group or internucleosidic linkage. In some embodiments, the term specifies an unmodified nucleotide building block. In some embodiments, the term also includes a modified nucleotide building block. The term "C" as used herein particularly denotes a nucleotide building block comprising a cytosine nucleobase, a ribose a phosphate group or internucleosidic linkage. In some embodiments, the term specifies an unmodified nucleotide building block. In some embodiments, the term also includes a modified nucleotide building block. The term "U" as used herein particularly denotes a nucleotide building block comprising an uracil nucleobase, a ribose a phosphate group or internucleosidic linkage. In some embodiments, the term specifies an unmodified nucleotide building block. In some embodiments, the term also includes a modified nucleotide building block, e.g. a nucleotide building block including a thymine nucleobase. The term "polynucleotide" includes, for instance, cDNA, RNA, DNA / RNA hybrid, antisense RNA, siRNA, mRNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatised, synthetic, or semi-synthetic nucleotide bases. The gRNAs may comprise a nucleoside or nucleosides that have been modified, for instance the modifications may be to the sugar moiety. In some examples, the 2’-position of the sugar moiety may be modified. A modification may be to any moiety that is not “-OH”, for instance to a -OMe. In some examples, the 4’ position of the sugar moiety may be modified. The gRNA may comprise one, two, three, four, or more types of nucleoside. The gRNA may comprise a combination of modified nucleosides and unmodified nucleosides. The gRNA may comprise only modified nucleosides. The nucleotides of the gRNA may all be modified in the same manner or may be modified in two or more different manners. The gRNA may comprise a modification to one or more internucleoside linkages. The gRNA of the present disclosure may include one or more naturally occurring nucleobase and / or one or more modified nucleobase. A modified nucleobase is a nucleobase that is capable of base pairing with a nucleobase of a nucleic acid but is structurally different from a naturally occurring nucleobase. In an example, the gRNA comprises phosphorothioate bonds and / or internal 2'-OMe residues. A gRNA or sgRNA of the invention may comprise in 5' to 3' direction: (i) a target-specific spacer sequence, (ii) a repeat / anti-repeat structure comprising a lower stem, optionally a bulge with a directional kink and optionally an upper stem, (iii) optionally a nexus sequence and (iv) a hairpin sequence comprising at least one locked hairpin secondary structure. In certain embodiments, the hairpin sequence (iv) of the gRNA may include in 5' to 3' direction a first hairpin and optionally a second hairpin wherein at least the first hairpin has a locked secondary structure. In certain aspects, the locked hairpin of a gRNA or sgRNA of the present invention forms a secondary structure comprising a contiguous stem having a melting temperature of above 65°C as determined by e.g. differential scanning calorimetry and UV absorbance spectroscopy. In some aspects, the gRNA is an sgRNA, a plurality of RNA molecules, crRNA or tracrRNA. In certain aspects, the gRNA of the invention consists of a single RNA molecule, e.g. a single guide RNA (sgRNA) molecule. In further aspects, the gRNA of the invention comprises at least two separate RNA molecules, e.g. a spacer-containing CRISPR RNA (crRNA) molecule and a trans- activating CRISPR RNA (tracrRNA) molecule. In still further embodiments, the gRNA of the invention comprises three or more separate RNA molecules, e.g. a spacer-containing CRISPR RNA (crRNA) molecule, and a trans-activating CRISPR RNA (tracrRNA) molecule comprised of two separate RNA molecules. In some aspects of the invention, the guide RNA or sgRNA is of a sequence according to any one of SEQ ID NOs: 2, 3, 5, 6 or 7 and further comprises one or more insertions, deletions, replacements, or substitutions. The gRNA of the invention may have a sequence according to SEQ ID NOs: 2, 3, 5, 6 or 7 and also comprises one to five, one to three, one or two, or one insertions, substitutions, replacements, or deletions. In some examples, the gRNA is of a sequence according to any one of SEQ ID NOs: 2, 3, 5, 6 or 7 and further comprises one or more insertions, deletions, replacements, or substitutions, wherein the one or more insertions, deletions, replacements, or substitutions do not alter the modifications recited herein. The gRNA of the invention may have a sequence according to SEQ ID NOs: 2, 3, 5, 6 or 7 and also comprises one to five, one to three, one or two, or one insertions, substitutions, replacements, or deletions, wherein the one or more insertions, deletions, replacements, or substitutions do not alter the modifications recited herein. The gRNA of the invention may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 2, 3, 5, 6 or 7 wherein the modifications recited herein are maintained. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 2. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 3. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 4. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 5. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 6. The guide RNA or sgRNA may comprise or be according to the sequence of SEQ ID NO: 7. In an aspect of the present disclosure, there is provided one or more nucleic acid molecule encoding a gRNA as provided herein. In an embodiment, there is provided a nucleic acid molecule encoding an sgRNA as provided herein. A further aspect provided herein is a library comprising a plurality of gRNAs or sgRNAs as described above comprising different target-specific spacer sequences and a constant gRNA sequence, particularly a common constant gRNA sequence comprising at least one locked hairpin secondary structure at a position that does not interfere with oligonucleotide binding and / or editing. For example, the target-specific spacer sequences of individual gRNAs may be directed against different portions of a single target oligonucleotide sequence, e.g. a single gene, against different target sequences, e.g. different genes or any combination thereof. The library may comprise different sgRNAs comprising individual target-specific spacer sequences and common constant gRNA sequences, particularly a common locked hairpin secondary structure, optionally a common repeat / anti-repeat structure, and / or a common nexus sequences. Alternatively, the library may comprise different gRNAs composed of crRNAs comprising individual target-specific spacer sequences and a common tracrRNAs. In another aspect, there is provided a complex comprising a Cas enzyme and a gRNA disclosed herein. Preferably, the Cas enzyme is Cas9. In certain aspects, the Cas enzyme is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, and Cas14 including any recombinant variant thereof and particularly from Cas9 including any recombinant variant thereof. In another aspect, there is provided an engineered extracellular vesicle (EV) for delivery of a bioactive cargo. The EV may also comprise the gRNA as disclosed herein and optionally further comprise an endosomal escape enhancer. The terms “extracellular vesicle” or “EV” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable from a cell in any form. The size of EVs may vary considerably, but an EV typically comprises a volume defined by a bi-lipid membrane and having a nano-sized hydrodynamic radius, i.e., a radius below 1000 nm. The different types of EVs are defined by different morphologies, structure, messages and function. EVs can be broadly divided into two categories, (1) ectosomes and (2) exosomes. Clearly, EVs may be derived from any cell type, whether in vivo, ex vivo or in vitro (further details of suitable source or producer cells are herein described below). Exosomes, microvesicles and ARMMs are just some examples of the different subtypes that fall under the hereinbefore broader description of EVs and represent particularly preferable EVs, but it will be appreciated that other EVs may also be advantageous in certain circumstances. Advantageously, the EV is an exosome. Furthermore, the said terms can be understood to also relate to, in some embodiments, extracellular vesicle mimics, cellular membrane vesicles obtained through membrane extrusion, sonication or other techniques, etc. Exosomes, microvesicles and ARMMs are just some examples of the different subtypes that fall under the hereinbefore broader description of EVs and represent particularly preferable EVs, but it will be appreciated that other EVs may also be advantageous in certain circumstances. Advantageously, the EV is an exosome. The terms “exosome” or “exosomes” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable or derivable from the endosomal, lysosomal and / or endo-lysosomal pathway and / or from inward budding of the plasma membrane and / or cell membrane. Exosomes often have a size of from about 30 to about 300 nm, typically in the range of from about 40 to about 250 nm, and sometimes from about 40 to about 160nm, which is a highly suitable size range. Clearly, EVs may be derived from any cell type, both in vivo, ex vivo, and in vitro. For instance, EVs may be obtainable from amnion epithelial (AE) cells, from mesenchymal stromal cells (MSCs), from placenta-derived cells, from HEK cells. The terms “producer cell”, “cell source”, EV-producing cells” and “EV- producing cell source” are used interchangeably herein and shall be understood to relate to any cell from which the EVs of the present invention may be obtainable or derivable. Generally, EVs may be derived from essentially any cell source, be it a primary cell source or an immortalized cell line. The EV source cells may thus be any embryonic, fetal, or adult somatic stem cell types, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method, as well as any adult cell source. The source cells per the present invention may be selected from a wide range of cells and cell lines, for including but not limited to mesenchymal stem or stromal cells (obtainable from e.g., bone marrow, adipose tissue, Wharton’s jelly, perinatal tissue, chorion, placenta, tooth buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amnion cells and more specifically amnion epithelial (AE) cells optionally expressing various early markers, myeloid suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. The term “genetically modified cargo” can mean any molecule that is physically and chemically able to be carried by an EV, for instance a Cas9 RNP. It is suitably a molecule that does not naturally occur in an EV, i.e., the EV is engineered so as to contain the cargo. The term “genetically modified cargo” shall be understood to relate to either a therapeutic cargo or any combination thereof. The term “bioactive” can mean having a biological effect. Thus, with respect to diagnostic cargo, the term “bioactive” can mean any molecule that enables the identification of one or more properties of living matter. With respect to therapeutic cargo, the term “bioactive” can mean any molecule that has a therapeutic effect on living matter, including both treatment and prophylaxis. Living matter includes living beings, organs, tissues and cells. The terms “cargo” and “bioactive cargo” are used interchangeably throughout the specification and either a cargo or a bioactive cargo can be employed in any and all embodiments of the disclosure. Cargo in accordance with the present disclosure may include, but are not limited to, the following: In a preferred aspect, the genetically modified cargo is a protein, preferably a Cas9 ribonucleoprotein (RNP). An “endosomal escape enhancer” is a generally understood term in the art and may include any protein, (poly)peptide, or fragment, including, but not limited to, virally- and / or bacterially- derived components that enable, facilitate and / or enhance the release of an EV (e.g., exosome) and / or lysosome from the endo- lysosomal pathway and / or architecture and in doing so greatly increase the cytoplasmic delivery in vitro and in vivo. The endosomal escape enhancer may not exist naturally in an EV. Rather, the EV is engineered so as to contain the endosomal escape enhancer. In one embodiment, the endosomal escape enhancer is any polypeptide exhibits fusogenic activity and / or that facilitates the fusion of viral and / or cellular membranes. In a preferred embodiment, the endosomal escape enhancer is VSV-G or a variant thereof. An endosomal escape enhancer as described herein can comprise or consist of an amino acid sequence of any one of SEQ ID NOs: 11, 12 and 13 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 11, 12 and 13, wherein the polypeptide exhibits fusogenic activity as described herein and / or binds to LDL-R on the surface of a cell as described herein, or a functional fragment or variant thereof. In a preferred aspect, an endosomal escape enhancer as described herein can comprise or consist of an amino acid sequence of SEQ ID NO: 11 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 11, wherein the polypeptide exhibits fusogenic activity as described herein and / or binds to LDL-R on the surface of a cell as described herein, or a functional fragment or variant thereof. An endosomal escape enhancer as described herein can be encoded by a polynucleotide sequence comprising or consisting of any one of SEQ ID NOs: 8, 9 and 10 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 8, 9 and 10, wherein the sequence encodes a polypeptide exhibiting fusogenic activity and / or that binds to LDL-R on the surface of a cell as described herein, or a functional fragment or variant thereof. In various aspects, the polynucleotide constructs described herein, for example according to the second aspect, can comprise or consist of a polynucleotide sequence comprising or consisting of any one of SEQ ID NOs: 8, 9 and 10 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 8, 9 and 10, wherein the sequence encodes a polypeptide exhibiting fusogenic activity and / or that binds to LDL-R on the surface of a cell as described herein, or a functional fragment or variant thereof. In a preferred aspect, an endosomal escape enhancer as described herein can be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 8 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8, wherein the sequence encodes a polypeptide exhibiting fusogenic activity and / or that binds to LDL-R on the surface of a cell as described herein, or a functional fragment or variant thereof. In some embodiments, an endosomal escape enhancer is not required and may be optional. The engineered EV may also comprise a release system. The release system may be any system capable of physically linking components in a manner whereby the components may subsequently be separated and may be positioned for the release of the genetically modified cargo, preferably the Cas9 RNP. Release systems in accordance with the disclosure may include, but are not limited to, the following: Cis-cleaving sequences (or self-cleaving) such as inteins, and mini-inteins, light induced monomeric or dimeric release domains such as Kaede, KikGR, EosFP, tdEosFP, mEos2, PSmOrange, the GFP-like Dendra proteins Dendra and Dendra2, CRY2-CIBN, etc. Alternatively, nuclear localization signal (NLS) – nuclear localization signal-binding protein (NLSBP) (NLS-NLSBP) release system may be employed. Protease cleavage sites may also be incorporated into the protein constructs and / or fusion proteins for spontaneous release, etc., depending on the desired functionality of the fusion polypeptide. It is to be appreciated that in certain embodiments the protein construct may be a fusion protein. In the case of nucleic acid cargos specific nucleic acid cleaving domains may be included. In particular examples, the release system may be a cis-cleaving sequence. The cis-cleaving sequence may be an amino acid sequence positioned between at least two domains of a protein construct or a fusion protein. For instance, the cis-cleaving sequence may be positioned between a cargo and an endosomal escape enhancer, a multimerization domain or an EV protein. A portion of the cis-cleaving sequence may also form part of the protein to which it is attached (i.e., a cargo, and an endosomal escape enhancer, a multimerization domain or an EV protein). The cis-cleaving sequence may be a self-cleaving protein, for example an intein. The intein may be a slow-cleaving or a fast-cleaving intein. The intein may be a mini-intein, such as a mini-intein that has been modified to optimise the cleavage rate. The intein may be a delta-intein-CM. Thus, cis-cleaving sequences (or self-cleaving) in