gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Adeno-associated virus shRNA Knockdown Vector
Our Adeno-associated virus (AAV) shRNA Knockdown vector system is an efficient method for stably knocking down expression of a target gene in a wide variety of mammalian cell types, in vitro or in vivo. Due to the low immunogenicity and cytotoxicity of AAV, this is the ideal shRNA vector for many animal studies.
An AAV vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. After the viral genome is delivered into the target cells, it enters the nucleus and remains as episomal DNA. The shRNA is stably expressed from a human U6 promoter, leading to degradation of target gene mRNA within infected cells.
A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.
Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The table below lists different AAV serotypes and their tissue tropism.
|AAV1||SM, CNS, lung, retina, pancreas, heart, liver|
|AAV2||SM, CNS, liver, kidney, retina|
|AAV4||CNS, retina, lung, kidney|
|AAV5||SM, CNS, lung, retina|
|AAV6||SM, heart, lung, adipose, liver|
|AAV7||SM, retina, CNS, liver|
|AAV8||SM, CNS, retina, liver, pancreas, heart, kidney, adipose|
|AAV9||SM, lung, liver, heart, pancreas, CNS, retina, testes, kidney|
|AAVrh10||SM, lung, liver, heart, pancreas, CNS, retina, kidney|
For further information about this vector system, please refer to the papers below.
|Methods in Enzy. 507:229-54 (2012)||Review of AAV virology and uses|
|Curr Opin Pharmacol. 24:59-67 (2015)||AAV use in gene therapy, and serotype differences|
Our AAV vector system is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.
Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient, and is not known to cause any human diseases.
Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.
Stable knockdown: AAV generally remains in a stable episomal state within the nucleus, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene is typically stable and long-lasting.
High viral titer: Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1011 genome copy per ml (GC/ml).
Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).
Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.
Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.
Stable knockdown: AAV generally remains in a stable episomal state within the nucleus, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the target gene cannot easily be reactivated once it is knocked down by the AAV shRNA Knockdown vector. This can be an advantage or a disadvantage, depending on experimental goals.
Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.
Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.
Terminator: Terminates transcription of the shRNA.
hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.