TABLE 2.
Studies using cell‐based EV loading strategies.
| Study | EV loading method | Donor cells | Cargo | EV validation | Target cells | Target genes/sequences | CRISPR Results | In vivo cell targeting mechanism |
|---|---|---|---|---|---|---|---|---|
| Passive packaging strategies | ||||||||
| (Luo et al., 2022) | Donor cells transfected with lentiviral particles containing plasmids encoding dCas‐VP64 (catalytically inactive Cas9 fused to a transcriptional activator), sgRNA, Lamp2b‐RBP4 and MS2‐P65. Cargo packaged into EVs by overexpression. | Hepatocyte AML12 cells | RNP (dCas9) |
NTA TEM Markers: CD63+, Lamp2B+ TSG101+ GM130‐ |
Hepatic stellate cells (HSC) |
HNF4α HGF1 FOXA2 |
In vitro gene activation: Conversion of HSCs to hepatocyte‐like cells (HLCs) |
Lamp2b‐RBP4 was incorporated into the EV membrane targeting HSCs In vivo: Intravenous injection (tail) |
| HSCs of CCL4‐induced hepatic fibrosis mouse model |
HNF4α HGF1 FOXA2 |
In vivo gene activation: ‐Conversion of HSC to HLCs ‐Decreased fibrosis ‐Reduced protein levels of profibr‐ ogenic mediators and collagen ‐Recovered liver counts |
||||||
| (Chen et al., 2019) | Donor cells transfected with CRISPR/Cas9 expression vectors targeting HPV (Human Papilloma Virus) or HBV (Hepatitis B Virus). Cargo packaged into EVs by overexpression. | HUH‐7 cells | RNP |
DLS TEM Markers: CD63+ CD81+ |
HUH‐7 cells transfected with HBV expression plasmid | HBV DNA (2 target sites) |
In vitro gene editing: Deletion in selected clones |
– |
| HeLa cells | RNP | HeLa cells transfected with HPV expression plasmid | HPV DNA (2 target sites) |
In vitro gene editing: Deletion in selected clones |
||||
| (He et al., 2020) |
Donor cells transfected with pLenti‐CRISPR‐V2 and pCMV‐VSV‐G. Cas9 packaged into EVs by overexpression. Loading of sgRNA expression plasmids by electroporation of isolated EVs. |
HEK293 cells Liver cancer HepG2 cells |
Cas9 protein + sgRNA plasmid |
DLS TEM Markers: CD40+ |
HepG2 cells | IQGAP1 |
In vitro gene editing: HepG2 EVs: 19.5% indels and 10.57% apoptosis HEK293 EVs: 15.9% indels and 12.07% apoptosis |
– |
| HepG2 xenograft mice | Iqgap1 |
In vivo gene editing: HepG2 EVs: Up to 25 % reduced IQGAP1 protein level HEK293 EVs: Up to 50% reduced IQGAP1 protein level |
Intratumoral injection, Tropism |
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| (Lainšček et al., 2018) |
GEDEX: Donor cells transfected with plasmids encoding Cas9 or dCas9‐VPR (catalytically inactive Cas9 fused to the transcriptional activator VPR) and sgRNA. Cargo packaged into EVs by overexpression (100 ng Cas9 protein each 10 ug EVs). |
HEK293T cells | RNP (Cas9) |
NTA DLS TEM Markers: CD63+ TSG101+ Calnexin‐ |
HEK293 cells | MYD88 |
In vitro gene editing (Cas9): 9.2‐10.1% indels |
– |
| RNP (dCas9) |
HEK293 cells Mouse Neuro2A cells |
ACTC1 Actc1 |
In vitro gene activation (dCas9): HEK293 (ACTC1): 7‐fold increase in mRNA levels Neuro2A (Actc1): 5‐fold increase in mRNA levels. |
– | ||||
| BALB/c mouse model with alpha‐naphthylisothiocyanate (ANIT) induced hepatotoxicity. | Hgf |
In vivo gene activation (dCas9): ‐ 1.5‐fold increased HGF protein levels in liver ‐ Reduced protein levels of biochemical liver damage markers ‐ Reduced liver damage morphology |