accordance with the disclosure may include, but are not limited to, the following: Inteins, mini-inteins, delta inteins and certain variants, mutations and domains thereof having a desired functionality (including, but not limited to self- cleaving instead of splicing), such as a mini-intein modified to optimise the cleavage rate. For example, a mini-intein having C1A, D24G, V67L and / or D150G substitution in the N-terminal portion (or an appropriate – C-extein position). For example, splicing is enabled by the +1 position of the intein, wherein the +1 is Cys. Substitution of Cys with Ala in ΔI-CM removes the splicing capability and supports cleavage only. It will be appreciated that other mutations, substitutions, and the like may also similarly work. As such the substitution hereinbefore mentioned, while exemplary, is not limiting in any way and may also differ from intein to intein. In certain instances, one may opt to utilize slow-cleaving inteins. A slow- cleaving system may be preferable when more time is required to ensure efficient loading of an EV with the desired cargo. Slow-cleaving inteins in accordance with the disclosure may include, but are not limited to, the following: mini-inteins, delta inteins, delta-intein-CM and a mini-intein and certain variants, mutations and domains thereof having a desired functionality (such as, but not limited to cleavage action instead of splicing and cleavage rate). In a preferred embodiment, the slow-cleaving cis-cleaving release system is based on an intein system, wherein the C-terminal portion of the intein may comprise the amino acid sequences Val-Val-Val-His-Asn, more preferably wherein the C-terminal portion of the intein is modified to comprise Val-Val-Val-His-Asn-Gly. Certain modifications at the +1 C-extein position, have been observed to slow the cleavage rate (i.e., are slow-cleaving). In certain instances, one may opt to utilize a fast-cleaving cis-cleaving release system (such as a fast-cleaving cis-cleaving intein). A fast-cleaving system may be preferable when EVs need to be harvested quickly. In a preferred embodiment, the fast-cleaving cis-cleaving release system is based on an intein system, wherein the C-terminal portion of the intein may comprise the amino acid sequences Val-Val-Val-His-Asn or Val-Val-Val-His-Asn-Cys. Certain modifications at the +1 C-extein position, such as the abovementioned example, have been observed to speed up the cleavage rate (i.e., are fast- cleaving). In a preferred embodiment of the present invention, the intein is a delta-ICM intein or more preferably an I-29 intein. In one embodiment, the delta-ICM intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 52. In a preferred embodiment, the I-29 intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 53. In one preferred embodiment the engineered EV also comprises an EV protein (or an EV polypeptide) that may be an exosomal protein when the EV is an exosome. In one embodiment an EV polypeptide is a polypeptide that is localised to an EV when expressed in an EV producer cell. Such EV protein (or exosomal protein) comprised in the engineered EVs may be selected from a wide variety of proteins, such as EV transmembrane proteins, EV membrane associated proteins, ICAMs, integrins, sydecans, syntenins, GPI anchor proteins, ATP transporters, members of the myristoylated alanine rich protein kinase C substrate (MARCKS) protein family, matrix metalloproteinases (MMPs), TNFRs known at the time of the disclosure to be associated with EVs (or exosomes), as well as any combinations, derivatives, domains, variants, mutants, or regions thereof. Transmembrane EV proteins may include, but are not limited to, the following non-limiting examples: CD9, CD53, CD63, CD81, CD82, CD54 (ICAM1), CD50 (ICAM3), CD49d (ITGA4), CD71, CD133, ALIX, Syntenin-1, Syntenin-2, Lamp2, Lamp2a, Lamp2b, TSN1, TSN3, TSN4, TSN5 TSN6, TSPAN8, TSN31, TSN10, TSN11, TSN12, TSN13, TSN14, TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN9, TSN32, TSN33, CD37, CD151, CD231 (TSN7; TSPAN7; TALLA-1, TM4SF2), NOTCH1, NOTCH2, NOTCH3, NOTCH4, Delta-Like Protein (Delta 1; DLL1), CD3 epsilon, CD34, CD40, CD47, CD86, CD115 (CSF1R), CD125, CD200, CD362 (syndecan 2), EGFR, GLUR2, GLUR3 (GRIA3), L1CAM, Syntaxin 3 (STX3), TFR1, UPK1A, UPK1B, VTI1A, VTI1B, CD184 (CXCR4), CD102, Integrin beta-5 (ITGB5), Integrin beta-6 (ITGB6), Integrin beta-7 (ITGB7), CD104, CD19, CD11a, CD11b, CD11c, CD235a, CD3zeta, CD41, CD49b (ITGA2), CD49c, CD49e, CD50, CD61, JAG2, CD18 (ITGB2), CD13, CD45, CD110, CD117, CD135, CD273, CD274, AGRN, HLA-DM, LFA-1,Mac-1alpha, CD36, CD279, TfR1, Syndecan 3, Syndecan 4, CD224, CLIC1, CLIC4, CD44, Prostaglandin F2 Receptor Negative Regulator (PTGFRN) (FPRP), GP130, HLAA, Limp2, MYOF, ATP2B2, ATP2B3, ATP2B4, IGSF2, IGSF3, IGSF8, ITGB1, ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3 (AT1B3), SLC3A2, MMP14,PDGFR, PRPH2, ROM1, CD55, or any single-pass or multi-pass transmembrane protein and / or tetraspanin, or any other EV protein and / or exosomal protein known at the time of the disclosure that spans the EV membrane (or exosomal membrane). Membrane associated EV proteins in accordance with the disclosure may include, but are not limited to, the following non-limiting examples: CD2, CD138 (Syndecan 1), CD40L, Delta-Like Protein (Delta 4; DLL4), Jaggard 1 (JAG1), MFGE8 (lactoadherin; LA), FLOT1, FLOT2, SLIT2, TCRA, CD117 (isoform 3), GAPDH, AT2B4, BASP1, BSG, ARRDC1, TCRA, TCRB, TCRD, TCRG, or any other EV protein and / or exosomal protein known at the time of the disclosure that is associated with the EV / exosomal membrane, plasma membrane or lysosomal membrane from which the EV / exosome has been created. Other EV proteins in accordance with the disclosure may include, but are not limited to, the following non-limiting examples: LGALS3BP, COL6A1, LAMB1, LAMC1, HSPG2, ANNEXIN, CD111, AAAT, BAS1, CD45RA, GTR1, ITA3, ITGA3, Mac-1beta, MARCKSL1, SSEA4, TCRB, TCRD, TCRG, or any other EV protein and / or exosomal protein known at the time of the disclosure that is particularly enriched in EVs and / or exosomes. Where the EV protein (or exosomal protein) is a transmembrane protein, it may be either a single-pass transmembrane protein or a multi-pass transmembrane protein. Single-pass transmembrane EV proteins in accordance with the disclosure may include, but are not limited to, the following non-limiting examples: AGRN, ALIX, CD11a, CD11b, CD11c, CD13, CD18 (ITGB2), CD19, CD102, CD104, CD110, CD115 (CSF1R), CD117, CD125, CD135, CD200, CD235a, CD273, CD274, CD3 epsilon, CD3zeta, CD34, CD362 (Syndecan 2), CD40, CD41, CD44, CD45, CD49b (ITGA2), CD49c, CD49d (ITGA4), CD49e, CD50 (ICAM3), CD51, CD54 (ICAM1), CD61, CD71, CD86, Delta-Like Protein 1 (Delta 1; DLL1), EGFR, Integrin beta-5 (ITGB5), Integrin beta-6 (ITGB6), Integrin beta-7 (ITGB7), L1CAM, Lamp2, Lamp2a Lamp2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Syntaxin (STX3), Syntenin 1, Syntenin 2, CD30, JAG2, HLA-DM, LFA-1, MAC-1alpha, VTI1A, VTI1B, AT1B3, BSG, TfR1, Syndecan 3, Syndecan 4, CD224, CLIC1, CLIC4, PTGFRN (FPRP), GP130, HLAA, MYOF, IGSF2, IGSF3, IGSF8, ITGB1, AT1B3 (ATP1B3), SLC3A2, MMP14, PDGFR, CD55, or any other EV protein and / or exosomal protein known at the time of the disclosure that spans the EV membrane (or exosomal membrane) only once. Multi-pass transmembrane EV proteins in accordance with the present disclosure may include, but are not limited to, the following non-limiting examples: GLUR2, GLUR3 (GRIA3), CD47, CD133, CD151, CD184 (CXCR4), CD231 (TSPAN7; TALLA-1; TM4SF2); CD37, CD53, CD63, CD81, CD82, CD9, TSN1, TSN3, TSN4, TSN5 TSN6, TSPAN8, TSN31, TSN10, TSN11, TSN12, TSN13, TSN14 (TSPAN14), TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN9, TSN32, TSN33, CD36, CD279, AT2B4, Limp2, ATP2B1, ATP2B2, ATP2B3, ATP2B4, ATP1A1, ATP1A2, ATP1A3, ATP1A4, PRPH2, ROM1, UPK1A, UPK1B, or any other EV protein and / or exosomal protein known at the time of the disclosure that spans the EV membrane (or exosomal membrane) more than once. When a multi-pass transmembrane protein is the EV protein (or exosomal protein) it may particularly include a tetraspanin. Tetraspanins in accordance with the present disclosure may include, but are not limited to, the following non-limiting examples: CD231 (TSPAN7; TALLA-1; TM4SF2); CD37, CD53, CD63, CD81, CD82, CD9, TSN1, TSN3, TSN4, TSN5 TSN6, TSPAN8, TSN31, TSN10, TSN11, TSN12, TSN13, TSN14 (TSPAN14), TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN9, TSN32, TSN33, ROM1 (TSN23), UPK1A (TSN21), UPK1B (TSN20), or any other EV protein and / or exosomal protein known at the time of the present disclosure that spans the EV membrane (or exosomal membrane) four times (i.e., 4 x membrane domains) and has two extravesicular loops displayed on the surface (or outer leaflet) of the EV membrane (or exosomal membrane) where one of the loops is a larger outward-facing loop and the second loop of the two loops is a smaller outward- facing loop. In one embodiment, the EV polypeptide is a polypeptide comprising a myristoylation site. As used herein the term “myristoylation site” refers to an amino acid sequence to which a myristic acid molecule is covalently attached when the polypeptide is expressed in a cell, preferably a mammalian cell. In one embodiment, the myristoylation site is Gly-X-X-X-Ser / Thr, where “X” represents any amino acid. Particularly advantageous EV proteins include the tetraspanins, TSN2, CD63, CD81, CD9, CD82, the single-pass transmembrane EV protein PTGFRN, a HIV GAG protein and VSVG. In a most preferred embodiment, the EV polypeptide is TSN2. Mutations may be introduced into the wild-type sequence of the EV protein to alter its function. A preferred mutant according to the disclosure is CD63(Y235A). Advantages of an EV protein (or exosomal protein) includes, but are not limited to, the following: • Including an EV protein (e.g., exosomal protein) in the protein construct helps to actively load the Cas9 RNP into the EV (or exosome). • The EV protein (e.g., exosomal protein) traffics a protein construct, which may comprise other protein domains with desired utility (e.g., a release system, preferably a self-cleaving intein), such as part of a fusion protein to an EV membrane. The use of EV proteins has the effect of driving loading of the protein construct into EVs such that protein construct is actively loaded into EVs; as a result, EV proteins are sometimes referred to as carrier proteins. In a particular embodiment, there is provided an engineered EV suitable for delivery of at least one bioactive cargo, for instance wherein the cargo is a Cas9 RNP comprising a gRNA or sgRNA disclosed herein. The EV comprises a protein construct or a fusion protein which comprises a cargo wherein the cargo is a Cas9 RNP comprising a gRNA or sgRNA disclosed herein, an EV protein, and a release system, wherein the release system is positioned in- between the cargo and the EV protein. The release system may be adjacent to the cargo, such that the cargo is released as either an isolated component or substantially free form. In a particular embodiment, the release system is a cis-cleaving amino acid sequence. The EV further optionally comprises an endosomal escape enhancer, for instance VSV-G. The EV may be an exosome and the EV protein may be an exosomal protein. In one embodiment the endosomal escape enhancer (e.g., VSV-G) may be displayed on the surface of the EV and the cargo may be loaded into the lumen of the EV. The invention further relates to the use of the gRNA or the sgRNA, nucleic acid molecule, the library, the complex or the EV as described above for CRISPR- mediated Oligonucleotide binding and / or editing, particularly for CRISPR / Cas- mediated Oligonucleotide binding and / or editing in vitro, ex vivo or in vivo, e.g. in a cell-free system, in an isolated cell, in an organ or in an organism. The use includes non-medical applications, e.g. as research tool or medical applications, e.g. for in vivo or ex vivo use. In particular embodiments, a library may be used in screening applications, e.g. in unbiased screening applications, in order to identify target sequences suitable for CRISPR- mediated Oligonucleotide binding and / or editing. In particular, the invention further relates to the use of the gRNA or sgRNA, nucleic acid molecule, the library, the complex or the EV as described above for CRISPR-mediated Oligonucleotide binding and / or editing in a eukaryotic cell or a eukaryotic organism or a part, e.g. organ of a eukaryotic organism. The eukaryotic cell may be selected from an animal cell, e.g. a mammalian cell including a human cell, or a plant cell. In particular embodiments, the cell is a stem cell including a pluripotent stem cell, e.g. a human pluripotent stem cell, or an immortalized cell, e.g. a tumor cell. The eukaryotic organism may be selected from an animal, e.g. a mammal including a non-human mammal and a human being. Still a further aspect relates to a preparation comprising the gRNA, nucleic acid molecule, the library, the complex or the EV as described above for use in medicine including human medicine and veterinary medicine. Further, the preparation may comprise a pharmaceutically acceptable excipient, e.g. a polycationic compound such as a polycationic lipid. The preparation may be administered by known routes, e.g. by injection or infusion. Still a further aspect relates to the use of the gRNA, nucleic acid molecule, the library, the complex or the EV as described above for genome editing in a plant cell, plant organism or part thereof. Some aspects also relate to compositions comprising the gRNA, or sgRNA, a library, complex or an EV according to the invention and a pharmaceutically acceptable excipient or diluent, vehicle, solvent or carrier. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The terms encompass any of the agents approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the therapeutic cargo. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe and non-toxic. Exemplary excipients include: Exemplary excipients include degradation or loss of activity stabiliser excipients such as proteins such as human serum albumin, polyols such as glycerol, sorbitol and erythritol, amino acids such as arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine and methionine, polymers such as polyvinylpyrrolidone and hydroxypropyl cellulose, surfactants such as polysorbate 80, polysorbate 20 and pluronicF68, antioxidants such as ascorbic acid and alpha-tocopherol (vitamin E), buffers such as acetate, succinate, citrate, phosphate, histidine, tris(hydroxymethyl)aminomethane (TRIS), metal ion / chelators such as Ca2+, Zn2+ and EDTA, Cyclodextrin based such as hydroxypropyl ß-cyclodextrin and others such as polyanions and salts, stabilisers or bulking agents such as lactose, trehalose, dextrose, sucrose, sorbitol, glycerol, albumin, gelatin, mannitol and dextran, or preservatives such as benzyl alcohol, m-cresol, phenol, 2-phenoxyethanol. The pharmaceutical compositions according to the present invention may be formulated by any known method of formulation including but not limited to: Oral drugs – Tablet, Capsule, Sustained release, liquid Intravenous Formulations Parenteral Formulations Topical Formulations - cutaneous administration: cream, ointment, gel, paste, powder Modified release Formulations – sustained release formulation Liquid or lyophilized formulations The gRNAs as per the present invention may be administered to a human or animal subject via various different administration routes, for instance auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra- arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracerebroventricular, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated and / or the characteristics of the EVs, the cargo molecule in question, or the EV population as such. The present invention also provides a composition comprising gRNA or sgRNA, a library, a complex or an EV for use as a medicament. The present invention also provides a composition comprising gRNA or sgRNA, a library, a complex or an EV for use in the treatment of disease. Non-limiting examples of disorders, some of which may be genetic disorders and conditions that are suitable targets include the following non-limiting examples: autoimmune diseases (such as celiac disease, Crohn’s disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus) ulcerative colitis, ankylosing spondylitis, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor- associated periodic syndrome (TRAPS), deficiency of the interleukin-1 receptor antagonist (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cord injury, vasculitis, Guillain- Barré syndrome, acute myocardial