Edit-R all-in-one lentiviral sgRNA non-targeting controls

Gene editing
Gene editing reagents
Edit-R All-in-one Lentiviral sgRNA Non-targeting Controls

Edit-R All-in-one lentiviral sgRNA non-targeting controls

All-in-one non-targeting constructs are bioinformatically designed and validated to not target any gene in human or mouse genomes.

These non-targeting controls should be used to establish experimental baselines and to distinguish sequence-specific biological effects from non-specific effects. Available as high-titer purified lentiviral particles and glycerol stock formats.

CRISPR-Cas9 gene editing experiments require the use of negative control samples in order to accurately distinguish biological effects of sgRNA-induced editing of targeted genomic DNA from non-specific effects.

Edit-R All-in-one lentiviral sgRNA non-targeting controls are:

CRISPR guide RNA sequences and Cas9 cloned into the same optimized lentiviral expression backbone as Edit-R All-in-one gene targeting sgRNAs
Available as glycerol stocks and as purified, concentrated lentiviral particles (50 µL at ≥1 x 10⁷ TU/mL)
Designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in the human or mouse genomes.

Five different negative control CRISPR RNA sequences are available. Due to the biological variability inherent in different cells, time points, assays and conditions, we recommend testing at least two non-targeting sgRNAs to identify the best negative control for establishing an effective baseline for your particular experiment.

*Bulk volumes of all-in-one lentiviral sgRNA control particles are available upon request

Edit-R All-in-one lentiviral system percent editing (TIDE)

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.

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 system, wild-type cells were transduced with the All-in-one were selected with 2 µg/mL puromycin for 3 days. For the All-in-one 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 Puro^R 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.

Schematic diagram of the Edit-R All-in-one lentiviral sgRNA vector

In the Edit-R All-in-one lentiviral sgRNA vector backbone, the gene-specific guide RNA is expressed under the control of a human U6 promoter, while expression of the Cas9 and puromycin resistance marker (Puro^R) or EGFP marker is driven from either the human EF1α or the mouse CMV promoter. The plasmid contains the Amp^R resistance marker for growth and selection in E. coli.

Vector Element	Utility
Cas9	Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with a sgRNA
T2A	Self-cleaving peptide allows for simultaneous expression of puromycin resistance and Cas9 protein from a single transcript
Puro^R	Puromycin resistance marker permits antibiotic selection of transduced mammalian cells
EGFP	Fluorescent marker permits FACS selection of transduced mammalian cells
mCMV	Mouse cytomegalovirus immediate early promoter
hEF1α	Human elongation factor 1 alpha short promoter
U6	Human RNA polymerase III promoter U6
sgRNA	Optimized single guide RNA, a fusion of gene-specific crRNA with the tracrRNA scaffold
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
Amp^R	Ampicillin resistance gene for vector propagation in E. coli cultures