AAV2 CRISPR/Cas9 system

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The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines

The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines. The indicated cell types (1.0 x 10⁵ cells) were seeded in 12-well plates one day before transduction. Cells were transduced with 1.0 x 10⁵ MOI (genomic titer) each of AAV2-Up and AAV2-Down viral particles targeting the CCR5 gene. After 72 hours, cells were harvested and analyzed using the Guide-it Mutation Detection Kit. As a control, cells were transfected with a plasmid (P: 2.5 µg) encoding Cas9 and a guide sequence targeting CCR5, using Xfect Transfection Reagent.

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Production of full-length Cas9 and sgRNA

Production of full-length Cas9 and sgRNA. The pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors encode truncated upstream and downstream portions of the Cas9 gene respectively, with a 1.6-kb overlapping region of homology. A user-defined single guide RNA (sgRNA) can be cloned into pAAV-Guide-it-Down (see Vector Maps). Separate AAV2-Up and AAV2-Down particles are generated by transfecting HEK 293T cells with either pAAV-Guide-it-Up or pAAV-Guide-it-Down along with pRC2-mi342 and pHelper vectors. The resulting viral particles are isolated using AAV Extraction Solution. Target cells are cotransduced with both AAV2-Up and AAV2-Down particles. The 1.6-kb homologous overlapping region results in recombination during AAV genome processing giving rise to a full-length Cas9 gene that is transcribed and translated into protein.

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AAV vector recombination to produce full-length Cas9 and sgRNA

AAV vector recombination to produce full-length Cas9 and sgRNA. The large size of the Cas9 gene precludes its packaging into a single AAV particle. pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors contain truncated upstream and downstream portions of Cas9, respectively, with a shared 1.6-kb overlapping region of homology. Separate viruses are generated with each plasmid. Transduction of target cells with both viruses results in recombination to generate the full-length Cas9 (4.1 kb) gene. Subsequent expression in target cells results in functional Cas9 protein guided to the appropriate genomic site by the sgRNA.
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Consistent recovery of equally high genomic titers of AAV2-Up and AAV2-Down viral particles

Consistent recovery of equally high genomic titers of AAV2-Up and AAV2-Down viral particles. AAV2-Up and AAV2-Down viral particles were extracted from HEK 293T cells using the AAVpro Extraction Solution. Genomic titer was measured using the AAVpro Titration Kit (for Real Time PCR). Similar genomic titers were obtained for AAV2-Up and AAV2-Down viral particles in three independent experiments.

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Vector map of pAAV-Guide-it-Up

Vector map of pAAV-Guide-it-Up. The structure of the pAAV-Guide-it-Up vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free System, a kit that produces AAV particles for cell delivery of Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV particle. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids; the pAAV-Guide-it-Up vector contains a truncated portion of the gene (3,106 bp) that encodes the N-terminal 1,035 amino acids of Cas9 under the control of the CMV promoter.

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Vector map of pAAV-Guide-it-Down

Vector map of pAAV-Guide-it-Down. The structure of the pre-linearized pAAV-Guide-iT-Down vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free system, a kit that produces AAV particles for cell delivery of the Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV virus. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids. The pAAV-Guide-it-Down vector contains a truncated downstream portion of the gene (2,616 bp) that encodes the C-terminal 872 amino acids of Cas9. A user-defined single guide RNA (sgRNA) can be cloned into the pAAV-Guide-it-Down vector downstream of the human U6 promoter. To construct the vector, a pair of oligos corresponding to the genomic target (guide sequence) are annealed to form a duplex. The duplexed DNA is then cloned into the prelinearized vector.

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632609: AAVpro CRISPR/Cas9 Vector System

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The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines

The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines. The indicated cell types (1.0 x 10⁵ cells) were seeded in 12-well plates one day before transduction. Cells were transduced with 1.0 x 10⁵ MOI (genomic titer) each of AAV2-Up and AAV2-Down viral particles targeting the CCR5 gene. After 72 hours, cells were harvested and analyzed using the Guide-it Mutation Detection Kit. As a control, cells were transfected with a plasmid (P: 2.5 µg) encoding Cas9 and a guide sequence targeting CCR5, using Xfect Transfection Reagent.

Back

Production of full-length Cas9 and sgRNA

Production of full-length Cas9 and sgRNA. The pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors encode truncated upstream and downstream portions of the Cas9 gene respectively, with a 1.6-kb overlapping region of homology. A user-defined single guide RNA (sgRNA) can be cloned into pAAV-Guide-it-Down (see Vector Maps). Separate AAV2-Up and AAV2-Down particles are generated by transfecting HEK 293T cells with either pAAV-Guide-it-Up or pAAV-Guide-it-Down along with pRC2-mi342 and pHelper vectors. The resulting viral particles are isolated using AAV Extraction Solution. Target cells are cotransduced with both AAV2-Up and AAV2-Down particles. The 1.6-kb homologous overlapping region results in recombination during AAV genome processing giving rise to a full-length Cas9 gene that is transcribed and translated into protein.