infarction, ARDS, sepsis, meningitis, encephalitis, liver failure, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), kidney failure, heart failure or any acute or chronic organ failure and the associated underlying etiology, graft-vs-host disease, Duchenne muscular dystrophy and other muscular dystrophies, In-born errors of metabolism including: Disorders of carbohydrate metabolism e.g.,  G6PD deficiency galactosemia, hereditary fructose intolerance, fructose 1,6- diphosphatase deficiency and the glycogen storage diseases, Disorders of organic acid metabolism (organic acidurias) such as alkaptonuria, 2- hydroxyglutaric acidurias, methylmalonic or propionic acidemia, multiple carboxylase deficiency, Disorders of amino acid metabolism such as phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, Aminoacidopathies e.g., hereditary tyrosinemia, nonketotic hyperglycinemia, and homocystinuria,  Hereditary tyrosinemia, Fanconi syndrome, Primary Lactic Acidoses e.g., pyruvate dehydrogenase, pyruvate carboxylase and cytochrome oxidase deficiencies, Disorders of fatty acid oxidation and mitochondrial metabolism such as short, medium, and long- chain acyl-CoA dehydrogenase deficiencies also known as Beta-oxidation defects, Reye’s syndrome, Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD.), MELAS, MERFF, pyruvate dehydrogenase deficiency, Disorders of porphyrin metabolism such as acute intermittent porphyria, Disorders of purine or pyrimidine metabolism such as Lesch–Nyhan syndrome, Disorders of steroid metabolism such as lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, Disorders of mitochondrial function such as Kearns–Sayre syndrome, Disorders of peroxisomal function such as Zellweger syndrome and neonatal adrenoleukodystrophy, congenital adrenal hyperplasia or SmithLemli-Opitz, Menkes syndrome, neonatal hemochromatosis, Urea cycle disorders such as N-Acetylglutamate synthase deficiency, carbamoyl phosphate synthetase deficiency, ornithine transcarbamoylase deficiency, citrullinemia (deficiency of argininosuccinic acid synthase), argininosuccinic aciduria (deficiency of argininosuccinic acid lyase), argininemia (deficiency of arginase), hyperornithinemia, hyperammonemia, homocitrullinuria (HHH) syndrome (deficiency of the mitochondrial ornithine transporter), citrullinemia II (deficiency of citrin, an aspartate glutamate transporter), lysinuric protein intolerance (mutation in y+L amino acid transporter 1, orotic aciduria (deficiency in the enzyme uridine monophosphate synthase UMPS), all of the lysosomal storage diseases, for instance Alpha-mannosidosis, Betamannosidosis, Aspartylglucosaminuria, Cholesteryl Ester Storage Disease, Cystinosis, Danon Disease, Fabry Disease, Farber Disease, Fucosidosis, Galactosialidosis, Gaucher Disease Type I, Gaucher Disease Type II, Gaucher Disease Type III, GM1 Gangliosidosis Type I, GM1 Gangliosidosis Type II, GM1 Gangliosidosis Type III, GM2 - Sandhoff disease, GM2 - Tay-Sachs disease, GM2 - Gangliosidosis, AB variant, Mucolipidosis II, Krabbe Disease, Lysosomal acid lipase deficiency, Metachromatic Leukodystrophy, MPS I -Hurler Syndrome, MPS I - Scheie Syndrome, MPS I Hurler-Scheie Syndrome, MPS II - Hunter Syndrome, MPS IIIA - Sanfilippo Syndrome Type A, MPS IIIB - Sanfilippo Syndrome Type B, MPS IIIB - Sanfilippo Syndrome Type C, MPS IIIB - Sanfilippo Syndrome Type D, MPS IV Morquio Type A, MPS IV - Morquio Type B, MPS IX - Hyaluronidase Deficiency, MPS VI -Maroteaux-Lamy, MPS VII - Sly Syndrome, Mucolipidosis I - Sialidosis, Mucolipidosis IIIC, Mucolipidosis Type IV, Mucopolysaccharidosis, Multiple Sulfatase Deficiency, Neuronal Ceroid Lipofuscinosis T1, Neuronal Ceroid Lipofuscinosis T2, Neuronal Ceroid Lipofuscinosis T3, Neuronal Ceroid Lipofuscinosis T4, Neuronal Ceroid Lipofuscinosis T5, Neuronal Ceroid Lipofuscinosis T6, Neuronal Ceroid Lipofuscinosis T7, Neuronal Ceroid Lipofuscinosis T8, Neuronal Ceroid Lipofuscinosis T9, Neuronal Ceroid Lipofuscinosis T10, Niemann-Pick Disease Type A, Niemann-Pick Disease Type B, Niemann-Pick Disease Type C, Pompe Disease, Pycnodysostosis, Salla Disease, Schindler Disease and Wolman Disease, etc. cystic fibrosis, primary ciliary dyskinesia, pulmonary alveolar proteinosis, ARC syndrome, Ret syndrome, neurodegenerative diseases including Alzheimer´s disease, Parkinson´s disease, GBA associated Parkinson’s disease, Huntington’s disease and other trinucleotide repeat-related diseases, prion diseases, dementia including frontotemporal lobe dementia, ALS, motor neuron disease, multiple sclerosis, cancer-induced cachexia, anorexia, diabetes mellitus type 2, AD, PD, Friedrichs, FTD, ALS, HD, Angelman’s syndrome, heart disease or disorder, cardiovascular disease or disorder and various cancers. In a preferred embodiment, the disease or disorder is a genetic disease or disorder. In a further preferred embodiment, the disease or disorder is a paediatric disease or disorder and / or a disease or disorder wherein symptoms are exhibited during childhood. In a further preferred embodiment, the disease or disorder is a heart disease or disorder or a cardiovascular disease or disorder. In a further preferred embodiment, the disease or disorder is a neurological disease or disorder, such as ALS or Angelman’s syndrome. In a particularly preferred embodiment, the disease or disorder is Angelman’s syndrome. In a preferred embodiment, the subject to be treated is a child. In one embodiment, a child is under the age of 21 years old, under the age of 18 years old, under the age of 16 years old or under the age of 14 years old. In a preferred embodiment, the child is pre-pubescent. Finally, the invention also provides a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the gRNA or sgRNA, a library, a complex or an EV. In some aspects, there is provided a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the composition comprising the guide and a CRISPR enzyme. This may include a method of treating a subject with a condition correctable by genome editing. Such conditions are numerous, and are further discussed herein, but may include conditions resulting from SNPs or trinucleotide repeat disorders. The system may also be used to transform and thereby modify the genome of, somatic tissue. These methods may preferably be ex vivo, for example, creating a modified cell line. Another example is Chimeric Antigen Receptor (CAR)T cells which can be modified ex vivo and re-infused into a patient to target cancers. Thus, methods of treating suitable cancers are provided. Such methods may include delivering CRISPR / Cas complex(es) to a cancer cell to stop tumor growth. Additionally, it should be noted that embodiments and features described in connection with one of the aspects and / or embodiments of the present disclosure also apply mutatis mutandis to all the other aspects and / or embodiments of the disclosure. Moreover, any and all features can be freely combined with any and all other features. Furthermore, any polypeptide or polynucleotide or any polypeptide or polynucleotide sequences (amino acid sequences or nucleotide sequences, respectively) of or relating to the present disclosure may deviate considerably from the original polypeptides, polynucleotides, and sequences, provided any given molecule retains the ability to carry out the desired technical effect associated therewith. Provided their biological properties are maintained, the polypeptide and / or polynucleotide sequences according to the present disclosure may deviate by typically as much as 50% and in some instances as much as 30% (calculated using, for instance, BLAST or ClustalW) as compared to the native sequence, although a sequence identity or similarity that is as high as possible is preferable (for instance at least 60%, at least 70%, at least 80%, or at least 90% or higher). Standard methods in the art may be used to determine sequence identity or homology. For example, PILEUP and BLAST algorithms can be used to calculate homology or line up sequences. Any SEQ ID NOs mentioned herein in connection with peptides, polypeptides and proteins shall only be seen as examples and for information only, and all peptides, polypeptides and proteins shall be given their ordinary meaning as the skilled person would understand them. Thus, as above-mentioned, the skilled person will also understand that the present disclosure encompasses not merely the specific SEQ ID NOs and / or accession numbers referred to herein, but also variants and derivatives thereof. All proteins, polypeptides, peptides, nucleotides, and polynucleotides mentioned herein are to be construed according to their conventional meaning as understood by a skilled person, unless otherwise defined. The invention is further defined in the following numbered embodiments: 1. A CRISPR / Cas system guide RNA, optionally a chimeric single guide RNA molecule (sgRNA), comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and capable of effecting the manipulation of a target nucleic acid within a prokaryotic or eukaryotic cell when in complex within the cell with a CRISPR protein, wherein the guide RNA comprises the following modifications: - a “G” at a residue corresponding to position 25 of SEQ ID NO: 1; - an insertion of “AGCUG” between residues corresponding to position 32 and 33 of SEQ ID NO: 1; - an insertion of “CAGCU” between residues corresponding to position 36 and 37 of SEQ ID NO: 1; - a “C” at a residue corresponding to position 46 of SEQ ID NO 1; and a replacement of residues corresponding to positions 73 to 76 of SEQ ID NO: 1 with “GGACUUCGGUCC”. 2. The guide RNA of embodiment 1, further comprising: - “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; - An insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; and - An insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. 3. The guide RNA of the previous embodiments, further comprising: - An insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; and - An insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1. 4. The guide RNA of the previous embodiments, further comprising: - An insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1. guide RNA of embodiment 1, further comprising: - An insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; - An insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1; - “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; - An insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; and - An insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. guide RNA of embodiment 1, further comprising: - “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; - An insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; - An insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1; and - An insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. guide RNA of embodiment 1, further comprising; - An insertion of a “C” between residues corresponding to position 53 and 54 of SEQ ID NO: 1; - An insertion of a “G” between residues corresponding to position 63 and 64 of SEQ ID NO: 1; - “UA” at residues corresponding to positions 65 and 66 of SEQ ID NO: 1; - An insertion of “C” between residues corresponding to position 86 and 87 of SEQ ID NO: 1; - An insertion of “CUUCG” between residues corresponding to position 88 and 89 of SEQ ID NO: 1; and - An insertion of “G” between residues corresponding to position 91 and 92 of SEQ ID NO: 1. The guide RNA of the preceding embodiments, wherein the guide RNA or sgRNA comprises a scaffold that comprises: - A protospacer sequence; - An extended stem loop, optionally wherein the extended stem loop is according to SEQ ID NO 14; - A nexus sequence, optionally wherein the nexus is according to SEQ ID NO 15; - A first hairpin, optionally wherein the first hairpin is a locked hairpin, and optionally wherein the locked hairpin is according to SEQ ID NO 16; - A second hairpin, optionally wherein the second hairpin is according to SEQ ID NO 17; and - A terminator sequence, optionally wherein the terminator sequence is according to SEQ ID NO 18. The guide RNA of any of the preceding embodiments, wherein the guide RNA is of the +85 sgRNA architecture. The guide RNA of any of the preceding embodiments, wherein the guide RNA is capable of forming a complex with a Cas9 protein. The guide RNA of any of the preceding embodiments, wherein the guide RNA has a sequence according to SEQ ID NO: 1 and comprises at least the recited modifications. 12.The guide RNA of any of the preceding embodiments, wherein the guide RNA has a sequence according to SEQ ID NO: 1 and comprises only the recited modifications. 13.The guide RNA of embodiment 8, wherein the locked hairpin further comprises a super stable loop. 14.The guide RNA of embodiment 8 or 13, wherein the locked hairpin has a melting temperature (Tm) of above 65 degrees. 15.The guide RNA of the preceding embodiments, wherein the guide RNA is of a sequence according to any one of SEQ ID NOs: 2, 3, 5, 6 or 7 and comprising one or more insertions, deletions, replacements, or substitutions. 16. The guide RNA of the preceding embodiments, comprising the sequence of SEQ ID NO: 2. 17.The guide RNA of the preceding embodiments, comprising the sequence of SEQ ID NO: 3. 18.The guide RNA of the preceding embodiments, comprising the sequence of SEQ ID NO: 5. 19.The guide RNA of the preceding embodiments, comprising the sequence of SEQ ID NO: 6. 20.The guide RNA of the preceding embodiments, comprising the sequence of SEQ ID NO: 7. 21.At least one nucleic acid molecule encoding a guide RNA of any one of embodiments 1-20. 22.The at least one nucleic acid of embodiment 21, wherein the nucleic acid is one nucleic acid encoding a sgRNA. 23.A library comprising a plurality of guide RNAs of any one of embodiments 1-20 comprising different target-specific spacer sequences and a constant gRNA backbone. 24.A complex comprising a Cas enzyme and a guide RNA of any one of embodiments 1-20. 25.The complex according to embodiment 24 wherein the Cas enzyme is Cas9. 26. In vitro or ex vivo use of a guide RNA of any one of embodiments 1- 20, a library of embodiment 23 or a complex of embodiment 24 or embodiment 25 for CRISPR-mediated oligonucleotide binding and / or editing, particularly for genome editing. 27.An engineered extracellular vesicle (EV) for delivery of a bioactive cargo, the engineered EV comprising: a. the guide RNA of any one of embodiments 1-20, or the complex of embodiment 24 or embodiment 25; and optionally, b. an endosomal escape enhancer. 28.A composition comprising a guide RNA of any one of embodiments 1- 20, a library of embodiment 23 or a complex of embodiment 24 or embodiment 25, or an EV of embodiment 27, and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier. 29.A composition comprising a guide RNA of any one of embodiments 1- 20, a library of embodiment 23 or a complex of embodiment 24 or embodiment 25, or an EV of embodiment 27, for use as a medicament. 30.A composition comprising a guide RNA of any one of embodiments 1- 20, a library of embodiment 23 or a complex of embodiment 24 or embodiment 25, or an EV of embodiment 27, for use in the treatment of disease. 