sgRNA-shRNA Structure Design and Activity Verification

We have reported the novel Drosha-mediated sgRNA-shRNA structure for multiplex genome targeting in previous study (Yan et al., 2016). Here, the LIG4.shRNA-1, which showed the highest activity for silencing the porcine LIG4 gene in the preceding experiment, was used for the sgRNA-shRNA structure design. For further improvement, a pair of more efficient Drosha-processing sequences from miR-30 (Zeng and Cullen, 2005) were used to replace the former Drosha recognition sites. As designed in Figure 3A, the LIG4.shRNA sequence flanked by the Drosha-processing sequences (as shown in Supplementary Figure S3) was synthesized directly and inserted into the middle of two identical IGF2.sgRNA sequences. And then the IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA cassette was cloned into the pll3.7-mU6-CMV-hStCas9 vector (Xu et al., 2015a), generating the sgRNA-shRNA/StCas9 all-in-one expression vector, which was supposed capable for the simultaneous IGF2 gene targeting and LIG4 gene silencing.

sgRNA-shRNA structure design and activity verification. (A) The sgRNA-shRNA structure was designed with two identical sgRNAs targeting the IGF2 gene and one shRNA against the LIG4 gene as we previously did (Yan et al., 2016). (B) The schematic of the SSA-based single-fluorescent eGFP surrogate reporter. The DsRed expression cassette was removed from the dual-fluorescent DsRed-eGFP (RG) surrogate reporter to avoid the interference of the robust red fluorescence on the green fluorescence reporter. (C) Representative pictures and flow cytometric counting results of eGFP⁺ cells. Negative control: cells transfected with IGF2.eGFP reporter and single StCas9 expression vector; Positive control: cells transfected with linearized IGF2.eGFP reporter and single StCas9 expression vector. (D) Comparison of the sgRNA activities driven by IGF2.sgRNA/StCas9 and sgRNA-shRNA/StCas9 (n = 3, P = 0.265). The percentage of eGFP⁺ cells was used as an indirect measurement for the IGF2.sgRNA activity. (E) The relative expression of LIG4 gene down-regulated by different shRNAs or structures (n = 3, ^∗P < 0.05 compared with SC). SC, the non-specific shRNA control; Sg-SC, sgRNA-shRNA structure with non-specific shRNA control; Sg-Sh, sgRNA-shRNA structure with LIG4.shRNA-1; Sh-1, LIG4.shRNA-1.

Surrogate reporter assay was conducted to verify the sgRNA activity as described above. In brief, HEK293T cells were co-transfected with the IGF2.eGFP surrogate reporter and the sgRNA-shRNA/StCas9, the IGF2.sgRNA/StCas9, or the single StCas9 (pll3.7-mU6-CMV-hStCas9 with no sgRNA as the negative control) expression vector. The linearized IGF2.eGFP surrogate reporter, which was supposed to repair spontaneously in cells after the transfection, was used as the positive control to co-transfect the cells with the single StCas9 expression vector as we previously did (Xu et al., 2015a). After transfected for 2 days, the cells were photographed and cells from each parallel well were harvested independently for flow cytometric analysis. The percentage of eGFP positive cells was used as an indirect measurement for the IGF2.sgRNA activity.

In addition to the IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA (Sg-Sh) cassette, an IGF2.sgRNA-SC-IGF2.sgRNA (Sg-SC) cassette was generated by replacing the LIG4.shRNA-1 with the non-specific shRNA control. To verify the shRNA activity driven by the sgRNA-shRNA structure, the Sg-SC cassette, as well as Sg-Sh, was further cloned into the pLenti-H1 vector. Then, the pLenti-H1 based shRNA control (SC), LIG4.shRNA-1(Sh-1), Sg-SC and Sg-Sh expression vectors were used to transfect the PK15 cells, respectively, along with the pB-CBh-Puro vector. The transfected cells were enriched by puromycin selection for about 2 days as above, and then were harvested for detecting the relative expression of the LIG4 gene by qRT-PCR analysis.