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Gene editing, also called genome editing, is a group of technologies to change the sequence of DNA in the genome. Several approaches to genome editing have been developed, including Zinc Finger, TALEN, and CRISPR/Cas9. Compared with the other gene editing tools, CRISPR/Cas9 system is faster, cheaper, more accurate, and more efficient, showing unprecedented advantages in gene therapy.

CRISPR/Cas9 related products
Adeno Associated Virus (AAV) CRISPR/Cas9 Servicepdf download
Adenovirus CRISPR/Cas9 Service
Lentivirus CRISPR/Cas9 Service

CRISPR/Cas9 system can be delivered in vitro by the non-viral reagents (such as polymer, liposome, nano-particle, electroporation) or viral vectors (such as lentivirus, adenovirus and AAV) and in vivo by adenovirus or AAV vectors.

1)In vitro non-viral vectors CRISPR-Cas9 delivery system

In vitro non-viral vectors CRISPR/Cas9 delivery system can be mediated by non-viral reagents, such as polymer [51], liposome [52], nano-particle [53], electroporation [54]. The characteristics of these methods are summarized in the following table 2. For more detailed information about the transfection reagents, you can refer to the website: www.genemedi.net/i/transfection-in-vitro-in-vivo.

Table 2 - Comparison between polymer, liposome, nano-particle.

Comparison

polymer

liposome

nano-particle

electroporation

Principle

endocytosis

endocytosis

membrane fusion/impale cells

 transient increase in the permeability of cell membrane

Integration

No

No

No

No

Time to peak expression

48h-72h

48h-72h

24h-48h

48h-72h

Sutainable time

transient expression

transient expression

transient expression

transient expression

Cell Type

a number of cell types

 adherent and suspension cell lines

Almost all the cells

not suitable for sensitive cell types

Particle diametre

75 to 520 nm

50-200nm

100-300nm

——

Animal experiment

low effiency

low effiency

target delivery

target delivery

Cytotoxicity

high

low

 non-toxic

——

Immune Response

No

No

No

No

Efficiency

low effiency (<10%)

depend on cell type

high efficiency

high efficiency

Price

 inexpensive

medium

expensive

most expensive (electroporation device)

2) In intro viral vectors CRISPR-Cas9 delivery system


a. lentiviral

With the ability to mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells, lentiviral is a good vector to deliver CRISPR system [55,56]. More useful information of lentivirus can be found on this website: www.genemedi.net/i/lentivirus-packaging.

b. adenoviral

Based on human adenovirus type 5 (Ad5), recombinant adenovirus (Ad) a replication-defective adenoviral vector system, is widely used for CRISPR-Cas9 system in most cell types [57-59]. Genemedi got a rich experience in adenovirus packaging, you could find more information on www.genemedi.net/i/adenovirus-packaging.

3) In vivo CRISPR-Cas9 delivery system-AAV-SaCas9


With mild immunogenicity, adeno-associated viruses (AAV) have superior biosafety rating and broad range of infectivity and mediate long-term and stable expression of CRISPR system in vivo. Considering the loading capacity of AAV (4.7kb), SaCas9 variant has been identified, referred to as KKH SaCas9, displaying robust genome editing activities at target sites with NNNRRT PAMs and showing comparable off-target effects between wild-type and KKH SaCas9 [60]. Combining CRISPR/SaCas9 system and AAV vector, AAV-SaCas9 has been widely applied for gene editing in vivo [61-63]. Genemedi is good at AAV production, please find more information on this website: www.genemedi.net/i/aav-packaging.

Table 3 - Comparison between Retrovirus, Lentivirus, Adenovirus and AAV vectors.

Comparison

Retrovirus

Lentivirus

Adenovirus

AAV

Genome

ss RNA

ss RNA

ds DNA

ss DNA

Integration

Yes

Yes

No

No

Packaging Capacity

3kb

4kb

5.5kb

2kb

Time to peak expression

72h

72h

36h-72h

cell: 7 days; animals: 2 weeks

Sutainable time

about 3 weeks

stable expression

transient expression

> 6 months

Cell Type

Most Dividing

Most Dividing/Non-Dividing Cells

Most Dividing/Non-Dividing Cells

Most Dividing/Non-Dividing Cells

Titeration

10^7 TU/ml

10^8 TU/ml

10^11 PFU/ml

10^12  v.g./ml

Animal experiment

suitable

low efficiency

lowest efficiency

most suitable

Immune Response

high

medium

medium

ultra-low



View Crispr Knowledge Base>>

Other knowledge bases

AAV Knowledge Basepdf download
CRISPR/Cas9-gRNA system Knowledge Base
Adenovirus Knowledge Basepdf download
Transfection Knowledge Base
Lentivirus Knowledge Basepdf download
Advanced cell therapy