31.A method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the guide RNA of any one of embodiments 1-20, a library of embodiment 23 or a complex of embodiment 24 or embodiment 25, or an EV of embodiment 27. Sequence Listing SEQ ID NO:1 Unmodified gRNA sequence NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO:2 M1L gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAG UCCGUUAUCAACUUGGACUUCGGUCCAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO:3 M1LR gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAG UCCGUUUACAACUUGGACUUCGGUCCAAGUGGCACCCGAGUCGGGUGCUUUUUU SEQ ID NO:4 CR3 gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUCAGAGCUAAGCACAAGAGUGCAUAGCAAGUUGAAAUAAGGCUAG UCCGUUUACAACUUGAAAAAGUGGCACCCGAGUCGGGUGCUUUUUU SEQ ID NO:5 MN1LR gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGCGCUA GUCCGUGUUACAACUUGGACUUCGGUCCAAGUGGCACCcGAGUCGGGUGCUUUUUU SEQ ID NO:6 M2LR gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAG UCCGUUUACAACUUGGACUUCGGUCCAAGUGGCACCcGACUUCGGUCGGGUGCUUUUUU SEQ ID NO:7 MN2LR gRNA scaffold sequence NNNNNNNNNNNNNNNNNNNNGUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGCGCUA GUCCGUGUUACAACUUGGACUUCGGUCCAAGUGGCACCCGACUUCGGUCGGGUGCUUUUUU SEQ ID No 8: VSV-G ATGAAGTGTCTCCTTTATCTCGCCTTTCTCTTTATAGGGGTCAACTGCAAATTTACGATCGTTTTTCC GCACAATCAGAAAGGAAACTGGAAAAATGTGCCATCTAACTACCATTACTGCCCTTCTAGCTCTGACC TTAACTGGCATAATGATTTGATCGGGACAGCGCTCCAAGTCAAAATGCCGAAATCCCACAAGGCGATC CAAGCGGATGGCTGGATGTGTCATGCGTCCAAATGGGTTACAACTTGCGATTTCCGGTGGTACGGGCC CAAATACATCACCCACAGTATTAGAAGCTTTACACCTAGTGTAGAACAGTGCAAAGAGAGCATCGAAC AAACCAAACAGGGCACTTGGCTCAATCCAGGCTTTCCCCCGCAGTCTTGCGGCTACGCCACTGTGACC GATGCGGAGGCCGTGATCGTCCAAGTCACCCCCCATCACGTCCTGGTTGACGAATATACAGGGGAGTG GGTAGATAGTCAATTTATCAACGGTAAATGCAGTAATTATATATGCCCCACCGTGCATAATTCTACCA CATGGCACAGCGATTACAAGGTGAAAGGGTTGTGCGATTCAAACCTCATATCAATGGACATCACCTTC TTTTCAGAAGATGGTGAGCTCAGTTCTCTTGGGAAGGAAGGTACTGGGTTTCGGAGTAATTACTTCGC CTACGAAACGGGTGGCAAGGCCTGCAAGATGCAGTACTGCAAACACTGGGGGGTCCGCCTGCCAAGTG GGGTTTGGTTCGAAATGGCGGACAAGGATTTGTTTGCGGCGGCGCGGTTCCCAGAGTGTCCAGAAGGG TCATCCATATCTGCGCCTTCTCAAACGTCTGTGGACGTGTCTCTTATCCAAGACGTGGAGAGGATTTT GGATTACTCACTTTGCCAAGAAACGTGGAGTAAGATAAGAGCGGGTCTGCCAATAAGTCCGGTGGATC TTAGTTACCTGGCACCAAAAAACCCAGGCACCGGGCCCGCCTTCACTATCATAAACGGGACTCTGAAA TATTTTGAGACGAGATATATCCGGGTTGATATCGCTGCGCCGATACTCTCCAGGATGGTCGGCATGAT TAGTGGTACGACGACTGAGCGAGAACTGTGGGATGACTGGGCTCCGTACGAAGACGTTGAGATCGGTC CAAACGGGGTCTTGCGCACATCTAGTGGTTATAAGTTCCCGCTGTATATGATTGGTCACGGTATGCTT GACTCAGACCTCCATCTCAGTTCCAAGGCTCAAGTGTTTGAGCACCCACACATCCAAGATGCGGCAAG TCAACTTCCTGACGATGAGTCACTGTTCTTTGGGGACACTGGACTCTCCAAGAATCCAATTGAGTTGG TTGAAGGATGGTTTTCATCATGGAAAAGTAGTATAGCCTCTTTTTTCTTCATAATTGGACTTATCATA GGTTTGTTCTTGGTACTCAGAGTCGGTATACACCTGTGTATAAAATTGAAGCACACTAAGAAAAGGCA AATCTATACGGACATAGAAATGAATCGATTGGGGAAG SEQ ID No 9: CVG ATGAATTTTCTCCTTCTGACATTTATCGTACTGCCACTGTGTTCTCATGCTAAGTTCAGTATCGTGTT CCCCCAGAGTCAGAAGGGTAATTGGAAAAATGTGCCCTCCTCATACCACTACTGCCCCAGTTCATCAG ACCAGAACTGGCATAACGACCTGCTTGGGATCACAATGAAGGTTAAAATGCCCAAAACACACAAGGCT ATCCAGGCCGATGGCTGGATGTGCCATGCTGCCAAGTGGATCACCACCTGCGATTTTCGCTGGTACGG GCCAAAATACATCACTCATTCAATTCACTCAATACAGCCCACCTCTGAACAGTGCAAGGAGTCTATTA AGCAAACCAAGCAGGGCACATGGATGAGCCCCGGCTTTCCACCTCAGAATTGCGGGTACGCAACAGTG ACCGACTCTGTGGCAGTCGTTGTGCAAGCGACCCCCCATCATGTCCTGGTGGACGAATACACCGGCGA GTGGATAGATTCTCAGTTCCCAAATGGCAAATGCGAGACTGAAGAGTGTGAGACAGTGCATAACTCAA CAGTGTGGTACAGCGACTATAAGGTAACCGGTTTGTGTGACGCAACCCTGGTGGATACCGAAATCACG TTCTTCTCTGAAGACGGAAAGAAGGAATCCATCGGCAAGCCCAACACAGGTTACCGTTCCAACTACTT CGCATATGAAAAGGGCGATAAAGTATGTAAGATGAACTACTGTAAGCATGCAGGCGTTCGACTTCCTA GTGGCGTGTGGTTCGAATTTGTGGATCAGGATGTTTATGCTGCCGCCAAGTTACCTGAATGCCCCGTG GGCGCCACTATTAGTGCCCCAACACAGACATCCGTGGATGTGTCATTAATCCTTGACGTGGAAAGAAT CCTGGACTACTCACTCTGTCAGGAGACCTGGTCTAAGATCCGGAGCAAGCAGCCTGTAAGTCCCGTAG ACCTCTCTTATCTGGCCCCAAAAAACCCGGGCACAGGGCCTGCCTTCACCATAATCAACGGCACACTG AAGTACTTCGAGACTCGCTATATTAGGATTGACATCGACAACCCAATAATCTCAAAAATGGTGGGGAA GATTAGCGGATCACAGACAGAGAGGGAGCTGTGGACAGAGTGGTTTCCTTACGAAGGCGTAGAAATTG GCCCGAACGGAATCCTCAAGACCCCTACTGGATATAAGTTTCCTCTGTTTATGATCGGACACGGCATG CTTGACAGCGACCTGCATAAGACCTCTCAGGCCGAGGTCTTCGAGCACCCCCACCTGGCGGAGGCTCC CAAGCAGCTGCCAGAGGAAGAGACATTGTTCTTCGGCGACACAGGGATAAGCAAAAACCCTGTCGAAC TGATTGAGGGCTGGTTCAGCTCATGGAAATCAACCGTCGTGACATTCTTCTTCGCAATAGGTGTGTTC ATACTGCTCTATGTCGTCGCTCGGATTGTTATCGCTGTTAGATACCGCTACCAAGGCTCAAACAATAA GAGAATCTACAACGACATCGAGATGAGCAGGTTTAGAAAGGTGAGCGGCTGGCGGCTGTTCAAGAAGA TTAGC SEQ ID No 10: PFV ATGGCCCCTCCCATGACCCTGCAGCAGTGGATCATCTGGAAGAAGATGAACAAGGCCCACGAGGCCCT GCAGAACACCACCACCGTGACCGAGCAGCAGAAAGAGCAGATCATCCTGGACATCCAGAACGAGGAAG TGCAGCCCACCAGGCGGGACAAGTTCAGATACCTGCTGTACACCTGCTGCGCCACCTCCAGCCGGGTG CTGGCCTGGATGTTCCTGGTGTGCATCCTGCTGATCATCGTGCTGGTGTCCTGCTTCGTGACCATCAG CCGGATCCAGTGGAACAAGGACATCCAGGTGCTGGGCCCCGTGATCGACTGGAACGTGACCCAGCGGG CCGTGTACCAGCCCCTGCAGACCCGGCGGATCGCCCGGTCCCTGCGGATGCAGCACCCCGTGCCCAAG TACGTGGAGGTGAACATGACCAGCATCCCCCAGGGCGTGTACTACGAGCCCCACCCCGAGCCCATCGT GGTGAAAGAAAGAGTGCTGGGCCTGAGCCAGATCCTGATGATCAACAGCGAGAACATCGCCAACAACG CCAACCTGACCCAGGAAGTGAAGAAACTGCTGACCGAGATGGTGAACGAAGAGATGCAGAGCCTGAGC GACGTGATGATCGACTTCGAGATCCCCCTGGGCGACCCCAGGGACCAGGAACAGTACATCCACCGGAA GTGCTACCAGGAATTTGCCAACTGCTACCTGGTGAAGTACAAAGAGCCCAAGCCCTGGCCCAAAGAGG GCCTGATCGCCGACCAGTGCCCCCTGCCCGGCTATCACGCCGGCCTGACCTACAACCGGCAGAGCATC TGGGACTACTACATCAAGGTGGAGAGCATCAGGCCCGCCAACTGGACCACCAAGAGCAAGTACGGCCA GGCCCGGCTGGGCAGCTTCTACATCCCCAGCAGCCTGCGGCAGATCAACGTGAGCCACGTGCTGTTCT GCAGCGACCAGCTGTACAGCAAGTGGTACAACATCGAGAACACCATCGAGCAGAACGAGCGGTTCCTG CTGAACAAGCTGAATAACCTGACCAGCGGCACCAGCGTGCTGAAGAAGAGAGCCCTGCCCAAGGACTG GTCCAGCCAGGGCAAGAACGCCCTGTTCCGGGAGATCAATGTGCTGGACATCTGCAGCAAGCCCGAGA GCGTGATCCTGCTGAATACCAGCTACTACAGCTTCAGCCTGTGGGAGGGCGACTGCAACTTCACCAAG GACATGATCAGCCAGCTGGTGCCCGAGTGCGACGGCTTCTACAACAACTCCAAGTGGATGCACATGCA CCCCTACGCCTGCCGGTTCTGGCGGAGCAAGAACGAGAAAGAGGAAACCAAGTGCCGGGACGGCGAGA CCAAGCGGTGCCTGTACTACCCCCTGTGGGACAGCCCTGAGAGCACCTACGACTTCGGCTACCTGGCC TACCAGAAGAACTTCCCCAGCCCCATCTGCATCGAACAGCAGAAGATCCGGGACCAGGACTACGAGGT GTACAGCCTGTACCAGGAATGCAAGATCGCCAGCAAGGCCTACGGCATCGACACCGTGCTGTTCAGCC TGAAGAATTTCCTGAACTACACCGGCACCCCCGTGAACGAGATGCCCAACGCCAGGGCCTTCGTGGGC CTGATTGACCCCAAGTTCCCCCCCAGCTACCCCAACGTGACCCGGGAGCACTACACCAGCTGCAACAA CCGGAAGCGGCGGAGCGTGGACAACAACTACGCCAAGCTGCGGAGCATGGGCTACGCTCTGACAGGCG CCGTGCAGACCCTGTCCCAGATCAGCGACATCAACGACGAGAACCTGCAGCAGGGCATCTACCTGCTG CGGGACCACGTGATCACCCTGATGGAAGCCACCCTGCACGACATCAGCGTGATGGAAGGCATGTTCGC CGTGCAGCACCTGCACACCCACCTGAATCACCTGAAAACCATGCTGCTGGAACGGCGCATCGACTGGA CCTACATGAGCAGCACCTGGCTGCAGCAGCAGCTGCAGAAAAGCGACGACGAGATGAAGGTGATCAAG CGGATCGCCAGATCTCTGGTGTACTACGTGAAGCAGACCCACAGCAGCCCCACCGCCACCGCCTGGGA GATCGGCCTGTACTATGAGCTGGTGATCCCCAAGCACATCTACCTGAACAACTGGAATGTGGTGAACA TCGGCCACCTGGTGAAAAGCGCCGGACAGCTGACCCACGTGACCATCGCCCACCCCTACGAGATCATC AACAAAGAATGCGTGGAGACCATCTATCTGCACCTGGAAGATTGCACCCGGCAGGACTACGTGATCTG CGACGTGGTGAAGATCGTGCAGCCCTGCGGCAACAGCAGCGACACCAGCGACTGCCCCGTGTGGGCCG AGGCCGTGAAAGAACCCTTCGTGCAGGTGAACCCCCTGAAGAACGGCTCCTACCTGGTGCTGGCCAGC AGCACCGACTGCCAGATCCCCCCCTACGTGCCCAGCATCGTGACCGTGAATGAGACCACCTCCTGCTT CGGCCTGGACTTCAAGCGGCCCCTGGTGGCCGAGGAAAGACTGAGCTTCGAGCCCCGGCTGCCCAACC TGCAGCTGAGGCTGCCCCACCTGGTGGGCATCATCGCCAAGATCAAGGGCATCAAGATCGAGGTGACC AGCAGCGGCGAGAGCATCAAAGAACAGATCGAGCGGGCCAAGGCCGAGCTGCTGCGGCTGGATATCCA CGAGGGCGACACACCCGCCTGGATCCAGCAGCTGGCCGCCGCCACCAAGGACGTGTGGCCCGCTGCAG CCAGCGCCCTGCAGGGCATCGGCAACTTTCTGAGCGGCACCGCCCAGGGCATCTTCGGCACCGCCTTC TCCCTGCTGGGCTACCTGAAGCCCATCCTGATCGGCGTGGGCGTGATTCTGCTGGTGATTCTGATCTT CAAGATCGTGAGCTGGATCCCCACCAAGAAAAAGAACCAG SEQ ID No 11: CVG MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKA IQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATV TDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEIT FFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPV GATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTL KYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGM LDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVF ILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRKVSGWRLFKKIS SEQ ID No 12: PFV MAPPMTLQQWIIWKKMNKAHEALQNTTTVTEQQKEQIILDIQNEEVQPTRRDKFRYLLYTCCATSSRV LAWMFLVCILLIIVLVSCFVTISRIQWNKDIQVLGPVIDWNVTQRAVYQPLQTRRIARSLRMQHPVPK YVEVNMTSIPQGVYYEPHPEPIVVKERVLGLSQILMINSENIANNANLTQEVKKLLTEMVNEEMQSLS DVMIDFEIPLGDPRDQEQYIHRKCYQEFANCYLVKYKEPKPWPKEGLIADQCPLPGYHAGLTYNRQSI WDYYIKVESIRPANWTTKSKYGQARLGSFYIPSSLRQINVSHVLFCSDQLYSKWYNIENTIEQNERFL LNKLNNLTSGTSVLKKRALPKDWSSQGKNALFREINVLDICSKPESVILLNTSYYSFSLWEGDCNFTK DMISQLVPECDGFYNNSKWMHMHPYACRFWRSKNEKEETKCRDGETKRCLYYPLWDSPESTYDFGYLA YQKNFPSPICIEQQKIRDQDYEVYSLYQECKIASKAYGIDTVLFSLKNFLNYTGTPVNEMPNARAFVG LIDPKFPPSYPNVTREHYTSCNNRKRRSVDNNYAKLRSMGYALTGAVQTLSQISDINDENLQQGIYLL RDHVITLMEATLHDISVMEGMFAVQHLHTHLNHLKTMLLERRIDWTYMSSTWLQQQLQKSDDEMKVIK RIARSLVYYVKQTHSSPTATAWEIGLYYELVIPKHIYLNNWNVVNIGHLVKSAGQLTHVTIAHPYEII NKECVETIYLHLEDCTRQDYVICDVVKIVQPCGNSSDTSDCPVWAEAVKEPFVQVNPLKNGSYLVLAS STDCQIPPYVPSIVTVNETTSCFGLDFKRPLVAEERLSFEPRLPNLQLRLPHLVGIIAKIKGIKIEVT SSGESIKEQIERAKAELLRLDIHEGDTPAWIQQLAAATKDVWPAAASALQGIGNFLSGTAQGIFGTAF SLLGYLKPILIGVGVILLVILIFKIVSWIPTKKKNQ SEQ ID No 13: CD63 MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVA FVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASIL DRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVL VVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM SEQ ID NO 14: Extended Stem Loop GUUUGAGAGCUAUGCUGGAAACAGCAUAGCAAGUUCAAAU SEQ ID NO 15: Nexus AAGGCUAGUCC SEQ ID NO 16: First Hairpin of M1L including region between nexus and hairpin 1 UUAUCAACUUGGACUUCGGUCCAAGUG SEQ ID NO 17: Second Hairpin of M1L GCACCGAGUCGGUGCU SEQ ID NO 18: Terminator Sequence UUUUUU SEQ ID NO 19: crRNA part of repeat / anti-repeat RNA duplex without 4Us with M1L / M1LR upper stem GUUUVAGAGCUAAGCUG SEQ ID NO 20: tracrRNA part of repeat / anti-repeat RNA duplex of without 4Us with M1L / M1LR upper stem CAGCUUAGCAAGUUBAAAU SEQ ID NO 21: sgRNA repeat / anti-repeat RNA duplex of M1L and M1LR GUUUGAGAGCUAAGCUGGAACAGCUUAGCAAGUUCAAAU SEQ ID NO 22: First hairpin loop with superstable hairpin sequence ACUUGGACUUCGGUCCAAGU SEQ ID NO 23: First Hairpin of M1LR including region between nexus and hairpin 1 UUUACAACUUGGACUUCGGUCCAAGUG SEQ ID NO 24: Second Hairpin of M1LR GCACCCGAGUCGGGUGCU SEQ ID NO 25: First Hairpin loop of M1L and M1LR without region between nexus and hairpin 1 ACUUGGACUUCGGUCCAAGUG SEQ ID NO 26: Nexus of M1L and M1LR AAGGCUAGUCCG SEQ ID NO 27: Constant region of M1L GUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGGACUU CGGUCCAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 28: Constant region of M1LR GUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAGUCCGUUUACAACUUGGACUU CGGUCCAAGUGGCACCCGAGUCGGGUGCUUUUUU SEQ ID NO 29: Exemplary spacer sequence GGACAGUACUCCGCUCGAGU SEQ ID NO 30: sgRNA repeat / anti-repeat double stranded RNA without 4Us with M1L / M1LR upper stem GUUUVAGAGCUAAGCUGGAAACAGCUUAGCAAGUUBAAAU SEQ ID NO 31: cH1 promoter sequence GGCACCAGACCCATTCACACCCCCAACCCCCCAAGCTGAGGCTGCCCGTGGCCCCATCCATGGCCTTG GGGACCTCGACGGATGGGGCACGCGCAACTCCTGCACCCTCCCAACCGCTCCGTATACCGCCGCTccC TCACTCCCAGCACGAGGCGGGGCGGGGCCGCCGTCATTAGCATACACCGCAGAGCATGCCGGGTAACT CGCCCTACTCTAAAAGGGTTCCGGAACCAGAGCTGTATATAGGCACTTCCACAAAGTGCGTCACCg SEQ ID NO 32: hH1 promoter sequence GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGC GCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGT CGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCtTATAAGttCTG TATGAGACCACCACCg SEQ ID NO 33: mH1 promoter sequence CGCTCTTGAAGGACGACGTCATCATCCCTTGCCCGGATGCGCGGGCTTCTTGTCTGGCACAGAGCCTG GGATAGAGCACATGCAAATTACGCGCTGTGCTTTGTGGGAAATCACCCTAAACGTAAAATTTATTCCT CTTTCGAGCCTTATAGTGGCGGCCGGTCTACACCCTCACCg SEQ ID NO 34: cU6 promoter sequence GCGCcGCCGCTCCTTCAGGCACTGCCGACGACAGCCCAGGCGGAGGTCCTGAGCGCCGGCGCTAAATT TGCATAAAGAACTACCCAGGAGCCCTCGCGCGCGGAAACGGGCAAAAAGGGGCTTCTAATATGGAAAT ATTACGCCGAATCGCGTTACAAATCGGCTAAGCGGGCCTAAGAGTTAACAAGATGTGCTATTAAGCGG AGCCTTTTGGTGGGAAGAAATGGAGTAGTCACTGTGTTCTAAAAGAACTTGCAGAATGAGCCTTTAAA TACCGCAGTCTCGATGCTCTTAGTCGCACCg SEQ ID NO 35: mU6 promoter sequence ATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTAC TCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAG ACAGATAATCTGTTCTTTTTAATACTAGCTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGA GATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAAT TGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGCACCg SEQ ID NO 36: hU6 promoter sequence AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGT TAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAA AGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACC GTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAAcaccg SEQ ID NO 37: SCR1T promoter sequence CCCCAGTTGCAAAAGTTGACACAACTCTAGATCTGCTTCCAAATATAGAATCATAACAAGGGTTAGGG TGTGATTATATAATATTGGTCTTAATTGATGTGCTAGGGCTTTAAAAGTTGGTTAAAATAACGCTCTA ATGCCTTTTTAATATATTGTCTTTTTCAAAATCTCAAATCGGACACTTCTTCGTGTATGAGACTCCAT TTTTTGGCTCCGTCACGTGATATGTATTATCAGCTATAGTGGTGTAAACAAAGTTTTTTACTAGCTGT AATGGCATTTTGTCGGAGTGGTAAATCGCCTTCTTGTTGTGCGTTCGAGTTCTGGACTCTGCACTGGG CTACTTTGAAAAATACCTCTAATGCGCCGATGGTTTAGTGGTAAAATCCATCGTTGCCATCGATGGGC CCCCGGTTCGATTCCGGGTCGGCGCAGGTTGACGTCACCg SEQ ID NO 38: cU6.3T promoter sequence GCGCcGCCGCTCCTTCAGGCACTGCCGACGACAGCCCAGGCGGAGGTCCTGAGCGCCGGCGCTAAATT TGCATAAAGAACTACCCAGGAGCCCTCGCGCGCGGAAACGGGCAAAAAGGGGCTTCTAATATGGAAAT ATTACGCCGAATCGCGTTACAAATCGGCTAAGCGGGCCTAAGAGTTAACAAGATGTGCTATTAAGCGG AGCCTTTTGGTGGGAAGAAATGGAGTAGTCACTGTGTTCTAAAAGAACTTGCAGAATGAGCCTTTAAA TACCGCAGTCTCGATGCTCTTAGTCGTGAAAAATACCTCTAATGCGCCGATGGTTTAGTGGTAAAATC CATCGTTGCCATCGATGGGCCCCCGGTTCGATTCCGGGTCGGCGCA SEQ ID NO 39: hU6T promoter sequence AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGT TAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAA AGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACC GTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAATGAAAAATACCT CTAATGCGCCGATGGTTTAGTGGTAAAATCCATCGTTGCCATCGATGGGCCCCCGGTTCGATTCCGGG TCGGCGCA SEQ ID NO 40: reference sequence of constant region GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUUACAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUUUUU SEQ ID NO 41: Unmodified nexus-first hairpin sequence UUAUCA SEQ ID NO 42: region between nexus and the first hairpin in M1LR UUUACA SEQ ID NO 43: constant region of RNAFE GUUUAAGAGCUAAGCUGGAAACAGCUUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 44: constant region of gRNAFE4C GUUUCAGAGCUAAGCUGGAAACAGCUUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 45: constant region of gRNAFE4G GUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 46: constant region of gRNAT-lock GUUUUAGAGCUAAGCUGGAAACAGCUUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUU CGGUCCAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 47: constant region of T-lock T4C GUUUCAGAGCUAAGCUGGAAACAGCUUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGGACUU CGGUCCAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 48: constant region of T-lock T4G GUUUGAGAGCUAAGCUGGAAACAGCUUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGGACUU CGGUCCAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID NO 49: crRNA part of repeat / anti-repeat RNA duplex without 4 Us with upper stem of RNAFE GUUUVAGAGCUAUGCUG SEQ ID NO 50: tracrRNA part of repeat / anti-repeat RNA duplex without 4 Us with upper stem of RNAFE CAGCAUAGCAAGUUBAAAU SEQ ID NO 51: sgRNA repeat / anti-repeat RNA duplex without 4 Us with upper stem of RNAFE GUUUVAGAGCUAUGCUGGAACAGCAUAGCAAGUUBAAAU SEQ ID NO 52 - ΔI-CM intein ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGLRIA GGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALADALDDKF LHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHN SEQ ID NO 53 - I-29 intein ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLRARPVVSWFDQGTRDVIGLRIA GGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQALADALDDK FLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVHN SEQ ID NO 54 – upper stem crRNA sequence of RNAFE GCUAUGCUG SEQ ID NO 55 – upper stem tracrRNA sequence of RNAFE CAGCAUAGC SEQ ID NO 56 – upper stem of RNAFE GCUAUGCUGGAAACAGCAUAGC SEQ ID NO 57 – upper stem crRNA sequence of CR3 GCUAAGCAC SEQ ID NO 58 – upper stem tracrRNA sequence of CR3 GUGCAUAGC SEQ ID NO 59 – upper stem of CR3 GCUAAGCACAAGAGUGCAUAGC SEQ ID NO 60 – upper stem crRNA sequence of M1L / M1LR GCUAAGCUG SEQ ID NO 61 – upper stem tracrRNA sequence of M1L / M1LR CAGCUUAGC SEQ ID NO 62 – upper stem of M1L / M1LR GCUAAGCUGGAACAGCUUAGC SEQ ID NO 63: constant region of CR3 GUUUCAGAGCUAAGCACAAGAGUGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUUACAACUUGAAAAA GUGGCACCCGAGUCGGGUGCUUUUUU SEQ ID NO 64: lower stem crRNA modification to remove 4 Us UUUVA SEQ ID NO 65: lower stem travRNA modification to remove 4 Ts UBAAA SEQ ID NO 66: crRNA part of repeat / anti-repeat RNA duplex without 4 Us with upper stem of CR3 GUUUVAGAGCUAAGCAC SEQ ID NO 67: tracrRNA part of repeat / anti-repeat RNA duplex without 4 Us with upper stem of CR3 GUGCAUAGCAAGUUBAAAU SEQ ID NO 68: sgRNA repeat / anti-repeat RNA duplex without 4 Us with upper stem of CR3 GUUUVAGAGCUAAGCACAAGAGUGCAUAGCAAGUUBAAAU SEQ ID NO 69: crRNA part of repeat / anti-repeat RNA duplex without 4Us with M1L / M1LR upper stem GUUUVAGAGCUAAGCUG SEQ ID NO 70: tracrRNA part of repeat / anti-repeat RNA duplex without 4Us with M1L / M1LR upper stem CAGCUUAGCAAGUUBAAAU SEQ ID NO 71: Unmodified / reference crRNA sequence of upper stem GCUA SEQ ID NO 72: Unmodified / reference tracrRNA of upper stem UAGC SEQ ID NO 73: Unmodified / reference sequence of upper stem of sgRNA GCUAGAAAUAGC SEQ ID NO 74: Unmodified / reference crRNA sequence of lower stem UUUUA SEQ ID NO 75: Unmodified / reference tracrRNA of lower stem UUAAAAU SEQ ID NO 76: Unmodified crRNA bulge sequence GA SEQ ID NO 77: Unmodified tracrRNA bulge sequence AAGU SEQ ID NO 78: Unmodified tracrRNA bulge sequence AAGU SEQ ID NO 79: SKIPPED SEQ ID NO 80: Unmodified / reference first hairpin sequence ACUUGAAAAAGUG SEQ ID NO 81: crRNA lower stem without 4Us GUUUVA SEQ ID NO 82: tracrRNA lower stem without 4Us UBAAAU SEQ ID NO 83: sgRNA intervening nucleotide sequence in M1L / M1LR GAAA SEQ ID NO 84: sgRNA intervening nucleotide sequence in CR3 AAGA SEQ ID NO 85: constant region of T-lock T4A GTTTAAGAGCTAAGCTGGAAACAGCTTAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGGACTT CGGTCCAAGTGGCACCGAGTCGGTGCTTTTTT Examples The materials, methods, and examples described hereinbelow detail embodiments according to the present disclosure and support the understanding thereof. