CRISPR Activation (SAM-Mediated) AAV Vector

Overview

CRISPR/Cas9 vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN).

Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome.

The Synergistic Activation Mediator (SAM) system is a powerful tool for transcriptional activation of genes within their endogenous genomic loci. This system is derived from CRISPR/Cas9 genome-editing systems, but rather than mediating genome editing, a modified type of gRNA directs the assembly of a multi-component transcriptional activation complex (SAM complex) at targeted sites. In general, assembly of the SAM complex is sufficient to induce very strong transcriptional activation of the target site.

The complete AAV-based SAM system consists of three components, SagRNA/MS2, MS2/P65/HSF1 and dSaCas9/VP64 which are expressed using two separate AAV vectors. The target site-specific SagRNA sequence selected by the user is cloned into the AAV msSagRNA expression vector. In this vector the gRNA is modified to include two 138-nt hairpin RNA aptamers which form binding sites for the bacteriophage MS2 coat proteins. These hairpin RNA aptamers linked to the SagRNA facilitate the efficient recruitment of MS2-fusion proteins. Additionally, the AAV msSagRNA vector drives the expression of a three-domain fusion protein consisting of MS2, p65 (the trans-activation subunit of NF-kB), and HSF1 (the activation domain of human heat shock factor 1). The AAV dSaCas9/VP64 helper vector on the other hand drives the expression of a fusion protein consisting of a catalytically inactive variant of SaCas9 and the synthetic VP64 transactivation domain.

When cells are co-transduced with these two AAV vectors, the user-selected SagRNA can potentially recruit both MS2/P65/HSF1 (via MS2-binding hairpin aptamers attached to the gRNA) and dSaCas9/VP64 (via CRISPR/Cas9 complex assembly) to SagRNA target sites, thereby assembling powerful SAM complexes. These SAM complexes can achieve robust transcriptional activation of the target sites through synergistic interactions among the VP64, p65 and HSF1 activation domains.

Our AAV msSagRNA vector is designed to work with SaCas9 derived from Staphylococcus aureus, which is >1 kb shorter in comparison to the conventional SpCas9 derived from Streptococcus pyogenes. SaCas9 provides a distinct advantage over SpCas9, which has limited use in AAV vector-based applications due its larger size and the restrictive cargo capacity of AAV vectors. SaCas9 functionally differs from SpCas9 in two major aspects – first, SaCas9 requires a different gRNA scaffold sequence from the one required by SpCas9. Secondly, the PAM sequence for SaCas9 is NNGRRT, whereas the PAM for SpCas9 is NGG. 

This vector system is designed primarily for use in large-scale screens of genomic loci, using libraries of gRNA sequences to generate gRNA/MS2 expression vector libraries. However, this system can also be used to activate transcription of individual genes, or small sets of genes.

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. Due to their low immunogenicity in host organisms, our AAV msSagRNA expression vectors are the perfect tools for in vivo CRISPR-based applications.

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.

Serotype Tissue tropism
AAV1 Smooth muscle, CNS, brain, lung, retina, inner earpancreas, heart, liver
AAV2 Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes
AAV3 Smooth muscle, liver, lung
AAV4 CNS, retina, lung, kidney, heart
AAV5 Smooth muscle, CNS, brain, lung, retina, heart
AAV6 Smooth muscle, heart, lung, pancreas, adipose, liver
AAV6.2 Lung, liver, inner ear
AAV7 Smooth muscle, retina, CNS, brain, liver
AAV8 Smooth muscle, CNS, brain, retina, inner earliver, pancreas, heart, kidney, adipose
AAV9 Smooth muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose
AAVrh10 Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
AAV-DJ Liver, heart, kidney, spleen
AAV-DJ/8 Liver, brain, spleen, kidney
AAV-PHP.eB CNS
AAV-PHP.S PNS
AAV2-retro Spinal nerves 
AAV2-QuadYF Endothelial cell
AAV2.7m8 Retina, inner ear

Tissue type Recommended AAV serotypes
Smooth muscle AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
CNS AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
PNS AAV-PHP.S
Brain AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
Retina AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8
Inner ear AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
Lung AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
Liver AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Pancreas AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
Heart AAV1,AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
Kidney AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Adipose AAV6, AAV8, AAV9
Testes AAV2, AAV9
Spleen AAV-DJ, AAV-DJ/8
Spinal nerves AAV2-retro
Endothelial cells AAV2-QuadYF

For further information about this vector system, please refer to the papers below.

References Topic
Cell. 154:442 (2013) Characterization of CRISPRa and CRISPRi systems
Nature. 517:583 (2015) Description of the SAM system
Genome Biol. 16:257 (2015) Characterization of Staphylococcus aureus Cas9
Nature. 520:186 (2015) In vivo genome editing with SaCas9-based AAV vectors

Highlights

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.

Advantages

Endogenous genomic context: The SAM system can activate transcription of target sites within their endogenous genomic loci. This is unlike transgenic or genome-editing methods which involve alterations to the genomic context of the gene of interest.

Orthogonal to physiological regulation: Targeted transcriptional activation of a gene using the SAM vector system does not require prior knowledge of how the gene of interest is naturally regulated. However, accurate DNA sequence information of the target site is necessary.

Strong activation: Transcriptional activation of genes using the SAM system can often achieve very high-level gene expression.

Disadvantages

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 be transduced by any serotype.

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 technically demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector. 

PAM requirement: Our AAV msSagRNA vector is designed to work with SaCas9 derived from Staphylococcus aureus. SaCas9-mediated CRISPR targeting is dependent on the presence of the PAM sequence, NNGRR (NNGRRT preferred) on the immediate 3’ end of the gRNA recognition sequence.

Specificity: The SAM based approach for targeted activation of genes is relatively new, and detailed information regarding the specificity of targeting using SagRNA/MS2 RNAs is currently not available.

Key components

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 downstream SagRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

SagRNA: Specifies the target sequence for SaCas9 nuclease. Scaffold gRNA sequence is included.

MS2 scaffold: This hairpin aptamer sequence binds robustly to fusion proteins containing the MS2 bacteriophage coat proteins.

Terminator: Terminates transcription of the SagRNA.

EF1A promoter:  Human eukaryotic translation elongation factor 1 α1 promoter. It drives the ubiquitous expression of the downstream MS2/P65/HSF1 regulatory protein.

Regulatory protein: Allows users to add MS2/P65/HSF1 which is a fusion protein of MS2 bacteriophage coat protein, NF-kappaB trans-activating subunit p65 and human heat-shock factor 1 activation domain.

BGH pA: Bovine growth hormone 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.

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