Back

AAV vector recombination to produce full-length Cas9 and sgRNA

AAV vector recombination to produce full-length Cas9 and sgRNA. The large size of the Cas9 gene precludes its packaging into a single AAV particle. pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors contain truncated upstream and downstream portions of Cas9, respectively, with a shared 1.6-kb overlapping region of homology. Separate viruses are generated with each plasmid. Transduction of target cells with both viruses results in recombination to generate the full-length Cas9 (4.1 kb) gene. Subsequent expression in target cells results in functional Cas9 protein guided to the appropriate genomic site by the sgRNA.
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Consistent recovery of equally high genomic titers of AAV2-Up and AAV2-Down viral particles

Consistent recovery of equally high genomic titers of AAV2-Up and AAV2-Down viral particles. AAV2-Up and AAV2-Down viral particles were extracted from HEK 293T cells using the AAVpro Extraction Solution. Genomic titer was measured using the AAVpro Titration Kit (for Real Time PCR). Similar genomic titers were obtained for AAV2-Up and AAV2-Down viral particles in three independent experiments.

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Vector map of pAAV-Guide-it-Up

Vector map of pAAV-Guide-it-Up. The structure of the pAAV-Guide-it-Up vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free System, a kit that produces AAV particles for cell delivery of Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV particle. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids; the pAAV-Guide-it-Up vector contains a truncated portion of the gene (3,106 bp) that encodes the N-terminal 1,035 amino acids of Cas9 under the control of the CMV promoter.

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Vector map of pAAV-Guide-it-Down

Vector map of pAAV-Guide-it-Down. The structure of the pre-linearized pAAV-Guide-iT-Down vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free system, a kit that produces AAV particles for cell delivery of the Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV virus. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids. The pAAV-Guide-it-Down vector contains a truncated downstream portion of the gene (2,616 bp) that encodes the C-terminal 872 amino acids of Cas9. A user-defined single guide RNA (sgRNA) can be cloned into the pAAV-Guide-it-Down vector downstream of the human U6 promoter. To construct the vector, a pair of oligos corresponding to the genomic target (guide sequence) are annealed to form a duplex. The duplexed DNA is then cloned into the prelinearized vector.

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The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

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632608: AAVpro CRISPR/Cas9 Helper Free System (AAV2)

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The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector

The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector. pAAV-Guide-it-1 is supplied pre-linearized and ready for insertion of the sgRNA target sequence (sgRNA shown in orange). When target cells are transduced with a virus packaged with the vector, the cells will express both SaCas9 and the sgRNA in order to achieve genome editing.

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Guide-it Mutation Detection Kit

Guide-it Mutation Detection Kit for side-by-side genomic DNA modification comparison using the AAVpro CRISPR/Cas9 System (SpCas9) and the AAVpro CRISPR/SaCas9 System. Two different genomic regions in CYP2 Exon 1 were targeted and the results are shown in wells 1 and 2 for each enzyme, and the target regions are shown below the gel. The indel percentage is indicated below each well for each cell type. To carry out the modification, 1 x 10⁵ cells of three different cell lines were seeded in each well of 12-well plates one day before transduction. Cells were transduced with either the two-vector AAVpro CRISPR/Cas9 System (SpCas9) or the one-vector AAVpro CRISPR/SaCas9 System at 1 x 10⁵ MOI for each vector as determined by genomic titer. After 72 hr, cells were harvested and analyzed using the Guide-it Mutation Detection Kit.

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632619: AAVpro CRISPR/SaCas9 Helper Free System (AAV2)

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The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector

The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector. pAAV-Guide-it-1 is supplied pre-linearized and ready for insertion of the sgRNA target sequence (sgRNA shown in orange). When target cells are transduced with a virus packaged with the vector, the cells will express both SaCas9 and the sgRNA in order to achieve genome editing.

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Guide-it Mutation Detection Kit

Guide-it Mutation Detection Kit for side-by-side genomic DNA modification comparison using the AAVpro CRISPR/Cas9 System (SpCas9) and the AAVpro CRISPR/SaCas9 System. Two different genomic regions in CYP2 Exon 1 were targeted and the results are shown in wells 1 and 2 for each enzyme, and the target regions are shown below the gel. The indel percentage is indicated below each well for each cell type. To carry out the modification, 1 x 10⁵ cells of three different cell lines were seeded in each well of 12-well plates one day before transduction. Cells were transduced with either the two-vector AAVpro CRISPR/Cas9 System (SpCas9) or the one-vector AAVpro CRISPR/SaCas9 System at 1 x 10⁵ MOI for each vector as determined by genomic titer. After 72 hr, cells were harvested and analyzed using the Guide-it Mutation Detection Kit.

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632618: AAVpro CRISPR/SaCas9 Vector System