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting in any way. Although alternative methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Example 1 – Materials and Methods Construct building All the designed transgenes were synthesized and purchased from IDT (Integrated DNA Technologies, USA). The gRNA constructs were generated by inserting purchased gRNA transgene fragments into the hU6 promoter vector (backbone), using restriction enzymes XhoI and BamHI for digestion and T4 DNA ligase for ligation. For the VSVG-Foldon-Intein-Cas9 construct, Cas9 was fused to VSVG-Foldon-Intein through digestion of our previous VSVG-Foldon-Intein-Cre plasmid with BamHI and XhoI sites to remove Cre and insert Cas9 by ligation. Cell Culture HEK-293T cells were utilized for the functional EV production in this study and were being kept in complete Dulbecco’s Modified Eagle Media (DMEM) (high glucose), with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Antibiotic- Antimycotic (Anti-anti) (Gibco, USA) added. The reporter cell line, HEK-SL, was maintained in the same medium. All the cells were cultured at 37℃ and at humidified air atmosphere with 5% CO2. Plasmid transfection Before transfection, HEK-293T cells were first seeded into 15-cm dishes. The number of dishes was decided by the amount of EVs to be used in indicated experiments. Transfection was then processed with Polyethylenimine (PEI, Polysiences) under the manufacturer’s protocol. The PEI to plasmid ratio was controlled at 2:1 in this study. For single plasmid transfection and multiple plasmid co-transfection, 30 µg plasmid was needed for single plasmid transfection for each dish; 2-plasmid co-transfection needs 20 µg of each plasmid while 15 µg of each plasmid was used for 3-plasmid transfection. EV production EVs was produced in transiently transfected HEK-293T cells using polyethyleneimine. Specifically, HEK-293T cells were seeded into 15-cm dishes with the density of cells counting to 5 million cells per dish, cultured in the complete DMEM medium at the previously mentioned condition. When the cells were completely seeded after 2 days, they were transfected with the plasmids and the culture media was then changed into Opti-MEM (Gibco, USA) with 1% Anti-anti post 6-hour transfection. Being cultured for another 48 hours, the conditioned medium (CM) was harvested and centrifugated at differential rates. (700 x g for 5 mins, followed by 2,000 x g for 10 mins). The supernatant was subsequently collected and filtered through a 0.22 µm filter system. EV isolation Isolating EVs from filtered CM was achieved by tangential flow filtration (TFF, MicroKross, 20 cm2, Spectrum labs). In the TFF system, particles with a size greater than 300 kDA (the particle size cutoff in the TFF) were retained and concentrated. Finished by TFF, these EV particles were further concentrated in Amicon Ultra-15100 kDA (Millipore) spin filters and were centrifugated at 4,000 x g for at least 30 mins to longer hours at 4°C. In the last step, the concentrated EVs were collected in maxirecovery 1.5 ml Eppendorf tubes (Axygene, USA) and they would be quantified in Nanopaticle Tracking Analysis (NTA) system. Nanoparticle Tracking Analysis (NTA) The EV samples after isolation were diluted with fresh 0.22 µm-filtered PBS. Then the particles sizes and concentrations of all the samples were measured in the NanoSight NS500 instruments with the same setting in the NTA 2.3 software. In the system, the particles were recorded in five videos, each duration of which was longer than 30 seconds with the camera level setting at 15 in light scatter mode. Concentrations of each sample were recorded and calculated for the further use in EV-adding assay. EV-adding assay in reporter cells 96 well-plates were seeded with the reporter cell line HEK-SL at the density of 8×103 cells per well for the first day. In the following day, EVs were directly added to each of 96-well plate with the designed dose gradient: 1E10, 1E9 and 1E8 for each sample and incubate at the same mentioned condition. The plates would be individually checked for the positive fluorescent protein positive cells either in the florescent microscope or MACSQuant flow cytometer, at three incubation time points: 24h, 48h, and 96h after EVs adding. MACSQuant flow cytometry For each time point in the EV-adding assay, MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec, Germany) was used to quantify the GFP expression in the reporter cells. Before sampling in the cytometry, cells in 96- well plates were washed once using PBS and trypsinized for 5 mins at 37°C. Cell culture media with 10% FBS was subsequently added to neutralize trypsin and DAPI was also added for checking cell viability in the sample. The processed samples were available for flow cytometry to detect. The data generated in the cytometry was further analysed in FlowJo software (version 10.6.2). The percentage of GFP positive cells for each sample was calculated and displayed within the software. Virus production Our lenti-viral vectors were used for transgene subcloning (Transfer plasmid, 22.5 µg / T175 flask), co-transfeted with pcoPE01 (Envelope plasmid, 7 µg / T175 flask) and pCD / NL-BH (Helper plasmid, 22.5 µg / T175 flask) into HEK293T cells and incubating overnight. In the following morning, the cell medium was replaced by complete DMEM medium supplemented with sodium butyrate (Sigma-Aldrich). Lasting for 6 to 8 hours, the previously replaced medium was changed back to complete DMEM medium with no additional chemicals. Viruses were harvested in Nalgene® Oak Ridge Centrifuge Tubes (Thermo Scientific) during 22 to 24-hour incubation with the normal medium. Subsequently, medium with virus particles was collected and filtered through a 0.45 µm syringe filter (VWR), centrifugated at 25,000 x g for 90 min at 4°C. The supernatant was aspirated, and the virus pelleted were resuspended in freshly prepared medium, IMEM with 20% FBS. The viruses were either directly added into target cells or stored at -80°C for long-term use. Stable reporter cell generation HEK293T cells were first seeded into 6-well plate and the viruses were added in the next day. The titration of viruses followed three doses: 2 µl, 10 µl, and 50 µl per well. Incubating virused with target cells for one day, the culture media was replaced to normal complete DMEM medium. Two days after virus transduction, the cells were treated with trypsin and resuspended in fresh media.2 µg / ml Puromycin were added for resistance selection of transduced cells whereas the unsuccessfully transduced cells would not survive. The cells continued to be under puromycin selection for approximately one week before being utilized for downstream experiments. Co-culture of EV-producing cells with reporter cells For EV-producing cells, HEK-293T cells were first seeded into 6-well plate, the density of cell each well reaching 0.5 million. Next day, the cells (reaching 60- 70% confluence) were transfected with corresponding construct plasmid through Lipofectamine2000 (Invitrogen, USA), following the manufacturer’s protocol. Six hours after transfection, the culture media was replaced by fresh complete DMEM media, to minimize the effect of toxicity of Lipofectamine 2000 to cells. The plasmid transfection lasting for 24 hours, the producing cells as well as reporter cells were digested with Trypsin and the number of cells were counted. The ratio of producing cell to its corresponding reporter cell were kept at 1:1 or 1:5, seeding into a 96-well plate. After co-culturing for 24 h, cells were trypsinized and under measurement of percentage of GFP positive cells in the MACSQuant flow cytometer. Fluorescent microscopy The GFP positive cells were observed under a fluorescent microscope for EVs adding and co-culture assay. Shooting areas were randomly selected for each picture, with parameters setting up for the groups using one experiment. All images were further processed using the Fiji software under the same parameter setting. Western blot analysis For whole cell samples, the protein in samples was isolated in RIPA buffer, containing a protease inhibitor cocktail, and mixed with sample buffer (4X). The stock was heated at 70℃ for 10 mins. After isolation, samples were then loaded onto a NuPAGE™ 4-12% Bis-Tris Protein Gel (Thermo Scientific), running at 120 V for 2 hours in NuPAGE™ MES SDS running buffer (Thermo Scientific). iBlot™ 2 Transfer Stacks (Thermo Scientific) were used for protein transferring and the membrane from the transferring process were being blocked in InterceptTM blocking buffer (LI-COR Biosciences), shaking for 1 h at room temperature. After fully blocking, the membrane was incubated with the primary antibodies overnight at 4℃. The membrane was washed using TBS-T buffer three times, for 5 min each time, incubating with corresponding secondary antibodies for 1 h at room temperature in a shaker afterwards. Washed with TBS-T buffer three times and with PBS once, the membrane was scanned and observed using the Odyssey infrared imaging system (LI-COR). Statistics Statistical tests for the biological replicates in our study are shown in each figure legend. Statistical analysis was under process by GraphPad software, with data presenting as +SD. The difference between two individual groups were accessed through two-tailed unpaired T-test. One-way ANOVA or Two- way ANOVA test was done for the analysis of multiple groups. Statistical significance was set up as * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. Example 2 – Delivery of Cas9 by engineered EVs The inventors initially identified that only Cas9 protein, not Cas9 / gRNA RNP complex could be successfully delivered by engineered EVs. This in turn resulted in poor to low genome editing in a target gene as the gRNA did not bind to Cas9 inside of the EVs. The inventors found they could improve the genome editing efficiency of target genes by optimizing the gRNA designs to allow improved encapsulation of Cas9 / gRNA into engineered EVs. To address these problems, the inventors have identified a number of modified gRNA, optionally sgRNA, that are able to bind to Cas9 inside of engineered EVs and achieve delivery of the large RNP complex to reporter cells. Upon gRNA transfection, optionally sgRNA, the editing efficiency in reporter cells significantly increased, indicating the gRNA, optionally sgRNA, loading in EVs may be the limitation for high gene editing efficiency in recipient cells. The inventor’s further sought to improve gRNA loading into EVs by improving gRNA expression, the stability of the gRNA and the binding capacity of the gRNA to Cas9. To investigate delivery of Cas9 / sgRNA ribonucleoprotein (RNP) complexes by engineered extracellular vesicles (EVs), the constructs and reporter system of Figure 4A were designed to assay for Cas9 / sgRNA RNP delivery. In this assay, no GFP is expressed because there is one stop codon before GFP, resulting in only mCherry expression in reporter cells without editing. However, after Cas9 / sgRNA delivery to cleave the linker between mCherry and GFP, a frameshift mutation led to the removal of the stop codon and thus GFP is expressed to report successful editing. As can be seen in Figure 4B, when the sgRNA was transfected into the reporter cells and the isolated EVs were added, around 3% gene editing efficiency was achieved. If VSVG-Foldon-Intein-Cas9 with sgRNA was co- transfected and then the isolated EVs were added to the reporter cells, no editing was seen. This means that the Cas9 / sgRNA complex cannot be incorporated into EVs. The reason for this failure is the sgRNA design which has 25 additional nucleotides at the 5’ and this resulted in the failure of incorporation into EVs. Once the 25 additional nucleotides were removed, it was able to bind to Cas9 inside of engineered EVs and successfully delivered to reporter cells as shown in Figure 5. Figures 5A-C shows the percentage of GFP positive cells in the reporter cell system after adding the isolated EVs for 48 to 96 hours determined by FACS. CD63-Intein-Cas9+sgRNA groups served as the negative control (no endosomal escape and thus no editing without VSV-G). The skilled person will however appreciate that any endosomal escape moity can be used to improve the editing efficiency of extracellular vesicles loaded with Cas9 and guide RNA. Figure 5D shows the percentage of GFP positive cells in reporter cells achieved by transient transfection of the respective plasmids by using lipofactamine2000. Figure 6A shows the percentage of GFP positive cells in reporter cells that were achieved by adding different groups of isolated EVs. The EVs from the first 3 groups were not co-transfected with sgRNA, but instead were transfected with sgRNA in reporter cells 6 hours before adding the EVs. This way, only the EVs were used to deliver the Cas9 protein, not the Cas9 / sgRNA complex. This approach still works but the efficiency is not as good as that achieved by EVs after co-transfection of VSVG-Foldon-Intein-Cas9 and sgRNA (incoporate Cas9 / sgRNA complex into isolated EVs). Figure 6B shows the percentage of GFP positive cells in reporter cells achieved by transient transfection of the corresponding plasmids, indicating all the plasmids used are functional. Figure 6C shows the percentage of GFP positive cells in reporter cells achieved by adding the corresponding EVs without sgRNA transfection into the reporter cells. This can be compared with A (with sgRNA transfection), indicating only the Cas9 / sgRNA complex could edit the reporter cells. In order to improve genome editing efficiency, it is possible to modify the scaffold of the gRNA and it would be advantageous to design an alternative sgRNA that is also capable of being incorporated and delivered to a target by an engineered EV. Example 3 – Modification of the gRNA scaffold to improve editing efficiency Various different modifications were made to the original gRNA as summarised in the table below to produce a series of test gRNAs. Table 1: Summary of the different gRNA designs that were tested in Figures 7 and 8, and the modifications they contain. As demonstrated in Figure 7A-D, the percentage of GFP positive cells in reporter cells after adding EVs from different sgRNA modification groups was assessed. The sgRNAFE and sgRNA T-lock design were based on previously published designs as outlined in Table 1 and shown in Figure 7E and 7F respectively, while the Pseudoknot and MALAT1 designs were newly generated. The sgRNAFE and sgRNA T-lock designs enhanced gene editing while Pseudoknot and MALAT1 designs decreased the efficiency. This demonstrates that whilst the 3’ end of the gRNA can be modified by adding additional structures, it does not produce gRNA that have optimal editing efficiency. As described above and shown in Figure 7E, the RNAFE modification includes an extension to the upper stem of the repeat / anti-repeat structure as well as a U-A to A-U base pair flip in the lower stem of the repeat / anti-repeat structure. The inventors went on to investigate the affects of instead replacing this base pair in the lower stem with a C-G or a G-C. The inventors designed constructs that included either an A-U, a C-G or a G- C base pair in the place of the fourth consecutive U-A base pair of the lower stem. In one set of designs, alongside this modification, the inventors also included the extension to the upper stem of the repeat / anti-repeat structure of the known RNAFE modification (as shown in Figure 7G). Figure 7G shows that, when used in combination with the extension to the upper stem of the repeat / anti-repeat structure of the known RNAFE modification, a C-G or G-C base pair can be used, rather than the known A-U flip, and comparable results are achieved. The inventors sought to investigate whether the gRNA scaffold could be further optimised to enhance genome editing efficiency. Table 2: Summary of the different gRNA designs that were compared in Figures 9 and 10. The gRNAs containing the modifications as detailed in Table 2 were assayed using the reporter assay for editing efficiency. As can be seen in Figure 9A, the percentage of GFP positive cells in reporter cells after adding the different EVs was assessed (co-transfection of VSVG- Foldon-Intein-Cas9 with different sgRNA designs). After 48hrs, CR3 looked to be the best gRNA, after 72hrs and 96hrs, respectively, CR3 and M1L appeared to be the best demonstrated by highest percentage GFP positive cells when compared to the other modifications. Figure 9 indicates that, when used in combination with the known T-Lock modification, using either a C-G or G-C base pair, rather than the known A-U flip, results in a slight improvement in editing (compare column 3 or 4 to column 2). In addition, the new M1L design (shown in Figure 1), which combines using a G-C base pair, rather than the known A-U flip, with the known T-Lock modification and an