Back

Knockout of cell-surface proteins in human iPS cells.

Knockout of cell-surface proteins in human iPS cells. In this experiment, human iPS cells were electroporated with either competitors' Cas9 (Vendor X and Vendor Y)/sgRNA RNPs or Recombinant Cas9 Protein GMP grade (Cat. # T230)/sgRNA RNPs against cell-surface protein A (gene A) and/or protein B (gene B). Cells were stained with either anti-protein A antibody or anti-protein B antibody and then run on a FACS machine to detect each surface protein. Takara Bio's GMP-grade Cas9 protein showed high knockout efficiency at each condition tested. Data provided by Drs. Akitsu Hotta and Huaigeng Xu at the Center for iPS Cell Research and Application (CiRA), Kyoto University.

Back

Knockout of CD81 in 293T/17 cells.

Knockout of CD81 in 293T/17 cells. In this experiment, 293T/17 cells were electroporated with either Guide-it Recombinant Cas9 (Electroporation-Ready; Cat. # 632641)/sgRNA RNPs or Recombinant Cas9 Protein GMP grade (Cat. # T230)/sgRNA RNPs against CD81. Electroporation was performed at 1,300, 1,200, and 1,100 V for conditions #1, 2, and 3, respectively. Pulse interval was 20 ms, for each of two pulses, for each condition. Cells were stained with CD81 PE-labeled anti-CD81 antibody (BioLegend, Inc.; Cat. # 349506) and then run on a FACS machine to detect CD81 seven days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with RNPs for both Guide-it Cas9 (632641) and GMP Cas9 (T230), high editing efficiencies were observed.

Back

T230: Recombinant Cas9 Protein GMP grade

Back

Knock out of CD81 in induced pluripotent stem cells

Knock out of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated withCas9/sgRNA RNPs against CD81. Cells were stained for CD81 and then run on a FACS machine to detect CD81 ten days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with both Cas9 and sgRNA, very high editing efficiency was observed.

.

Back

Homology-directed repair at the AAVS1 or CXCR4 genes

Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. A ssdNA template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both HindIII and BamHI restriction sites was inserted into the CXCR4 gene. The homology arms were 90 bp on each side. Panel B. After electroporation of the repair template and Cas9/sgRNA RNPs, gene targets were amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing. We verified that 98% of the HSCs stained positive for CD34+ five days after editing.

.

Back

Gene knockouts in the Cellartis hiPSC-18 cell line

Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitative estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets when compared with other vendors’ recommended methods for producing guide RNAs. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.

Back

Knockout of therapeutically relevant genes in CD3+ Pan T cells

Knockout of therapeutically relevant genes in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to perform single (B2M or PD-1) or double knockouts (B2M+PD-1 or TRAC+TRBC). After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, following the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. For the double knockouts, RNP complexes targeting the respective genes were prepared independently and mixed with the cells just before electroporation. Seven days after electroporation, knockout frequencies were measured by flow cytometry.

Back

Knockout of B2M in CD3+ Pan T cells

Knockout of B2M in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to knockout B2M. After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, according to the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. Seven days after electroporation, knockout frequencies and cell viability were measured by flow cytometry.

Back

632678: Guide-it Recombinant Cas9 (10 µg/µl)

Back

Knock out of CD81 in induced pluripotent stem cells

Knock out of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated withCas9/sgRNA RNPs against CD81. Cells were stained for CD81 and then run on a FACS machine to detect CD81 ten days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with both Cas9 and sgRNA, very high editing efficiency was observed.

.

Back

Homology-directed repair at the AAVS1 or CXCR4 genes

Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. A ssdNA template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both HindIII and BamHI restriction sites was inserted into the CXCR4 gene. The homology arms were 90 bp on each side. Panel B. After electroporation of the repair template and Cas9/sgRNA RNPs, gene targets were amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing. We verified that 98% of the HSCs stained positive for CD34+ five days after editing.

.

Back

Gene knockouts in the Cellartis hiPSC-18 cell line

Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitative estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets when compared with other vendors’ recommended methods for producing guide RNAs. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.

Back

Knockout of therapeutically relevant genes in CD3+ Pan T cells

Knockout of therapeutically relevant genes in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to perform single (B2M or PD-1) or double knockouts (B2M+PD-1 or TRAC+TRBC). After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, following the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. For the double knockouts, RNP complexes targeting the respective genes were prepared independently and mixed with the cells just before electroporation. Seven days after electroporation, knockout frequencies were measured by flow cytometry.

