Knockout your target gene
The Edit-R All-in-one system offers the simplest CRISPR gene knockout workflow available – a single reagent, easy delivery and straightforward selection, for guaranteed gene editing.
Using a single vector to express both gene-specific CRISPR guide RNA and Cas9 nuclease eliminates the need to perform sequential transduction and selection steps.
Lentiviral packaging allows for easy transduction into nearly any cell type. Eliminating the need for toxic transfection reagents or electroporation techniques makes the All-in-one system particularly useful for generating knockouts in difficult-to-transfect or primary cell types.
Transduction-ready reagents also eliminate any cloning or in vitro transcription steps.
The choice of a puromycin resistance gene or EGFP marker allows for straightforward selection of cells that have successfully integrated the vector. EGFP is recommended for rapid enrichment of edited cells, as FACS analysis may be performed as soon as fluorescence is expressed.
To transduce, simply thaw the reagent and add the appropriate amount to your cells. Then easily select for edited cells using puromycin or enrich using FACS.
When using puromycin selection, growth medium is replaced with puromycin selection medium 24-48 hours post-transduction. Cells are then monitored daily until growing normally in the selection medium. Once growth is stable, isolation and/or expansion of the edited cell population(s) may occur.
Alternatively, using the EGFP fluorescent reporter option offers the ability to enrich for edited cells via FACS selection in as few as 72 hours post-transduction. The FACS enrichment workflow is especially useful for short-lived cell types, such as primary cells.
The All-in-one lentiviral system has shown to edit cells as effectively as methods using two separate lentiviral vectors to deliver Edit-R single guide RNA and Cas9 nuclease.
For the two-vector system, cells were previously transduced with a constitutive hEF1α- or mCMV-Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin for 10 days, followed by transduction with non-targeting control (NTC), DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3.
For the All-in-one (AIO) system, wild-type cells were transduced with the NTC, DNMT3B or PPIB All-in-one lentiviral particles that also express a constitutive hEF1α- or mCMV-Cas9 nuclease, and the PuroR gene at an MOI of 0.3.
All transduced cells were selected with 2 µg/mL puromycin for 3 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.
Lentiviral delivery makes the Edit-R All-in-one system ideal for performing targeted gene knockout in difficult-to-transfect cell types, such as primary cells, iPSCs, and immune cell lines.
Jurkat or K562 cells were transduced at a MOI of 0.3 using Edit-R All-in-one EGFP Lentiviral sgRNA vectors targeting either the CD3 or CD28 genes. All-in-one lentiviral particles express Cas9 nuclease constitutively under the hEF1α or mCMV promoters. Lentiviral particles expressing Non-targeting control (NTC) guides were used as negative controls. After lentiviral transduction and expansion, cells were analyzed as unsorted population (pool) or were subjected to cell sorting to enrich for the brightest (top) or dimmest (dim) EGFP expressing cell populations. The pool, top, and dim populations of cells were then subjected to TIDE analysis to assess gene editing frequency.
Edit-R All-in-one lentiviral sgRNA
Find lentiviral sgRNA specific to any human or mouse gene.
Edit-R All-in-one positive controls and detection primers
All-in-one lentiviral sgRNAs targeting well characterized genes used to verify DNA double-strand breaks and gene editing efficiencies.
Edit-R All-in-one non-targeting controls
All-in-one lentiviral sgRNA constructs bioinformatically designed and validated to not target any gene in human, mouse or rat genomes.