extension to the upper stem of the repeat / anti-repeat structure (this extension also having been further modified as compared to the extension disclosed as part of the known RNAFE, since it also includes a U-A to A-U base pair flip), displays a significant improvement in editing over the known T-Lock modification alone (compare column 7 to column 1) or the known T-Lock modification in combination with the A-U flip of the known RNAFE modification (compare column 7 to column 2). Figure 10 shows the fluorescent images showing the GFP positive cells in reporter cells after adding the different EVs (co-transfection of VSVG-Foldon- Intein-Cas9 with different sgRNA designs) for 4 days. The efficiency of TA5E (adding additional 5 nucleotides at the 5’) decreased significantly. Figure 11 A-C shows the percentage of GFP positive cells in reporter cells after adding EVs from different sgRNA modification groups (Table 2) for 48 h, 72 h and 96 h respectively detected by FACS. The efficiency of TA5E (adding additional 5 nucleotides at the 5’) decreased significantly. sgRNA T-lock was the most efficient when delivering Cas9-RNPs by EVs, reaching 95.17% of GFP positive reporter cells when adding 1E10 EVs and it remained at 83.3% with a lower dose of 1E9 EVs. sgRNA FE did moreover increase the editing efficiency, however, not as much as T-lock. Notably, sgRNA FE was not as efficient as T-lock when the dose was decreased. In summary, through optimization of the sgRNA scaffold, the editing efficiencies could be significantly enhanced in the EV-mediated when utilizing the VICas9 construct for Cas9 loading and simultaneous overexpression of the sgRNA. During testing of the already reported optimized sgRNAs, it is well-noted that optimization of the original sgRNA scaffold could exert a positive impact on the editing efficiency within the EV-mediated system. Thus, it was an object of the investigation to exploit the potential of known optimization features. To achieve this, more modifications were introduced to the original sgRNA. Inspired by the design of sgRNA FE, multiple bases at certain positions in the scaffold were chosen to be flipped or scrambled compared to the original sgRNA T-Lock as it did in sgRNA FE, which led to the designs of sgRNA Tx series (sgRNA T4A, sgRNA T4C, and sgRNA T4G). For sgRNA T7A, a 7-nt random sequence was added to the position between the spacer and the stem- loop. Furthermore, it was desirable to combine all positive features from the published engineered sgRNAs to obtain an improved scaffold. Moreover, a new previously reported sgRNA, Cr3 was simultaneously being evaluated, with most of the modifications distributed in the nexus-hairpin 1 linker and the second hairpin structure). Next, the newly modified sgRNAs was transfected together with the VICas9 system and the engineered EVs were added to recipient reporter cells. At the dose 5E8 EVs, there were similar enhancement in editing efficiencies for all the sgRNA in the Tx series. The efficiencies increased 10% to 15% for all Tx series sgRNAs after reporter cells were incubated with 5E8 EVs for 96 hours. Furthermore, at lower concentrations of EVs, sgRNA T4G was the lead candidate in the Tx series at all time points. When the concentration was further decreased to 1E7, all the optimized sgRNAs together with VICas9 lost all editing efficiency. Furthermore, it was expected that sgRNA TA7 was not functional with the additional random sequence. M1L showed an increased editing efficiency when applied with the EV-mediated system. As the EV concentration was set to 5E8 EVs, a further ~10% increase in its editing efficiency was noticed when compared to sgRNA T4C and sgRNA T4G at all time points measured. However, when the EV concentration decreased, the difference between mentioned sgRNA groups decreased as well. These experiments seek to demonstrate that the gRNA scaffold can be modified to enhance genome editing efficiency, resulting in the best two M1L and CR3. The experiments also demonstrate that the 3’ of gRNA can be modified, such as adding additional structure and whilst it is feasible it isn’t optimal. The 5’ of gRNA cannot be modified, such as adding additional nucleotides or additional secondary structures. Finally, any modifications of the part between spacer and scaffold is forbidden, such as adding additional nucleotides, or may be any structure. Example 4 – Further modification of the gRNA scaffold to improve editing efficiency and generation of M1LR To obtain the structure of a further improved gRNA scaffold, the designs of CR3 and M1L were combined and further enhanced to include a modified linker between the nexus and a locked hairpin, where the linker is modified to include an A-U to U-A modification; and a second hairpin comprising an insertion of C and G base pair in the stem. The structure of M1LR is demonstrated in Figure 1B. The editing efficiency of M1LR was compared to those gRNA modifications as summarised in Table 2. On the first attempt, as demonstrated in Figure 12, the percentage of GFP positive cells in reporter cells after adding Evs from different sgRNA modification groups for 48 h, 72 h and 96 h respectively detected by FACS was analysed. M1LR shows the most improved editing efficiency, even a little bit better than CR3 for different doses of EVs at different time points. After 48 hours post addition of the 5E9 E EVs, the efficiency achieved by M1LR reached 72.7% and it was 5.9 times more efficient compared to unmodified sgRNA (12.23%). It additionally, was almost twice as efficient as M1L, although M1L was unexpectedly performing less well in comparison with M1LR, since it was in previous experiments performing similar to sgRNA Cr3. Compared to reported sgRNA Cr3, M1LR still significantly outperformed Cr3 by 20% at the dose 5E9 EVs. A notable detail was that 4.52% and 4.56% of the HEK-SL cells expressed eGFP at the very low EV concentration of 5E7, which most likely corresponds to low picomolar levels of Cas9-RNPs added per well. On a second attempt, as demonstrated in Figure 13, the percentage of GFP positive cells in reporter cells after adding EVs from different sgRNA modification groups for 48 h, 72 h and 96 h respectively detected by FACS, was analysed. As with the first attempt, M1LR is the best candidate gRNA and clearly better than CR3 for different doses of EVs at different time points. In summary, M1LR outperforms the wild type guide and is the best sgRNA when applied to the EV-mediated VICas9 system in HEK-SL reporter cells to date. It is assumed that it conferred the advantages from both M1L and sgRNA Cr3. Example 5 – Efficiency of editing can be further increased through optimisation of the promoter driving guide expression in producer cells To assess whether gene editing activity could be improved further via modulation of transcription of the guide RNA in EV producer cells, inventors tested whether driving transcription of the M1L guide design using alternative Polymerase III promoters could yield EVs with improved editing potency. The following Polymerase III promoters were tested: - cH1: chicken H1 – SEQ ID NO 31; - hH1: human H1 – SEQ ID NO 32; - mH1: mouse H1 – SEQ ID NO 33; - cU6.3: chicken U6.3 – SEQ ID NO 34; - mU6: mouse U6 – SEQ ID NO 35; - hU6: human U6 – SEQ ID NO 36, - SCR1T: SCR1 promoter chimeric with tRNA – SEQ ID NO 37 - cU6.3T: chicken – SEQ ID NO 38 - hU6T: human U6 promoter chimeric with tRNA – SEQ ID NO 39 VSV-G-Foldon-Intein-Cas9 constructs were co-transfected with the M1L guide RNA driven by the different promoters (Mock, cH1, hH1, mH1, cU6.3, mU6 and hU6) into HEK-293T cells. 6 hours after transfection the medium was changed to Opti-MEM and 2 days later, the conditional medium was harvested. EVs were isolated using TFF, followed by concentration with 10 kD spin filters. Afterwards, the concentrations of isolated EVs were determined by Nanoparticle Tracking Assay (NTA). Different doses of the EVs were added into HEK-SL reporter cells and GFP positive cells were quantified by using FACS after adding EVs for 72 hours. Figure 14A shows that, of the promoters tested, the most effective was the human U6 promoter. Based on these results, testing of further promoters was carried out (SCR1T, cU6.3, tRNA (functions as Polymerase III promoter independently), hU6T and hU6) according to the same method. Figure 14B shows that the SCR1T, cU6.3, tRNA and hU6T promoters perform similarly to the human U6 promoter. Example 6 – M1L and M1LR guide designs show efficient editing of further gene locus To confirm that the new M1L and M1LR guide designs can efficiently edit other targets, HEK293T cells were transiently transfected with: (i) either M1L guide RNA comprising a spacer sequence targeting the DNMT1 gene locus or M1LR guide RNA comprising a spacer sequence targeting the DNMT1 gene locus; and (ii) VSVG-Foldon-Intein-Cas9 or GAG-Intein-Cas9 5.5 hours following transfection, the media in the plates was exchanged with OptiMEM and plates were returned to the incubator for 48hrs, prior to the harvesting of EVs. The resulting EVs were then added to N2A cells.72 hours later, the N2A cells were harvested and DNA was extracted. PCR amplification of the DNMT1 gene was then performed on the harvested DNA samples. TIDE was then used to to measure gene editing efficiency at the target locus (DNMT1). Samples were run in batch analysis mode and the following parameters were used: - Guide Sequence – AACAGCTCTGAACGAGACCC - Alignment window left boundary – 100 - Decomposition window: 239-676 - Indel size: 10. Figure 15A shows that efficient editing of the Dnmt1 locus was achieved with EVs comprising either M1L and VSVG-Foldon-Intein-Cas9 or M1LR and VSVG-Foldon-Intein-Cas9. EV dosage is shown on the X-axis. Figure 15B shows that efficient editing of the Dnmt1 locus was achieved with EVs comprising either M1L and GAG-Intein-Cas9 or M1LR and GAG-Intein- Cas9. This data also demonstrates that editing occurs when GAG is used as the endosomal escape domain, this indicates that any endosomal escape domain may be used. EV dosage is shown on the X-axis. No result is shown for M1L editing at the Dnmt1 locus when 1e10 EVs were added. The inventors additionally went on to test the editing efficiency in N2A cells of EVs that were instead endogenously loaded with M1L comprising a spacer sequence targeting the Ttr gene locus. Figure 15C shows that efficient editing of the Ttr gene locus was achieved. Example 7 – M1L and M1LR guide designs show efficient editing with different when Cas is fused to different EV polypeptides The inventors went on to test the efficiency of gene editing when Cas9 was fused to different EV polypeptides. The following constructs were expressed in EV producer cells, alongside an M1L guide comprising a spacer sequence targeting the Ttr gene locus: - Myr-Intein 2 (I-29)-Cas9 - Single pass EV membrane polypeptide-Intein 2-Cas9 - TSN2-Intein 2 (I-29)-Cas9 - GAG-Intein 2 (I-29)-Cas9 - GAG-Intein 1 (ΔI-CM)-Cas9 EVs were harvested from the producer cells, purified and added to N2A cells. Figure 16A shows that efficient editing of the Ttr locus is achieved with all of the different EV polypeptides and indicates that any EV polypeptide may be used. The inventors also confirmed that Cas9 may be fused to different EV polypeptides (CD63 and TSN2) when M1LR is used as the guide. CD63-Intein- Cas9 or TSN2-Intein-Cas9 was co-transfected with unmodified sgRNA (gRNA) or modified sgRNA (gRNA-M1LR) respectively in HEK-293T cells. 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. The concentrations of isolated EVs were determined by Nanoparticle Tracking Assay (NTA). And then the indicated doses of EVs were incubated with HEK-SL reporter cells in 96-well plates for 72 hours and GFP positive cells were detected by FACS. Figure 16B shows that efficient editing is achieved with both EV polypeptides and indicates that any EV polypeptide may be used with any modified guide RNA. Example 8 – EVs provide more efficient editing than other methods of non-viral delivery The inventors tested whether the EVs comprising the M1LR guide could be used in to produce efficient editing in vivo. Previous published studies have shown up to 6.1% editing with non-viral delivery of editors to the CNS. VSV-G-Foldon-Intein-Cas9 and VSV-G-Foldon-Intein-Cas9 plus CD63-Intein- Cas9 constructs were co-transfected with an M1LR guide RNA targeting the TtR locus into EV producer cells. The medium was replaced by Opti-MEM 6 hours after transfection. 2 days later, the Opti-MEM was collected and EVs were isolated by exploring TFF, concentrated by 10 kD spin filters. Nanoparticle Tracking Assay (NTA) was used to evaluate the concentrations of isolated EVs. For a constant ICV delivery of the EV preparations, Alzet® osmotic pumps (2001D) were used. These pumps were primed with 200 µl of the EV preparation at a concentration of 2x1012 EVs / ml. The pumps were then prepared and implanted as described by Sanchez-Mendoza and colleagues (Sanchez-Mendoza E.H., Carballo J., Longart M., Hermann D.M., Doeppner T.R. Implantation of miniosmotic pumps and delivery of tract tracers to study brain reorganization in pathophysiological conditions. J. Vis. Exp. 2016;107:e52932). Briefly, 15 weeks old C57BL / 6 mice were anesthetized with isoflurane and mounted on a stereotactic frame. A heating pad was used to maintain a constant body temperature of 37 °C. Next, a small incision in the skin was made from the base of the neck to up in between the eyes. The injection needle of the pump system was placed in the ventricles based on coordinates measured relative to the bregma intersection (anteroposterior 0.07 cm, mediolateral 0.1 cm, dorsoventral 0.2 cm). The cannula was fixed to the skull with Loctite 454. A subcutaneous pocket for the osmotic pump was made by using a blunted scissor to slide underneath the skin at the base of the neck. Next the pumps were inserted under the skin at the base of the neck and pushed to the back as far as was possible without resistance. The osmotic pump was connected to the cannula via a vinyl catheter (0.71 mm outer diameter). An immediate constant delivery of the EV preparations started upon implantation of the pumps at a flow rate of 8 µl / hour. After surgery the incision was sutured and 48hours after the implantation, the pumps were removed, and bone wax was used to close the skull again. After 9 days from implantation, the mice were sacrificed and different regions (including the hippocampus and cortex) of brains were harvested to Eppendorf tubes to be stored at -80 °C. Next, the tissues were homogenized by tissue lyser in lysis buffer. Genomic DNA was isolated to serve as the templates for PCR amplification of the target regions. The PCR products were purified and sent for Sanger sequencing. And at last, the gene editing efficiency was analysed by using Synthego ICE software (v3.0). Figure 17a shows that up to 40% editing of the Ttr locus is achieved in the brain of adult mice. Editing was observed in both contralateral or ipsilateral sides and in both the hippocampus and the cortex, indicating good distribution. Thus, EV mediated delivery of the M1LR guide and Cas9 results in a significant improvement in CNS editing compared to previously published data. The inventors also tested how the delivering Cas9 and the M1L guide with EVs compares to the gold standard for CRISPR / Cas9 delivery in vitro (CRISPRMax). Figure 17b shows that, on two different occasions, significantly more editing was observed when EVs comprising Cas9 and the M1L guide were added to cells, as compared with when Cas9 and the M1L guide were delivered to cells using the CRISPRMax transfection reagent. Thus, EV mediated delivery of the M1L guide and Cas9 results in a significant improvement in editing compared with delivery of the M1L guide and Cas9 with CRISPRMax. Example 9 – Different fusogens allow for efficient editing with EV- mediated delivery The inventors confirmed that efficient editing can be achieved using EV- mediated deliver of CRISPR / Cas9 complexes when different fusogens are used. TSPAN-Intein-Cas9 and constructs were co-transfected with an M1LR guide RNA into EV producer cells alongside different viral fusogenic proteins (VSV- G-Foldon, CNV-G, CNV-G-Foldon and RV-G).2 days after transfection, EVs were collected and purified using TFF and concentrated using 10kD spin filters. The concentrations of isolated EVs were measured by Nanoparticle Tracking Assay (NTA) Different doses of the EVs were added into HEK-SL reporter cells for 72 hours and then the GFP positive cells were measured by FACS. Figure 18 shows that efficient editing is achieved with all of the fusogens and indicates that any fusogenic protein or endosomal escape enhancer may be used for EV-mediated delivery.

