Edit-R Lentiviral Cas9 Nuclease Reagents

Lentiviral CRISPR-Cas9 components for robust gene editing in biologically relevant cell types


Edit-R Lentiviral Cas9 Nuclease Reagents
Purified lentiviral particles or plasmid DNA for generation of stable Cas9 nuclease-expressing cell populations
The Edit-R Gene Engineering platform includes a lentiviral Cas9 expression vector to facilitate rapid generation of cell lines that constitutively express Cas9 nuclease. These cell lines empower many gene editing applications that require the ability to observe a phenotype in a mixed population of cells where only a subset of cells contain the gene editing event. Additionally, rapid testing or screening of multiple guide RNAs targeting a single gene and/or multiple genes is enabled by the generation of Cas9-expressing stable cell lines prior to delivery of the guide RNA.

The Edit-R Lentiviral Cas9 Nuclease Expression vectors contain a human codon-optimized version of the S. pyogenescas9 (csn1) gene and the blasticidin resistance marker (BlastR). In the inducible format, the resistance marker and the tetracycline response element (Tet-On™ 3G) are under the control of the hEF1α proimoter and the Cas9 is under the tight regulation of the TRE3G inducible promoter. In the constitutive format, both are expressed as a bicistronic transcript with a 2A peptide sequence linker under the control of a single promoter. BlastR is placed upstream of the Cas9 coding region rather than the 3' end so that no extra amino acids are added to the C-terminus, thus preserving the full Cas9 endonuclease activity.

Lentiviral Cas9 vector maps

Highlights

  • Provided as concentrated, purified lentiviral particles for immediate transduction; 50 (2 x 25)µL, within 10% of minimum ≥ 1 × 107 TU/mL functional titer, by QPCR titering.
  • Also available as certified endotoxin-free plasmid DNA for direct transfection into a packaging cell line and production of your own lentiviral particles.
  • Customize your construct with one of six SMARTchoice constitutive promoters or select an inducible promoter to ensure optimal Cas9 expression in your cell line of interest.
  • Utilize the tight regulation of the inducible Cas9 vector when you require temporal control over the expression of Cas9 and to generate a stable cell line with minimal background expression.

Not all RNA pol II promoters are equally active in different cellular environments

The activity of any given promoter controlling the transcription of Cas9 nuclease can differ greatly from one biological context to another, resulting in variable Cas9 expression levels and thus varying levels of DNA cleavage. Choosing an optimal promoter for your cell line or type will therefore affect the degree of gene editing in your experimentation. The Edit-R Lentiviral Cas9 Nuclease expression reagents are offered with six different, well-characterized constitutive cellular promoters and a robust inducible promoter from which you can choose.

SMARTchoice promoter options for expressing Cas9 nuclease
Promoter Description
hCMV human cytomegalovirus immediate early promoter
mCMV mouse cytomegalovirus immediate early promoter
hEF1α human elongation factor 1 alpha promoter
mEF1α mouse elongation factor 1 alpha promoter
PGK mouse phosphoglycerate kinase promoter
CAG chicken beta actin hybrid promoter
TRE3G doxycycline inducible promoter

 

  
HazardousNo
Shipping ConditionDry Ice,Ambient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C,-20 C
Cas9 Nuclease Selection Guide

Which Cas9 nuclease is right for you?

Cas9 Nuclease Selection Guide

While the best Cas9 nuclease product for your experiment may heavily depend on the particular application or cell type, a few basic questions may help to point you in the right direction for product selection.


Lentiviral Cas9 vector maps

Lentiviral Cas9 vector maps

Lentiviral Cas9 vector maps

Vector Element Utility
Cas9 Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with a sgRNA
TRE3G Inducible promoter with Tetracycline Response Elements which is activated by the Tet-On 3G protein in the presence of doxycycline
hCMV Human cytomegalovirus immediate early promoter
mCMV Mouse cytomegalovirus immediate early promoter
hEF1α Human elongation factor 1 alpha promoter
mEF1α Mouse elongation factor 1 alpha promoter
PGK Mouse phosphoglycerate kinase promoter
CAG Human cytomegalovirus, chicken β-actin hybrid promoter
BlastR Blasticidin resistance marker permits antibiotic selection of transduced mammalian cells
T2A Self-cleaving peptide allows for simultaneous expression of blasticidin resistance and Cas9 proteins from a single transcript
Tet-On 3G Tetracycline-regulated transactivator protein that binds to TRE3G promoter only in the presence of doxycycline.
U6 Human RNA polymerase III promoter U6
sgRNA Chimeric form of the crRNA and tracrRNA that provides Cas9 with the DNA target site recognition
mCMV Mouse cytomegalovirus immediate early promoter
5' LTR 5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genome
Ψ Psi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems
RRE Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes
WPRE Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells
3' SIN LTR 3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles
SV40 pA Simian virus 40 polyadenylation signal
pUC ori pUC origin of replication
SV40 ori Simian virus 40 origin of replication
AmpR Ampicillin resistance gene for vector propagation in E. coli cultures


