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transEDIT CRISPR/Cas9

Lentiviral Vectors | Flexible Markers | Tet-Inducible Option

Ready-to-go CRISPR Kits for > 65,000 Genes covering the human, mouse and rat genomes

  • All-in-one or single guide RNA delivery - including inducible Cas9
  • Multiple vectors to enable dual or triple selection for enhanced efficiency
  • Single or paired guide RNA CRISPR/Nickase strategies

transEDIT CRISPR/Cas9 lentiviral reagents provide powerful tools for genome editing, offering optimized gRNA designs cloned into a choice of expression vectors and formats for engineering specific gene knockouts. The vectors are designed to select for high gene editing efficiency using direct indicators of Cas9 expression from fluorescent or selection markers as well as efficient delivery as plasmid or lentiviral particles. In addition, complementary markers for co-selection of Cas9 and gRNA ensure efficient expression of both components necessary for Cas9 based genome editing. transEDIT reagents include lentiviral expression vectors containing specific gRNA targeting your gene of interest in various formats:

  • Single gRNA plus Cas9 on one vector (all-in-one)
  • Single/paired gRNA vectors for co-delivery with a separate Cas9 nuclease/nickase vector (two-vector system)

All transEDIT sets are available as glycerol stocks or as lentiviral particles.


transEDIT Vectors

Functional evaluation of targeted DNA cleavage by transEDIT™ CRISPR/CAS9

The percentage of indel frequency as a measure of targeting efficiency can be determined using the Surveyor assay (Guschin et al., 2010). The mutations introduced by the CRISPR Cas9 reagents result in sequence differences between the chromosomes. PCR amplification of the targeted area amplifies both alleles. Amplicons consisting of one strand from each allele have a bulge where the sequences differ. Treating the PCR product with nuclease digests the amplicon into two fragments. Using a bioanalyzer (or agarose gel) the percent of digested product and percent of cells with indels can be estimated.

gDNA extracted from cells transduced with transEDIT lentiviral vectors (and 1 week of puromycin selection) was amplified with a high-fidelity polymerase and subsequently assayed with Surveyor nuclease. Cas9-mediated cleavage efficiency (indel percent) was calculated based on fraction of cleaved DNA as detected by Bioanalyzer, High Sensitivity DNA Assay. Indel frequency for targeted loci are reported.

CRISPR/Cas9

Surveyor assay for indel frequency analysis.
(A) HEK293T cells stably expressing Cas9 were transduced with gRNA sequence(s) targeting DYRK1A and Irak4.
(B) HEK293T cells transduced with All-in-One (gRNA plus Cas9) targeting TP53. (* denotes expected fragment sizes)

High Frequency InDels by Enriching for High Cas9 Expression with Fluorescent Markers

The level of Cas9 expression has been shown to impact the efficiency of indel creation. Duda et al. showed that vectors with fluorescent markers tied to Cas9 expression through a 2A peptide allow the selection of the high Cas9 expressing cells through FACS analysis. Similar selection strategies can be employed with transEDIT Cas9 expression vectors.

Supporting Data II

Red and Green fluorescent marker expression was tied directly to Cas9 nuclease or nickase expression.
The 2A peptide tag ensures that the Cas9 and marker proteins are expressed at equimolar amounts. Cells were electroporated and cultured for 72 hours. FACS analysis was used to separate the heterogenous populations of cells into groups with low, medium or high fluorescence expression. A, VEGFA was targeted with a vector expressing a single gRNA and Cas9 nuclease linked to GFP via a 2A peptide. The population with the highest fluorescence had approximately 10-fold more cells with an indel mutation detectable by the Surveyor assay. B, two vectors expressing both nickase and a gRNA targeting EMX1 were also tested in combination with a single-stranded donor oligonucleotides (ssODN) that would introduce an EcoRV restriction site. Comparing digestion from EcoRV versus surveyor assay showed that both homologous recombination and nonhomologous end joining were increased significantly in the high expressing population. (Figure adapted from Duda et al, 2014.)

References

  • Charpentier & Doudna, 2013. Biotechnology: Rewriting a genome. Nature. 2013 Mar 7;495(7439):50-1.
  • Cong et al. 2013. Multiplex Genome Engineering Using CRISPR/Cas Systems. Vol. 339 no. 6121 pp. 819-823.
  • Duda et al. 2014. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Res. Jun 1, 2014; 42(10): e84.
  • Guschin et al. 2010. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol. 649:247-56.
  • Hsu et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology 31, 827–832 (2013).
  • Jinek et al. 2012. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 17 August 2012: Vol. 337 no. 6096 pp. 816-821.
  • Ran et al. 2013. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 154, 1380–1389.
  • Wang et al. 2013. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153: 910–918.