Claims

Claims 1. A CRISPR / Cas system guide RNA, wherein the guide RNA comprises a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA); wherein the crRNA comprises from 5’ to 3’ a target-specific spacer sequence and a repeat region; wherein the tracrRNA comprises from 5’ to 3’ an anti-repeat region, a nexus sequence, a first hairpin loop and a second hairpin loop; wherein the repeat region of the crRNA and the anti-repeat region of the tracrRNA form a double stranded repeat / anti-repeat RNA duplex structure through complementary base pairing; wherein the double stranded repeat / anti-repeat RNA duplex structure comprises a lower stem, a bulge and an upper stem; wherein the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises fewer than four consecutive uracil nucleotides; wherein the upper stem of the double stranded repeat / anti-repeat RNA duplex has a length of at least 5 contiguous complementary base pairs; wherein the first hairpin loop comprises a stem having a length of at least 5 contiguous complementary base pairs and / or wherein at least 2 of the contiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs.

2. The CRISPR / Cas system guide RNA of claim 1, wherein (i) the target-specific spacer sequence is capable of hybridizing to a target sequence in a genomic locus of interest in a cell; further wherein the guide RNA is capable of effecting the manipulation of a target nucleic acid within a prokaryotic oreukaryotic cell when in complex within the cell with a CRISPR protein; and / or (ii) the guide RNA has increased stability and / or exhibits more stable binding to a CRISPR protein, as compared with a guide RNA having SEQ ID NO.

1.

3. The CRISPR / Cas system guide RNA of claim 1 or claim 2, wherein the guide RNA comprises in 5’ to 3’ direction: the target-specific spacer sequence, the double stranded repeat / anti-repeat RNA duplex structure, the nexus sequence, the first hairpin loop and the second hairpin loop.

4. The CRISPR / Cas system guide RNA of any one of claims 1 to 3, wherein the lower stem of the double stranded repeat / anti-repeat RNA duplex is proximal to the spacer and the nexus; wherein the upper stem of the double stranded repeat / anti-repeat RNA duplex is distal from the spacer and the nexus; wherein the bulge of the double stranded repeat / anti-repeat RNA duplex is positioned between the lower stem and the upper stem; wherein the lower stem is formed of 5 contiguous complementary base pairs; wherein the bulge is formed by nucleotides that are not complementary base paired.

5. The CRISPR / Cas system guide RNA of any one of claims 1 to 4, wherein upper stem is formed of 9 contiguous complementary base pairs, preferably wherein 5 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs.

6. The CRISPR / Cas system guide RNA of any one of claims 1 to 5, wherein the repeat region of the crRNA that forms the upper stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a repeat sequence having at least 80%, atleast 90% or having 100% sequence identity to the nucleotide sequence SEQ ID NO 60; further wherein the anti-repeat region of the tracrRNA that forms the upper stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a repeat sequence having at least 80%, at least 90% or having 100% sequence identity to the nucleotide sequence SEQ ID NO 61.

7. The CRISPR / Cas system guide RNA of any one of claims 1 to 6, wherein the repeat region of the crRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises the nucleotide sequence UUUVA (SEQ ID NO 64); further wherein the anti-repeat region of the tracrRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises the nucleotide sequence UBAAA (SEQ ID NO 65); further wherein (i) the “V” of the UUUVA (SEQ ID NO 64) is A and the “B” of the UBAAA is U, (ii) the “V” of the UUUVA (SEQ ID NO 64) is C and the “B” of the UBAAA is G, or (iii) preferably the “V” of the UUUVA (SEQ ID NO 64) is G and the “B” of the UBAAA is C.

8. The CRISPR / Cas system guide RNA of any one of claims 1 to 7, wherein the repeat region of the crRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 81;further wherein the anti-repeat region of the tracrRNA that forms the lower stem of the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or having 100% sequence identity to SEQ ID NO 82, wherein: (i) the “V” of the SEQ ID NO 81 is A and the “B” of the SEQ ID NO 82 is U (ii) the “V” of the SEQ ID NO 81 is C and the “B” of the SEQ ID NO 82 is G; or (iii) preferably the “V” of the SEQ ID NO 81 is G and the “B” of the SEQ ID NO 82 is C.

9. The CRISPR / Cas system guide RNA of any one of claims 1 to 8, wherein the double stranded repeat / anti-repeat RNA duplex structure comprises a repeat sequence having SEQ ID NO 19; further wherein the double stranded repeat / anti-repeat RNA duplex structure comprises an anti-repeat sequence having SEQ ID NO 20; further wherein (i) the “V” of the SEQ ID NO 19 is A and the “B” of the SEQ ID NO 20 is U, (ii) the “V” of the SEQ ID NO 19 is C and the “B” of the SEQ ID NO 20 is G, or (iii) preferably, the “V” of the SEQ ID NO 19 is G and the “B” of the SEQ ID NO 20 is C.

10. The CRISPR / Cas system guide RNA of any one of claims 1 to 9, wherein the guide RNA is a single guide RNA (sgRNA) molecule comprising a stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA.

11. The CRISPR / Cas system guide RNA of claim 10, wherein the stretch of intervening nucleotides between the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA comprises or consists of the nucleotide sequence GAAA (SEQ ID NO 83) or AAGA (SEQ ID NO 84) 12. The CRISPR / Cas system guide RNA of claim 10 or claim 11, wherein the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 30; further wherein (i) the “V” of the SEQ ID NO 30 is A and the “B” is U, (ii) the “V” of the SEQ ID NO 30 is C and the “B” is G, or (iii) preferably, the “V” of the SEQ ID NO 30 is G and the “B” is C.