Differential expression of Cas9 by different promoters induce varying levels of gene editing

Differential expression of Cas9 by different promoters induce varying levels of gene editing

Differential expression of Cas9 by different promoters induce varying levels of gene editing

A human recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) (A) and a mouse fibroblast (NIH/3T3) (B), were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters.. A population of cells with stably integrated Cas9-blastR was selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Human PPIB / mouse Ppib using DharmaFECT 1 and DharmaFECT 3 Transfection reagent, respectively. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay using T7EI on genomic DNA extracted from the transfected cells.


Functional protein knockout of VCP is observed in a cell-based assay using stably expressed Cas9 and Edit-R synthetic crRNA:tracrRNA in a recombinant U2OS line.

Functional protein knockout of VCP is observed in a cell-based assay using stably expressed Cas9 and Edit-R synthetic crRNA:tracrRNA in a recombinant U2OS line.

Functional protein knockout of VCP is observed in a cell-based assay using stably expressed Cas9 and Edit-R synthetic crRNA:tracrRNA in a recombinant U2OS line.


Gene editing activity of inducible Cas9 vectors after induction with Doxycycline for 7 days

Gene editing activity of inducible Cas9 vectors after induction with Doxycycline for 7 days

Gene editing activity of inducible Cas9 vectors after induction with Doxycycline for 7 days

Cells were transduced with a constitutive (CAG-Cas9) or an inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin for 2 days in tetracycline-free medium and split in two populations: uninduced (DOX-) and induced (DOX+) with 500 ng/mL doxycycline for 7 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.


Inducible Cas9 vector displays minimal leak after 21 days without doxycycline induction

Inducible Cas9 vector displays minimal leak after 21 days without doxycycline induction

Inducible Cas9 vector displays minimal leak after 21 days without doxycycline induction

Cells were transduced with a constitutive (CAG-Cas9) or an inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin and maintained in tetracycline-free medium for 21 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.


Dose response for doxycycline in inducible U2OS-Cas9 cells

Dose response for doxycycline in inducible U2OS-Cas9 cells

Dose response for doxycycline in inducible U2OS-Cas9 cells

Cells were transduced with the inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin tetracycline-free medium for 4 days, suspended with trypsin and seeded in a 96-well plate in medium containing increasing concentrations of doxycycline (0 to 1000 ng/mL). The cells were incubated for 72 hours, lysed and analyzed for indel formation using a DNA mismatch detection assay with T7EI. Upper panel, representative gel image of the DNA mismatch detection assay with T7EI for PPIB targeted amplicon; lower panel, mean ± standard deviation of the estimated percentage of gene editing from three independently treated wells.


Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease Expression particles with synthetic crRNA and tracrRNA.

Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease Expression particles with synthetic crRNA and tracrRNA

Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease Expression particles with synthetic crRNA and tracrRNA.


Gene knockout workflow using the Edit-R Lentiviral Cas9 nuclease with lentiviral sgRNA

Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease with Lentiviral sgRNA

Gene knockout workflow using the Edit-R Lentiviral Cas9 nuclease with lentiviral sgRNA

Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease with sgRNA system. Gene editing with Edit-R Lentiviral Cas9 Nuclease and sgRNA can be performed using a mixed cell population approach (left side) typically for gene knockout screening or an isolated clonal cell line approach (right side) when a more defined cell type is required for phenotypic analysis.


Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease with Edit-R Lentiviral sgRNA

Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease with Edit-R Lentiviral sgRNA

Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease with Edit-R Lentiviral sgRNA


Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease in a pooled lentiviral sgRNA screen

Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease in a pooled lentiviral sgRNA screen

Gene knockout workflow using the Edit-R Inducible Lentiviral Cas9 Nuclease in a pooled lentiviral sgRNA screen


References

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