Gene editing also known as genome editing is a set of techniques that are used to alter the DNA sequence of an organism. There are three main techniques of genome editing namely ZFN, TALENs, and CRISPR/Cas9. Among all the other gene editing tools, CRISPR/Cas9 system is faster, cheaper, more accurate and efficient and thus holds unprecedented potential for gene therapy.

CRISPR/Cas9 system can be administered either in vitro with the help of non-viral carriers (polymer, liposome, nano-particle, electroporation) or viral vectors (lentivirus, adenovirus, AAV) or in vivo with the help of adenovirus or AAV vectors.

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

In vitro non viral vectors of CRISPR/Cas9 delivery may include non viral carriers such as polymer [51], liposome [52], nano particle [53], electroporation [54]. Some of the features of the methods described are presented 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

Due to its capacity to transduce quiescent cells and maintain the expression of the introduced genes, lentiviral has potential for use in delivering the CRISPR system [55,56]. More useful information of lentivirus can be found on this website: www.genemedi.net/i/lentivirus-packaging.

b. adenoviral

Human adenovirus type 5 (Ad5) based recombinant adenovirus (Ad) is a replication defective adenoviral vector which is most commonly used for CRISPR-Cas9 system in almost all 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

Due to low immunogenicity AAV have high biosafety and wide tropism and provide long-term and stable expression of CRISPR system in vivo. Due to the cargo capacity of AAV being 4. 7kb, a new SaCas9 variant has been described, known as KKH SaCas9, which is capable of generating efficient genome editing at target sites having NNNRRT PAMs and has been found to have similar off-target effects as wild-type and KKH SaCas9[60]. Taking advantage of CRISPR/SaCas9 system and AAV vector, AAV-SaCas9 has been used for gene editing in vivo in many studies [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

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Other knowledge bases

AAV Knowledge Base CRISPR/Cas9-gRNA system Knowledge Base

Adenovirus Knowledge Base Transfection Knowledge Base

Lentivirus Knowledge Base Advanced cell therapy