13. The CRISPR / Cas system guide RNA of claim 10 or claim 11, wherein the double stranded repeat / anti-repeat RNA duplex structure comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 21.

14. The CRISPR / Cas system guide RNA of any one of claims 1 to 13, wherein the first hairpin loop is a locked hairpin.

15. The CRISPR / Cas system guide RNA of claim 14, wherein the locked hairpin comprises a super stable loop.

16. The CRISPR / Cas system guide RNA of claim 14 or 15, wherein the locked hairpin has a melting temperature (Tm) of above 65 degrees.

17. The CRISPR / Cas system guide RNA of any one of claims 1 to 16, wherein the first hairpin loop comprises a stem having a length of contiguous 8 complementary base pairs, preferably wherein 4 of thecontiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs.

18. The CRISPR / Cas system guide RNA of any one of claims 1 to 17, wherein the first hairpin loop comprises a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.22 or SEQ ID NO 25.

19. The CRISPR / Cas system guide RNA of any one of claims 1 to 18, wherein the nexus sequence comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 15 or SEQ ID NO 26.

20. The CRISPR / Cas system guide RNA of any one of claims 1 to 19, wherein the guide RNA further comprises a terminator sequence at its 3’ end, preferably wherein the terminator sequence comprises or consists of SEQ ID NO 18.

21. The CRISPR / Cas system guide RNA of claim 20, wherein the guide RNA (i) does not comprise any additional modifications between the second hairpin loop and the terminator sequence; or (ii) comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides, or preferably does not comprise any nucleotides, between the second hairpin loop and the terminator sequence.

22. The CRISPR / Cas system guide RNA of any one of claims 1 to 21, wherein the guide RNA (i) comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides or preferably does notcomprise any nucleotides to the 5’ of the spacer sequence; and / or (Ii) comprises fewer than 7 nucleotides, fewer than 6 nucleotides, fewer than 5 nucleotides, fewer than 4 nucleotides, fewer than 3 nucleotides, fewer than 2 nucleotides or preferably does not comprise any nucleotides between the spacer sequence and the repeat region of the crRNA.

23. The CRISPR / Cas system guide RNA of any one of claims 1 to 22, wherein the target-specific spacer sequence is between 18 and 52 nucleotides in length, preferably wherein the target-specific spacer sequence is between 17 and 30 nucleotides in length.

24. The CRISPR / Cas system guide RNA of any one of claims 1 to 23, wherein the guide RNA further comprises a region between the nexus and the first hairpin loop comprising or consisting of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.

41.

25. The CRISPR / Cas system guide RNA of claims 1 to 24, wherein the second hairpin loop comprises a stem having 6 contiguous complementary base pairs, preferably wherein 5 of the contiguous complementary base pairs of the stem of the second hairpin loop are C-G or G-C base pairs.

26. The CRISPR / Cas system guide RNA of claims 1 to 25, wherein the second hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.

17.

27. The CRISPR / Cas system guide RNA of any one of claims 1 to 26 comprising from 5’ to 3’ the spacer sequence, SEQ ID NO.21, SEQ ID NO. 26, SEQ ID NO.16; SEQ ID NO.17 and SEQ ID NO.18.

28. The CRISPR / Cas system guide RNA of any one of claims 1 to 27, wherein the guide RNA comprises from 5’ to 3’, a spacer sequence and a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 27; further wherein the guide RNA comprises (i) a G at a residue corresponding to position 5 of SEQ ID NO: 27; (ii) AAGCUG at residues corresponding to position 12 to 17 of SEQ ID NO: 27; (iii) CAGCUU at residues corresponding to position 22 and 27 of SEQ ID NO: 27; (iv) a C at a residue corresponding to position 36 of SEQ ID NO 27; and (v) GGACUUCGGUCC at residues corresponding to position 63 to 74 of SEQ ID NO 27.

29. The CRISPR / Cas system guide RNA of any one of claims 1 to 23, wherein the guide RNA further comprises a region between the nexus and the first hairpin loop comprising or consisting of a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO.

42.

30. The CRISPR / Cas system guide RNA of any one of claims 1 to 23 or claim 29, wherein the second hairpin loop comprises a stem having 7 contiguous complementary base pairs, preferably wherein 6 of the contiguous complementary base pairs of the stem of the second hairpin loop are C-G or G-C base pairs.

31. The CRISPR / Cas system guide RNA of claims 1 to 23 or claim 29 or 30, wherein the second hairpin loop comprises or consists of a nucleotide sequence having at least 90%, at least 92%, at least 94%,at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 24.

32. The CRISPR / Cas system guide RNA of any one of claims 1 to 23 or claims 29 to 31 comprising from 5’ to 3’ the spacer sequence, SEQ ID NO. 21, SEQ ID NO.26, SEQ ID NO.23; SEQ ID NO.24 and SEQ ID NO.

18.

33. The CRISPR / Cas system guide RNA of any one of claims 1 to 32 wherein the guide RNA comprises from 5’ to 3’, a spacer sequence and a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 28; further wherein the guide RNA comprises: (i) a G at a residue corresponding to position 5 of SEQ ID NO: 28; (ii) AAGCUG at residues corresponding to position 12 to 17 of SEQ ID NO: 28; (iii) CAGCUU at residues corresponding to position 22 and 27 of SEQ ID NO: 28; (iv) a C at a residue corresponding to position 36 of SEQ ID NO 28; (v) GGACUUCGGUCC at residues corresponding to position 63 to 74 of SEQ ID NO 28; (vi) UA at residues corresponding to positions 55 and 56 of SEQ ID NO: 28; (vii) a C at a residue corresponding to position 85 of SEQ ID NO: 28; and (viii) a G at a residue corresponding to position 91 of SEQ ID NO: 28.

34. The guide RNA of any of the preceding claims, wherein the guide RNA is of the +85 sgRNA architecture 35. The guide RNA of the preceding claims comprising or consisting of SEQ ID NO: 2 or SEQ ID NO:

3.

36. At least one nucleic acid molecule encoding a guide RNA of any one of claims 1 to 35.

37. A library comprising a plurality of guide RNAs of any one of claims 1- 36 comprising different target-specific spacer sequences and a constant gRNA backbone.

38. A complex comprising a Cas enzyme and a guide RNA of any one of claims 1-37, preferably wherein the Cas enzyme is Cas9.

39. A method of preparing an EV comprising introducing into an EV- producing cell (i) a guide RNA of the claims 1 to 35; or (ii) a construct encoding a guide RNA of the claims 1 to 35 and expressing the construct in the EV-producing cell; thereby generating EVs comprising a guide RNA of the present invention.

40. The method of claim 39, wherein the construct encoding the guide RNA is expressed under the control of the human U6 promoter.

41. The method of claim 39 or claim 40, wherein the method further comprises introducing into the EV-producing cell (i) Cas9 or (ii) a construct encoding a Cas9 and expressing the construct in the EV-producing cell, thereby generating EVs comprising a guide RNA of claims 1 to 35 and a Cas9, preferably wherein the Cas9 is fused to an EV polypeptide.

42. An EV directly obtained by any one of the methods of claim 39 to 41.

43. An engineered extracellular vesicle (EV) for delivery of a bioactive cargo, the engineered EV comprising: (i). the guide RNA of any one of claims 1-35, or the complex of claim 36; and optionally, (ii) an endosomal escape enhancer. 44 A composition comprising a guide RNA of any one of claims 1-35, a nucleic acid molecule of claim 36, a library of claim 37 or a complex of claim 38, or an EV of claim 42 or 43, and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier.

45. A composition comprising a guide RNA of any one of claims 1-35, a nucleic acid molecule of claim 36, a library of claim 37 or a complex of claim 38, or an EV of claim 42 or 43, for use as a medicament.

46. A composition comprising a guide RNA of any one of claims 1 to 35, a nucleic acid molecule of claim 36, a library of claim 37 or a complex of claim 38, or an EV of claim 42 or 43 for the preparation of a medicament for treatment or prevention of a disease in a subject.

47. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a guide RNA of any one of a guide RNA of any one of claims 1 to 35, a nucleic acid molecule of claim 36, a library of claim 37 or a complex of claim 38, or an EV of claim 42 or 43 to a subject suffering from or susceptible to the disease.

48. The use or method of claims 45 to 47, wherein the subject is a mammal, preferably a human.

49. A method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the guide RNA of any one of claims 1 to 35, a nucleic acid molecule of claim 36, a library of claim 37 or a complex of claim 38, or an EV of claim 42 or 43.

50. The method of claim 49, wherein the method is in vivo, in vitro or ex vivo.

51. A complex comprising a fusion protein and a guide RNA, wherein the fusion protein comprises an EV polypeptide and a CRISPR protein, wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification.

52. An EV comprising a guide RNA, wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification.

53. An EV comprising CRSIPR / Cas complex, wherein the CRISPR / Cas complex comprises a guide RNA bound to a CRSIPR protein, further wherein the guide RNA comprises a first structural component, wherein the first structural component is a functional module for CRISPR protein binding, further wherein the first structural component comprises a modification.

54. An EV comprising the complex of claim 51.

55. The complex or EV of claims 51 to 54 wherein the CRISPR protein is Cas12a or preferably Cas9.

56. The complex or EV of claims 51 to 55, wherein the modification changes the nucleotide sequence of the structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / or ii. a guide RNA having SEQ ID NO 1.

57. The complex or EV of claims 51 to 56, wherein the first structural component is a stem-loop or an RNA duplex.

58. The complex or EV of claims 51 to 57, wherein the modification is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component wherein the extension modification is an increase in the number of complementary base pairs present in the structural component.

59. The complex or EV of claims 51 to 58, wherein the guide RNA comprises, preferably from 5’ to 3’, a target-specific spacer sequence, a repeat / anti repeat RNA duplex, a nexus, a first hairpin and a second hairpin; wherein the repeat / anti repeat RNA duplex comprises an upper stem, a bulge and a lower stem.

60. The complex or EV of claims 51 to 59, wherein the first structural component comprising a modification is a modified upper stem, a modified first hairpin or a modified second hairpin.

61. The complex or EV of claims 51 to 60, wherein the guide RNA further comprises a second structural component, wherein the second structural component is a functional module for CRISPR protein binding, wherein the second structural component comprises a modification.

62. The complex or EV of claim 61, wherein the modification in the second structural component changes the nucleotide sequence of the second structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / orii. a guide RNA having SEQ ID NO 1.

63. The complex or EV of claim 61 or 62, wherein the second structural component is a stem-loop or an RNA duplex; preferably wherein i. the first structural component is an RNA duplex and the second structural component is a stem loop or ii. the first structural component is a stem loop and the second structural component is a stem loop.

64. The complex or EV of claims 61 to 63, wherein the modification in the second structural component is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component 65. The complex or EV of claims 61 to 64, wherein i. the first structural component comprising a modification is a modified upper stem and the second structural component comprising a modification is a modified first hairpin; ii. the first structural component comprising a modification is a modified upper stem and the second structural component comprising a modification is a modified second hairpin; or iii. the first structural component comprising a modification is a modified first hairpin and the second structural component comprising a modification is a modified second hairpin.

66. The complex or EV of claims 61 to 65 wherein the guide RNA further comprises a third structural component, wherein the third structuralcomponent is a functional module for CRISPR protein binding, wherein the structural component comprises a modification.

67. The complex or EV of claim 66, wherein the modification in the third structural component changes the nucleotide sequence of the third structural component, as compared with the same or a corresponding structural component present in: i. any naturally occurring guide RNA; and / or ii. a guide RNA having SEQ ID NO 1.

68. The complex or EV of claim 66 or 67, wherein the third structural component is a stem loop or an RNA duplex, preferably wherein i. the first structural component is an RNA duplex, the second structural component is a stem loop and the third structural component is a stem loop; or ii. the first structural component is a stem loop, the second structural component is a stem loop and the third structural component is a stem loop; 69. The complex or EV of claims 66 to 68, wherein the modification in the third structural component is i. an extension modification, ii an increase in the proportion of G-C or C-G base pairs present in the structural component; or iii preferably an extension modification and an increase in the proportion of G-C or C-G base pairs present in the structural component.

70. The complex or EV of claims 59 to 69 wherein the first structural component comprising a modification is a modified upper stem, the second structural component comprising a modification is a modifiedfirst hairpin and the third structural component comprising a modification is a modified second hairpin.

71. The complex or EV of claims 59 to 70 wherein the modified upper stem comprises at least 5 contiguous complementary base pairs, preferably wherein the modified upper stem comprises 9 contiguous complementary base pairs wherein 5 of the contiguous complementary base pairs of the upper stem are C-G or G-C base pairs.

72. The complex or EV of claims 59 to 71 wherein the modified first hairpin comprises a stem having a length of at least 5 contiguous complementary base pairs, preferably wherein the first hairpin comprises a stem having a length of 8 complementary base pairs wherein 4 of the contiguous complementary base pairs of the stem of the first hairpin loop are C-G or G-C base pairs.

73. The complex or EV of claims 59 to 72 wherein the modified second hairpin comprises a stem having 7 contiguous complementary base pairs, preferably wherein 6 of the contiguous complementary base pairs are C-G or G-C base pairs.

74. The complex or EV of claims 59 to 73, wherein the guide RNA comprises a modification in the lower stem; wherein the modified lower stem comprises fewer than four consecutive uracil nucleotides.

75. The complex or EV of claims 59 to 74 wherein i. the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides, or preferably does not comprise any nucleotides, between the second hairpin loop and the terminator sequence; ii. the guide RNA comprises fewer than 25 nucleotides, fewer than 20 nucleotides, fewer than 15 nucleotides, fewer than 10 nucleotides, fewer than 5 nucleotides or preferably does notcomprise any nucleotides to the 5’ of the target-specific spacer sequence; and / or iii. the modified guide RNA comprises fewer than 7 nucleotides, fewer than 6 nucleotides, fewer than 5 nucleotides, fewer than 4 nucleotides, fewer than 3 nucleotides, fewer than 2 nucleotides or preferably does not comprise any nucleotides between the spacer sequence and the repeat / anti-repeat region.

76. The complex or EV of claims 59 to 75, wherein the guide RNA exhibits more stable binding to the CRISPR protein as compared with (i) a guide RNA having SEQ ID NO.1 and / or (ii) a guide RNA comprising a constant region having SEQ ID NO.

40.

77. The complex or EV of claims 59 to 75 wherein the guide RNA comprises or consists, preferably from 3’ to 5’ of a target-specific spacer sequence and a constant region, wherein the cinstant region comprises a sequence having at least 92%, at least 94%, at least 96%, at least 98% or having 100% sequence identity to SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 63, SEQ ID NO 27 or SEQ ID NO 28.

78. The EV of claim 52 to 77, wherein the EV is an exosome or a micovesicle.

79. The EV of claim 52 to 79, wherein the EV is derived from a HEK cell, preferably a HEK293 cell.

80. The EV of claim 52 to 79, wherein the EV comprises a fusogenic protein or an endosomal escape enhancer.

81. A population of EVs, comprising a plurality of EVs of claims claim 52 to 80.

82. A composition comprising a complex, an EV or a population of EVs of claims 51 to 81 and one or more of a pharmaceutically acceptable excipient, diluent, vehicle, solvent or carrier.

83. A complex, an EV, a population of EVs or a composition of any one of claims 51 to 82 for use in treatment or prevention of a disease in a subject.

81. A complex, an EV, a population of EVs or a composition of any one of claims 51 to 82 for the preparation of a medicament for treatment or prevention of a disease in a subject.

82. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a complex, an EV, a population of EVs or a composition of any one of claims 51 to 82 to a subject.

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