Table 1 Loading methods for EV-delivered CRISPR/Cas components

Pre-loading methods

Genetic engineering

Liver AML12 cells

20%–30%

pDNA

Simple, quick, common application

The off-target effect, limited load efficiency, potential carcinogenic risk

[17]

HEK293T cells

/

pDNA

[177]

HEK293T cells

22.3 ± 8.5 copies of Cas9 mRNA/100 EVs

Cas9 mRNA

[29]

HEK293T cells

/

RNPs

High efficiency loading, high gene editing activity; versatile; intact structure

Difficulties in obtaining large quantities of EVs-RNPs

[63]

HEK293T cells

50%–71%

[65]

Retroviral virus

/

[61]

HEK293T cells

 ~ 23%

[40]

HEK293T cells

1.5–2%

[35]

HEK293T cells, CHME-5 cells

 < 1%

[36]

Human iPSCs, HEK293T cells

7.9 RNPs/EV

Cas9 protein, sgRNA

High efficiency loading, high gene editing activity; versatile; quantitative measurements of transfer activity

Tedious preparation, time consuming, lack of stability and specific targeting

[23]

HEK293T cells

/

[48]

HeLa cells, HuH7 cells, Vero cells, CHO cells

10%

[37]

U2OS cells, HEK293T cells

540 RNPs/EV

[47]

HEK293T, HepAD38, HeLa, Huh7 cells

100 ng Cas9 protein/10 µg EVs

[39]

Multiple cells

0.7%

[50]

Expi293F cells

25 Cas9 molecules/EV

[46]

All types of screened malignancy

/

[141]

Co-incubation

Dendritic cells

/

pDNA

Intact structure, simple, and quick

Unknown long-term safety, low gene editing efficiency

[9]

Post-loading methods

Electroporation

HEK293T cells

/

pDNA

High efficiency loading, operation reproducibility, avoids the risk of gene transfer

EV damage, EV or RNA aggregation, not suitable for clinical application

[19]

HEK293T cells, ovarian cancer cells

 ~ 2%

pDNA

[38]

Hepatic stellate cells

20%

RNPs

[41]

RBCs

18%

Cas9 mRNA

[30]

Freeze–thaw, sonication, co-incubation

HEK293T cells

40%

pDNA

High efficiency loading, simple operation

EV integrity disruption and aggregation

[18]

HEK293T cells

37.62%

RNPs

[22]

Transfection kit

HEK293T cells

10 μg DNA/10⁹ EVs

pDNA

Straightforward, convenient, and cost-effective

Lower efficiency, potential toxicity

[21]

Serum

/

RNPs

[42]

Cell nanoporation

All cell types

/

Cas9 mRNA

High efficiency loading

Needing special equipment

[7]

Co-incubation

MDA-MB-231 breast cancer cells

7.7 ng Cas9 protein/3.5 × 10⁸ EVs

Cas9 protein or sgRNA

Intact structure, simple, quick, and versatile operation

Low efficiency

[49]