Hydrodynamical intravenous injection (tail) | |||||
| (Montagna et al., 2018) |
VEsiCas: Donor cells transfected with plasmids encoding Cas9, pVAX‐T7‐sgRNA (for cytosolic transcription of sgRNA) and VSV‐G. Cargo packaged into EVs by overexpression. |
BSR‐T7/5 cells | RNP | – | HEK293T cells |
CXCR4 VEGFA site 3 |
In vitro gene editing: CXCR4: > 60% indels VEGFA site3: > 30% indels |
– |
| HEK293‐EGFP cells | EGFP (2 target sites) |
In vitro gene editing: 17% EGFP deletion |
– | |||||
| Cardiac muscle cells of EGFP transgenic mice. | EGFP (2 target sites) |
In vivo gene editing: 30% EGFP negative cardiomyocytes compared to 0 % in control mice. |
Intracardiac injection | |||||
| Active packaging strategies | ||||||||
| (Yao et al., 2021) |
ABP Com‐com: Donor cells transfected with plasmids encoding CD63 fused to Aptamer‐Binding Protein (ABP) Com (CD63‐Com), aptamer com‐sgRNA with Cas9 and VSV‐G. The Com‐com interaction increased loading of sgRNA:Cas9 into EVs by 2‐5‐fold. |
HEK293T cells | RNP |
NTA TEM Markers: CD9+ CD63+ RAB5B+ |
HEK293T GFP‐reporter cells | DMD exon 53 |
In vitro genome editing: 51.4% indels |
– |
| Tibialis anterior muscle cells of Del52hDMD/mdx mice | DMD exon 53 |
In vivo genome editing: 0.2 % indels |
Intramuscular injection | |||||
|
(Osteikoetxea et al., 2022) JEV |
CIBN‐CRY2: Donor cells transfected with plasmids encoding CIBN‐MysPalm and Cas9‐CRY2 with sgRNA. Blue light (488 nm) and FAD molecule‐induced CIBN‐CRY2 dimerization increased loading of sgRNA:Cas9 into EVs by up to 10‐fold. |
Expi293F cells | RNP |
NTA TEM Markers: Alix+ TSG101+ Flotilin+ Syntenin+ CD63+ CD81+ Calnexin‐ |
HEK293‐loxP‐GFP‐STOP‐LoxP‐RFP Cre reporter cells. | LoxP (excises GFP) |
In vitro gene editing: 42.0% RFP+ cells |
‐ |
| HepG2‐loxP‐tdTomato‐STOP‐loxP‐GFP Cre reporter cells. | LoxP (excises tdTomato) |
In vitro gene editing: 19.2% GFP+ cells |
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| HEK293T cells | PCSK9 |
In vitro gene editing: 4.4% indels |
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| (Ye et al., 2020) |
GFP‐GFPnanobody: Donor cells transfected with plasmids encoding CD63‐GFP, Cas9‐GFP‐nanobody and sgRNA. GFP and GFP‐nanobody binding increased loading of sgRNA into EVs 2‐fold. |
HEK293T cells | RNP |
NTA TEM Markers: CD63+ |
Lung adeno‐carcinoma cell line A549 with stop‐DsRed genomic sequence | 5′‐ and 3′‐ends of stop sequence (2 targets sites) |
In vitro genome editing: ‐ Deleted stop sequence ‐ Increased DsRed fluorescent cells |
– |
| (Gee et al., 2020) |
FKBP12‐FRB (NanoMEDIC): Donor cells transfected with plasmids encoding FRB‐Cas9, FKBP12‐GagHIV, and sgRNA flanked by self‐cleaving ribozymes and packaging signal that binds Gag. Addition of rapamycin analog facilitates dimerization of FKBP12 and FRB. The viral GAG peptide accumulates in EVs. Addition of rapamycin analog increased Cas9 loading by 2‐fold. |
HEK293T cells | RNP |
NTA TEM Markers: CD63+ CD81+ |
DMD patient‐derived Δex44 iPSC‐derived skeletal muscle cells | DMD exon 45 (both acceptor and donor splice sites) |
In vitro gene editing: 92% exon 45 skipping |
– |
| Gastrocnemius muscle cells in GAG‐Luc2‐hDMD Ex45(stop) KI mice | DMD exon 45 (both acceptor and donor splice sites) |
