Edit-R All-in-one lentiviral sgRNA negative controls

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Gene editing
Gene editing reagents
Edit-R All-in-one lentiviral sgRNA negative controls

Edit-R All-in-one lentiviral sgRNA negative 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.

AAVS1/Rosa26 cutting controls are designed to target regions of the genome that do not result in functional knockout of a protein or known phenotypic changes. These regions are sometimes known as safe harbor regions. These guides are validated for mismatch detection assays and are recommended for screening applications as an additional control that results in Cas9 cutting without a phenotypic readout.

Non-targeting controls are recommended as negative controls for experiments using lentiviral sgRNA. All Edit-R non-targeting controls are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in the human or mouse genomes. Changes in viability or gene expression levels in cells treated with these controls likely reflect a baseline cellular response that can be compared to the levels in cells treated with target-specific sgRNAs.

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

Our neutral cutting controls target an inactive region of the genome to provide a quantitative measure of cutting efficiency by mismatch detection assays, while maintaining a negative or negligible phenotype.

The neutral cutting controls target safe harbor genomic loci: human AAVS1 located on chromosome 19 and mouse Rosa26 on chromosome 6. Safe harbor loci are well-characterized, widely accepted, inactive genomic regions.

How is the neutral cutting control different from the non-targeting controls we offer?

Our non-targeting control guide RNAs do not target any region in the genome. Non-targeting designs control for phenotypic changes that may be caused by the delivery of the CRISPR-Cas9 reagents in the absence of cutting. However, the non-targeting controls cannot indicate changes caused by Cas9 cutting because they do not result in cutting by the Cas9 enzyme.

Our positive controls target established housekeeping genes to offer insight into phenotypic changes that might be due to Cas9 cutting but are not specific to cutting within the gene of interest. However, there may be cell types or culture conditions under which cutting at the housekeeping gene locus could result in a phenotype, or experimental situations where disrupting a gene is not desired. To overcome this potentially confounding effect, the neutral cutting control can be included to provide more robust results and increased confidence in the phenotypes observed following editing of the target gene.

Neutral cutting controls can be helpful if there is concern that an observed phenotype is not gene-specific. In particular, neutral cutting controls are helpful as an additional control in screening applications and when a phenotype is observed with a positive control guide RNA. Additionally, this guide can be used in discovery settings where disruption of a gene could affect downstream phenotypes and results.