Journal: PLoS ONE
Article Title: A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs
Figure Lengend Snippet: gRNA produced by in vitro transcription from the U6T7 hybrid promoter directs cleavage of target DNA. gRNA were produced by in vitro transcription from the T7 promoter in the pU6T7 or the pDR274 vectors. The gRNAs, in the presence of CAS9 protein (NEB), cleaves the ROSA26 target site in vitro . The DNA template provided to test for CRISPR activity is a PCR product obtained from amplification of the ROSA26 gene locus. As a control, the mouse Fos gene amplicon was used as a template. Expression vectors with gRNAs containing the ROSA26 target sequence are: pU6T7-C24, pU6T7G-C24, pDR274-C24. Expression vectors lacking the ROSA26 template sequence are: pU6T7, pU6T7G, pDR274. Lanes 7 and 15 are the PCR amplicons containing ROSA26 and Fos gene target sequences, respectively. M, DNA size marker (NEB). Boxed vectors identify the transcribed gRNAs that produced INDELs at the target site after incubation with CAS9 protein. Arrows indicate the T7 endonuclease assay products.
Article Snippet: Construction of the U6T7 hybrid promoter The U6T7 hybrid promoter together with a gRNA scaffold was synthesized as a 373 bp product (Genscript) with the following sequence: AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGCTAATACGACTCACTA TAGG AGAGACCGAGAGAGGGTCTCA GTTT TAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTAAA The fusion fragment was placed into the pUC57-Kan vector (Genscript) at the XbaI cloning site and the plasmid was designated pU6T7.
Techniques: Produced, In Vitro, CRISPR, Activity Assay, Polymerase Chain Reaction, Amplification, Expressing, Sequencing, Marker, Incubation