Back

Knockout of B2M in CD3+ Pan T cells

Knockout of B2M in CD3+ Pan T cells. In this experiment, CD3+ Pan T cells were electroporated with Cas9/sgRNA RNPs in order to knockout B2M. After thawing, cells were grown in LymphoONE medium and activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator for three days. Cells were then electroporated with RNP complexes in triplicate, according to the Guide-it Recombinant Cas9 (10 μg/μl) User Manual. Seven days after electroporation, knockout frequencies and cell viability were measured by flow cytometry.

Back

632679: Guide-it Recombinant Cas9 (10 µg/µl)

Back

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

Back

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

Back

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

Back

The use of gesicles decreases off-target effects

The use of gesicles decreases off-target effects. HEK 293T cells were either simultaneously cotransfected with plasmids encoding Cas9 DNA and a sgRNA against EMX1, or treated with gesicles loaded with Cas9-sgRNA ribonucleoprotein complexes. After 72 hr, the EMX1 gene and a potential off-target locus (off-target 4) were analyzed using the Guide-it Mutation Detection Kit (Cat. # 631443). With the gesicles, no off-target effect could be detected (top panel). Sequencing data for the different clones were aligned with the underlined wild-type sequence, revealing a range of deletions and insertions (indels; highlighted in red). Gesicles correctly edited the EMX1 gene with no off-target effects, whereas plasmid cotransfection resulted in indels in both the target site, EMX1, as well as off-target site 4 (bottom panel).

Back

Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles

Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles. Cell lines were created that contained an integrated ZsGreen1 fluorescent protein expression cassette. In this system, successful Cas9-mediated cleavage can be measured by loss of ZsGreen1 expression. These cell lines were treated with gesicles loaded with Cas9-sgRNA protein complexes (with the sgRNA generated against ZsGreen1), and then analyzed by flow cytometry. Cas9-sgRNA protein complex delivery and ZsGreen1 knockout via gesicles was efficient and comparable to plasmid-based delivery in easier-to-transfect cell types (left graph) and surpassed the results achieved via plasmid-based delivery in harder-to-transfect cell types (right graph).

Back

Gesicle production overview for delivery of Cas9-sgRNA

Gesicle production overview for delivery of Cas9-sgRNA. (Step 1) Gesicle formation is induced by glycoproteins on the surface of 293T producer cells that have been cotransfected with our gesicle packaging mix and a target-specific guide RNA plasmid. (Step 2) Utilizing the iDimerize system, a small ligand is added to load the Cas9-sgRNA ribonucleoprotein complex into the gesicle through interaction with the membrane-bound CherryPicker red fluorescent protein on the gesicle surface. (Step 3) Loaded and red fluorescent protein-labeled gesicles pinch off from the producer cells and are collected from the supernatant, yielding a concentrated stock of Cas9-sgRNA gesicles. (Step 4) Harvested gesicles can be applied to a broad range of target cell types, to which they fuse, transiently labeling the cells red and releasing the Cas9-sgRNA complex into the cell. The presence of an NLS on the Cas9 protein and the absence of the dimerizer ligand in your cell culture medium ensures that the complex is transported to the nucleus after dissociating from the red fluorescent protein.

Back

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).

Back

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles. HT1080 cells containing an integrated fluorescent protein expression cassette were transfected with a plasmid encoding for Cas9 and AcGFP1-specific sgRNA or treated with gesicles. Both delivery methods were tested using either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. The optimized sgRNA scaffold had no effect on editing efficiency for plasmid-based delivery. However, the optimized sgRNA scaffold increased knockout efficiency by 36.4% for gesicles. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Back

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect. HT1080 cells containing an integrated fluorescent protein expression cassette were treated with 5 µl, 10 µl, 20 µl, or 30 µl of gesicles produced with either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. Editing efficiency using the traditional scaffold was low, regardless of gesicle dose. However, gesicles using the optimized scaffold demonstrated a dose-dependent increase in knockout up to 59%. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Back

Optimal sgRNA design strategies when using gesicles

Optimal sgRNA design strategies when using gesicles. While evaluating potential sgRNAs for knockout of different genes, several optimal strategies were identified. First, having a guanine in the first position of the target sequence provided the highest knockout efficiency. Additionally, having either an adenine or thymine in the seventeenth position of the target sequence was beneficial. Utilizing these two design principles when producing gesicles can improve overall editing efficiency in target cells.

Back

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes. The cell-surface protein receptor CD81 was knocked out in Jurkat cells using either plasmid cotransfection of Cas9 DNA and sgRNA or gesicles preloaded with a Cas9-sgRNA ribonucleoprotein complex. The knockout efficiency was measured six days later via antibody labeling of the membrane receptor followed by flow cytometry analysis. Results for delivery via gesicles were significantly greater than results achieved with plasmid transfection.

