Fig 1.
Sequences of hU6, T7 and U6T7 hybrid promoters.
Sequence comparison of the hU6, T7 and the two bifunctional U6T7 and U6T7G hybrid promoters. (a) human U6 promoter (from plasmid pX330), (b) bacteriophage T7 promoter (from plasmid pDR274), (c) hU6T7 hybrid promoter containing both the human U6 and T7 bacteriophage promoters, (d) U6T7G hybrid promoter with the A nucleotide removed from the +3 position (e) U6T7 hybrid promoter with C24 sequence, (f) U6T7G hybrid promoter lacking a G nucleotide at the +2 position with C24 sequence, (g) pX330 based hU6 promoter with C24 sequence and GG at the +1 /+2 position, (h) pX330 based hU6 promoter with C24 sequence, (i) pDR274 T7 promoter with C24 sequence. TATA marks the hU6 TATA box; +1 marks the transcription start site (G); double underlined sequence (a-d) are 4 base pair cloning overhangs generated by restriction endonuclease BsaI in T7-based and U6T7 vectors or restriction endonuclease BbsI in hU6-based expression vectors. Recognition sites for BsaI and BbsI are indicated in bold; single underlined sequence, C24 target template.
Fig 2.
gRNA expressed from the U6T7 hybrid promoter creates INDELs in mouse 3T3 cells.
Expression vectors containing the pU6T7 hybrid promoter as well as vectors containing only a T7 promoter (pDR274) or hU6 promoter (pX330) were transfected into NIH-3T3 cells. Vectors containing the ROSA26 target sequence are: pU6T7-C24, pU6T7G-C24, pDR274-C24, pX330-C24 G, pX330-C24 GG. Vectors serving as controls lack the C24 sequence (pU6T7, pU6T7G, pDR274, and pX330). The pU6T7 and pDR274 vectors were cotransfected with a CAS9 plasmid whereas the pX330 vector contains its own CAS9 gene expressed via the CMV promoter. The CAS9 expression vector used for co-transfections is the hCAS9 plasmid. M, 100 bp ladder marker (NEB). + and–indicate the presence and absence of the components in each plasmid vector, respectively,. Boxed indicators represent 3T3 cells with INDELs detected at the ROSA26 locus. Arrows indicate T7 endonuclease cleavage products. Non-transfected cells were also assayed for INDELs (Lane 16).
Fig 3.
RNAs transcribed from the U6T7/U6T7G hybrid promoter.
Lane 1: DR274; Lane 2: DR274-C24; Lane 3: pU6T7, Lane 4: pU6T7-C24; Lane 5: pU6T7G; Lane 6: pU6T7G-C24. M, RNA size marker, 150 nt and 80 nt. Each lane contains 2.5 ul of purified in vitro transcribed RNA (50 ul total elution volume).
Fig 4.
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.
Table 1.
Frequency of INDEL formation in mice using gRNA synthesized from pU6T7 expression vectors.