In vivo gene editing: 6.9% exon 45 skipping |
Intramuscular injection | |||||
| Anterior tibialis muscle cells in NOG‐mdx mouse with nonsense mutation in Ex23 of hDMD gene | DMD exon 23 (both acceptor and donor splice sites) |
In vivo gene editing: 1.1% gene editing leading to 1.6% exon 23 skipping |
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| (Ilahibaks et al., 2023) |
FKBP12‐FRB (TOP‐EV): Donor cells transfected with plasmids encoding sgRNA, VSV‐G, FRB‐Cas9, FKBP12 with N‐myristoylation sequence. Rapamycin induces FKBP12‐FRB dimerization. VSV‐G and rapamycin together increased Cas9 protein packaging 9.5‐fold whereas VSV‐G itself increased packaging 7.8‐fold. |
HEK293FT cells | RNP |
NTA TEM Markers: Alix+ Syntenin+ Calnexin‐ |
Cas9 stoplight reporter cells | Linker region in stoplight cassette (1 and 2 nt indels result in eGFP expression) |
In vitro gene editing: Rapamycin only: 0–1% eGFP signal VSV‐G only: 11.6% eGFP signal VSV‐G + rapamycin: 29.4% eGFP signal |
– |
| (Campbell et al., 2019) |
DmrC‐DmrA (Gesicles): Donor cells transfected with plasmids encoding Cas9‐DmrC, Cherrypicker Red‐DmrA, A/C dimerization molecule, VSV‐G and sgRNA. DmrC‐DmrA dimerization increased loading of Cas9 into the gesicles by 2‐fold. |
HEK293‐FT cells | RNP |
NTA TEM |
HIV‐NanoLuc CHME‐5 microglial cells containing HIV provirus with NanoLuciferase Reporter | HIV LTR (long terminal repeat) 5′ region |
In vitro gene editing: 8% indels |
– |
| Wang, Yu et al. (2018) |
ARMMs: Donor cells transfected with plasmids encoding 4WW‐Cas9‐anti‐GFP‐sgRNA and ARRDC1. The WW domains coupled to Cas9 interacts with the PPXY motifs of ARRDC1 which increased sgRNA loading significantly, compared to Cas9 alone. |
HEK293T | RNP |
NTA TEM Markers: Flotillin + Vinculin ‐ |
GFP positive U2OS cells (1 gene copy) | GFP |
In vitro gene editing: The GFP negative cells increased from 4.9% to 13.4%. |
– |
| Li, Zhou et al. (2019) |
ARE‐HuR: Donor cells transfected with plasmids encoding dCas9‐ARE, CD9‐HuR (Human antigen R) and sgRNA. The addition of the ARE motif increased dCas9 mRNA loading by up to 10‐fold. |
HEK293T |
sgRNA and dCas9 mRNA |
DLS TEM Markers: CD9+ CD63+ TSG101+ Lamp2B+ GM130‐ |
Adipogenic stem cells | CEBPA |
In vitro gene repression (dCas9): More than 2‐fold decreased mRNA levels |
– |
| Liver cells of C56BL/6 mice |
In vivo gene repression (dCas9): Up to 2‐fold decrease in mRNA levels |
Intravenous injection (tail) | ||||||
| Li, Zhang et al. (2023) |
pTA‐CasRx: Donor cells transfected with a plasmid encoding pTA‐Cas13‐HA and sgRNA. The pTA extracellular secretion signal increased Cas13 loading into EVs by 8‐fold. |
HEK293T | RNP (Cas13) |
NTA TEM IEM Markers: CD63 |
LPS (lipopolysaccharide)‐activated bone marrow‐derived macrophages from mice |
Il‐6 Tnf Il‐1β |
In vitro mRNA inactivation (Cas13): Reduced NOS2 (marker for macrophage activation) mRNA levels by 4‐fold |
– |
| Lung tissue of LPS‐induced acute lung injury mice |
In vivo mRNA inactivation (Cas13): Reduced IL‐6, TNF‐α and IL‐1β mRNA levels by 2‐fold |
Four intratracheal injections | ||||||
Density light scatter (DLS), Nanoparticle tracking analysis (NTA), Transmission electron microscopy (TEM), Immunogold electron microscopy (IEM).