Back

6501_gesicle_tube

Back

Gesicles utilize an optimized sgRNA scaffold

Gesicles utilize an optimized sgRNA scaffold. The traditional sgRNA scaffold was modified by extending the Cas9-binding hairpin and removing four consecutive uracils. This optimized scaffold is critical for successful gesicle-based editing due to improved knockout efficiency.

Back

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

Back

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

Back

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

Back

632616: Guide-it CRISPR/Cas9 Gesicle Packaging Set

Back

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid. 1 x 10⁵ cells (HEK 293, HeLa, U2OS, or HT1080 cell lines) were seeded in 12-well plates 16 hr prior to transfection. Cells were transfected with 2.5 µg of pGuide-it-ZsGreen1 plasmid using the Xfect Transfection Reagent; the plasmid expressed Cas9, ZsGreen1, and an sgRNA targeting the either CCR5, C4BPB, or EMX1. After 48 hr, fluorescence was analyzed by microscopy (A), and by FACS (B, Transfection efficiency). The efficiency of gene modification was also evaluated using the Guide-it Mutation Detection Kit, a PCR-based method for testing for the presence of indels (B). The target sequence was amplified directly from cells, the amplicon was melted and hybridized to form mismatched targets that were cleaved with Guide-it Resolvase. Cleavage products were resolved on an agarose gel and quantified by densitometry to estimate the frequency of indels (B, Indel).

Back

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).

Back

632601: Guide-it CRISPR/Cas9 System (Green)

Back

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid. 1 x 10⁵ cells (HEK 293, HeLa, U2OS, or HT1080 cell lines) were seeded in 12-well plates 16 hr prior to transfection. Cells were transfected with 2.5 µg of pGuide-it-ZsGreen1 plasmid using the Xfect Transfection Reagent; the plasmid expressed Cas9, ZsGreen1, and an sgRNA targeting the either CCR5, C4BPB, or EMX1. After 48 hr, fluorescence was analyzed by microscopy (A), and by FACS (B, Transfection efficiency). The efficiency of gene modification was also evaluated using the Guide-it Mutation Detection Kit, a PCR-based method for testing for the presence of indels (B). The target sequence was amplified directly from cells, the amplicon was melted and hybridized to form mismatched targets that were cleaved with Guide-it Resolvase. Cleavage products were resolved on an agarose gel and quantified by densitometry to estimate the frequency of indels (B, Indel).

Back

632602: Guide-it CRISPR/Cas9 System (Red)

Back

Inducible knockout of CD81 in HEK293 cells

Inducible knockout of CD81 in HEK293 cells. Target cells were first transduced with Tet-On 3G lentivirus and selected with G418. 24 independent Tet-On 3G-positive clones were assayed for induction of the luciferase reporter, and one clone exhibiting the lowest residual expression was selected and transduced with Cas9 lentivirus at an MOI of 5. Target cells were selected with 1 µg/ml puromycin for two weeks, and six stable clones were randomly selected for analysis of Cas9 induction. Cells from each clone were divided between two wells of a 12-well plate and incubated either in the presence or absence of doxycycline (0.5 µg/ml) for two days. Cas9 induction was then assayed by Western blot using polyclonal anti-Cas9 antibody (Cat. No. 632607), diluted 1:1,000, and ECL reagent. Cells derived from three of the Tet-On 3G-Cas9-positive clones (C-1, C-2, and C-3) were then divided between two wells of a 12-well plate and transduced with CD81-sgRNA lentivirus at an MOI of 5. Eight hours after transduction, doxycycline was added to one well for each clone at a concentration of 0.5 µg/ml, and cells were cultured for six days. Cells were then harvested and stained with a FITC-tagged anti-human CD81 antibody and scanned for CD81 expression by FACS. Panel A. Western blot results for six independent Tet-On 3G-Cas9-positive cell populations cultured in the absence (–) or presence (+) of 0.5 µg/ml doxycycline, assayed with anti-Cas9 antibody. Cas9 expression is either undetectable or highly elevated in the absence or presence of doxycycline, respectively. Panel B. FACS results for three independent Tet-On 3G-Cas9-positive 293T cell populations transduced with CD81-sgRNA lentivirus and cultured in the absence (–Dox) or presence (+Dox) of 0.5 µg/ml doxycycline. Two of the populations (C-1 and C-2) exhibited no residual editing in the absence of doxycycline, and knockout of CD81 in either 58.6% or 30.7% of cells, respectively, while the third population (C-3) exhibited residual editing (27.9% of cells) in the absence of doxycycline.

