The Dharmacon™ All-in-one CRISPR platform

Single reagent, easy delivery, straightforward selection

CRISPR workflows are simplified with Dharmacon All-in-one reagents. The All-in-one platform combines the CRISPR nuclease and single guide RNA expression into a single lentiviral packaged vector – for the simplest gene knockout/modulation workflow available. One reagent CRISPR for any human or mouse gene.

All-in-one benefits	Ideal for
Single reagent – CRISPR nuclease + sgRNA from one vector	New CRISPR users – simplified workflow for fast results
Easy, ready-to-use – Simply thaw and add lentivirus to your cells, no transfection or electroporation necessary	Easily generating cell models to study gene function
Simple selection – Choose antibiotic resistance or fluorescent selection markers	Difficult-to-transfect cells – Lentiviral delivery can be used with nearly any cell type
Our All-in-one system gives efficient gene knockout, activation, or interference	Hit validation – follow-up target hits from pooled-lentiviral screens

All-in-one CRISPR gene knockout, activation, and interference workflow

The power of the All-in-one system lies in the simplicity of it's workflow

To transduce, simply thaw the reagent and add the appropriate amount to your cells. Then easily select for targeted 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 targeted 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.

For CRISPRa and CRISPRi, we recommend the mixed cell population workflow.

CRISPR knockout all-in-one lentiviral system

CRISPRko single reagent

CRISPR sgRNA + Cas9 in one vector for easy gene editing

CRISPRko whole genome library

All-in-one high titer pooled screening library covering the entire human genome

CRISPR activation all-in-one lentiviral system

CRISPRa single reagent

CRISPRa sgRNA + dCas9-VPR in one vector for easy gene activation

CRISPRa whole genome library

CRISPRa all-in-one high titer pooled screening library covering the entire human genome

CRISPR interference all-in-one lentiviral system

CRISPRi single reagent

CRISPRi sgRNA + dCas9- SALL1-SDS3 in one vector for easy transcriptional repression

CRISPRi whole genome library

CRISPRi All-in-one high titer pooled screening libraries covering the entire human genome

The Dharmacon^TM All-in-one system

The All-in-one system offers the simplest CRISPR workflow available – a single reagent, easy delivery and straightforward selection.

Single reagent

Using a single vector to express both CRISPR nuclease and gene-specific guide RNA eliminates the need to perform sequential transduction and selection steps.

Easy delivery

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 genome editing and transcriptional modulation in difficult-to-transfect or primary cell types.

Transduction-ready reagents also eliminate any cloning or in vitro transcription steps.

Straightforward selection

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 targeted cells, as FACS analysis may be performed as soon as fluorescence is expressed.

Promoter options

Choice of promoters allows you to select the most active promoter in your cell line.

All-in-one performs as well as 2-vector sgRNA and Cas9

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.

Comparison of Edit-R two-vector and All-in-one systems

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.

Gene knockout in primary cells & iPSCs

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.

Edit-R All-in-one editing data

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.

Knockout your target gene

Find All-in-one lentiviral sgRNA for guaranteed editing of any human or mouse gene

Efficient transcriptional activation with CRISPRmod CRISPRa All-in-one Puro-dCas9-VPR

Transcriptional Activation with CRISPRmod All-in-one Puro-dCas9-VPR

HEK293T or HCT-116 cells were plated in 96-well plates at 20,000 or 5,000 cells/well, respectively, and transduced with 0.3 MOI CRISPRa all-in-one virus containing sgRNAs targeting POU5F1, TTN, IL1R2, or Non-targeting control #2 (NTC2). Cells were selected with 2.5 µg/µL puromycin for 5 days, allowed to expand, and harvested two weeks post-transduction. Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

Increasing multiplicity of infection (MOI) improves target activation

Increasing MOI can improve target activation

HEK293T cells were plated in 96-well plates at 20,000 cells/well and transduced with denoted MOIs of CRISPRa all-in-one virus containing sgRNAs targeting POU5F1, TTN, or NTC2. Cells were selected with 2.5 µg/µL puromycin for 5 days, allowed to expand, and harvested one or two weeks post-transduction. Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

Induction of OCT4 protein expression in unsorted U2OS cells

U2OS cells were plated in 96-well plates at 10,000 cells/well and transduced with 0.5 MOI EGFP CRISPRa All-in-one virus containing sgRNAs targeting POU5F1 or NTC2. Cells were passaged and expanded into 96-well and 6-well plates. RNA was extracted from cells cultured in 96-well plates one-week post-transduction. Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

Activation of OCT4 protein in unsorted U2OS cells by EGFP mCMV CRISPRa All-in-one viral particles

U2OS cells were plated in 96-well plates at 10,000 cells/well and transduced with 0.5 MOI EGFP CRISPRa All-in-one virus containing sgRNAs targeting POU5F1 or NTC2.

Overexpress your target gene at the transcriptional level

Find All-in-one lentiviral sgRNA for CRISPR activation for any human gene

Efficient transcriptional repression with CRISPRmod CRISPRi All-in-one system

Transcriptional Repression with CRISPRmod All-in-one Puro-dCas9-SALL1-SDS3

HEK293T, U2OS, or HCT-116 cells were plated in 96-well plates at 20,000, 10,000, or 5,000 cells/well and transduced with 0.3 MOI CRISPRi all-in-one virus containing sgRNAs targeting PPIB, SEL1L, or NTC2. Cells were selected with 2.5 µg/µL puromycin for 5 days, allowed to expand, and harvested at denoted time points post-transduction. Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

With puromycin selection transcriptional repression is improved

Selection markedly improves transcriptional repression in U2OS cells

U2OS cells were plated in 96-well plates at 10,000 cells/well and transduced with 0.3 MOI CRISPRi all-in-one virus containing sgRNAs targeting PPIB, SEL1L, TFRC, or NTC2. Cells were selected with 2.5 µg/µL puromycin (where applicable) for 5 days, allowed to expand, and harvested one week or two weeks post-transduction. Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

Increased transcriptional repression in EGFP positive Jurkat cells

Transcriptional repression in EGFP+ Jurkat cells

Jurkat cells were plated at 25,000 cells/well and transduced with 0.3 MOI CRISPRi all-in-one virus containing sgRNAs targeting CD46 or NTC2 (CRISPRi). Cells were cultured for one week and harvested for mRNA extraction (1-week post-transduction, Pre-FACS) or cultured for 3 weeks, washed in PBS, and resuspended in FACS buffer (1X PBS containing 10 mM Tris-HCl - pH 7.5, 25 mM HEPES, and 1% FBS). EGFP positive cells were sorted via FACS into 24 well plates containing ~50,000 EGFP positive cells per well. Cells were expanded for 1 week, and RNA was harvested at four weeks post-transduction (Post-FACS). Relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control.

Knock down your target gene at the transcriptional level

Find All-in-one lentiviral sgRNA for CRISPR interference for any human gene