Back

Inducible knockout of CD81 in Jurkat cells

Inducible knockout of CD81 in Jurkat cells. Tet-On 3G-Cas9-positive Jurkat cells with low residual Cas9 expression were generated using the established protocol and transduced twice with CD81-sgRNA lentivirus at an MOI of 5 on successive days. Cells were then divided between two wells of a 12-well plate and cultured in the absence (–Dox) or presence (+Dox) of doxycycline at a concentration of 0.5 µg/ml for seven days. Cells were then treated with FITC anti-human CD81 antibody and analyzed by FACS. Only a small proportion of cells (0.4%) cultured in the absence of doxycycline (–Dox; top) exhibit residual editing. A significant proportion of cells (32.0%) cultured in the presence of doxycycline (+Dox; bottom) exhibit knockout of CD81.

Back

Inducible editing of AAVS1 in HEK293 cells

Inducible editing of AAVS1 in HEK293 cells. Induction of Cas9 expression in six stable Tet-On 3G Cas9-positive HEK293 cell lines was assayed by qRT-PCR. Cells from each line were then divided between two wells of a 12-well plate and transduced with AAVS1-sgRNA lentivirus at an MOI of 5. Eight hours after infection, doxycycline was added to one well for each cell line at a concentration of 0.5 µg/ml, and the cells were cultured for six days. The Guide-it Mutation Detection Kit (Cat. # 631443) was then used to screen for editing of AAVS1. Panel A. qRT-PCR results for Cas9 expression in uninduced (–Dox) and induced (+Dox) cells for each clone. Ct and ΔCt values for uninduced and induced clones are indicated in the corresponding columns, and the calculated fold-difference in expression for each clone is indicated in the column on the right. Of the various clones tested, Clone 1 (numbers in green) exhibited the highest-fold inducibility, while only Clone 3 (numbers in red) exhibited residual expression of Cas9 in the absence of doxycycline and low inducibility. Panel B. Results of Guide-it Resolvase assay for detection of AAVS1 gene editing in uninduced (–) and induced (+) clones. In contrast with the assay of the parental line, which yielded bands of equivalent size for uninduced and induced cells, assays of the induced clones yielded additional bands of smaller sizes, indicating the occurrence of gene editing at AAVS1 for all clones tested. Cleavage products are also visible for the uninduced cells from Clone 3, consistent with the residual expression of Cas9 observed in the qRT-PCR assay.

Back

Vector components for Lenti-X Tet-On 3G CRISPR/Cas9 System

Vector components for Lenti-X Tet-On 3G CRISPR/Cas9 System. pLVX-EF1a-Tet3G encodes the Tet-On 3G transactivator protein under the control of the constitutive EF-1 alpha promoter. In contrast with the commonly used CMV promoter, EF-1 alpha is less susceptible to silencing by certain cell types (including various types of stem cells), enabling the establishment of inducible cell lines that are suitable for long-term use. Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein, which was evolved to have a far higher sensitivity to doxycycline. pLVX-TRE3G-Cas9-puro encodes a modified version of the Cas9 endonuclease under the control of the inducible P_TRE3GV promoter. The Cas9 sequence in pLVX-TRE3G-Cas9-puro was originally derived from the bacterium S. pyogenes and has been codon optimized for expression in mammalian cells. Additional Cas9 modifications include the incorporation of a C-terminal nuclear localization signal (NLS). pLVX-hyg-sgRNA1 encodes an sgRNA (designed and inserted by the user) under the constitutive human U6 promoter.

Back

Choosing an optimal clone for inducible genome editing

Choosing an optimal clone for inducible genome editing. Clones may be selected by assaying Cas9 expression or genome editing in both uninduced and induced cell populations. However, while Western blot detection of Cas9 is helpful for prescreening clones, CRISPR/Cas9-mediated editing is so efficient that even when Cas9 protein is undetectable by Western blot, genome editing can still occur in corresponding clones. We recommend qRT-PCR for identifying clones in which the residual expression of Cas9 is low enough for editing to be avoided in the absence of doxycycline. Data in the upper row is from a desirable clone (C-1), which exhibits robust expression of Cas9 protein in induced cells (Western blot, +), and the lowest residual levels of Cas9 mRNA (qRT-PCR). Genome editing occurs at much higher frequencies upon induction of Cas9 expression in cells derived from this clone (+ or +Dox) relative to uninduced cells (– or –Dox), as evidenced by the increase in smaller-sized bands in the resolvase assay (blue arrows) and the greater proportion of cells in the “knockout” category as determined by FACS, with the lowest residual editing in uninduced cells. In contrast, data in the lower row is from an undesirable clone (C-3), which exhibits residual transcription of Cas9 (qRT-PCR) and detectable frequencies of genome editing (Resolvase, FACS) in the absence of doxycycline, even though Cas9 protein expression in uninduced cells (–) is undetectable by Western blot

Back

632633: Lenti-X Tet-On 3G CRISPR/Cas9 System