bicistronic expression vector px330 cas9  (Addgene inc)

Journal: Developmental cell

Article Title: Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract.

doi: 10.1016/j.devcel.2019.01.005

Figure Lengend Snippet: Figure 7. LGR5 Deletion in Human ESCs Abrogates ISL1 Induction and Cono-ventriculogenesis (A) Schema of the strategy for construction of human LGR5-KO ESCs using the CRISPR/Cas9 system. (B) Human ESCs (ESO3) were transiently co-transfected with pX330-gRNA1 and pX330-gRNA2. The panel indicates the genotyping of the transduced ESC clones, showing both homologous LGR5-KO clones (#1 and #2) and hetero-knockout clones (#3, #4, and #5). (C) RT-PCR confirmed the absence of LGR5 mRNA expression of the LGR5-KO ESC line-derived cells on day 3 in cardiac differentiation. (D) Human LGR5-KO ESCs exhibited their impaired cardiogenic phenotype. LGR5 deletion did not affect MESP1 expression but significantly attenuated LEF1 and ISL1 expression in the cardiac differentiating cells on day 3 and day 6, followed by lower CM (TNNT2+) induction ratios than in WT ESC-derived cells on day 6, 10, and 18. *p < 0.05 and **p < 0.01 between WT and LGR5-KO ESC-derived cells. (E) Among the induced ISL1+Lineage+ intermediate populations on day 6, the percentage and cell number of only the ISL1+TNNT2+ population were significantly decreased in the LGR5-KO than in WT ESC-derived cells, whereas other intermediate populations (ISL1+HCN4+, ISL1+SMMHC+, and ISL1+CD31+) were not affected. (F) The comparison of the CVM- and FVM-enriched genes’ (Figure 5B; Table S3) expression in between WT and LGR5-KO ESC-derived beating CMs harvested on day 14, analyzed by quantitative RT-PCR (qPCR). **p < 0.01 between WT and LGR5-KO ESC-derived cells. (G) qPCR gene expression heatmap of representative genes related to mesodermal and cardiac differentiation in various time stages between WT and LGR5-KO ESC-derived cells. (H) A top panel shows a novel and putative MESP1 binding site on the human LGR5 promoter locus and indicates the sequence divergences of the region among several mammalian species. Chromatin immunoprecipitation (ChIP) assays demonstrated that recruitment of MESP1 onto the above binding site on the LGR5 promoter was augmented on day 3 in cardiac differentiation (arrows, bottom). This recruitment was not affected by LGR5 deletion. **p < 0.01, NS, not significant. (I) A top panel shows a novel and putative LEF1 binding site on the human ISL1 promoter locus and indicates the sequence divergences of the region among several mammalian species. ChIP assays demonstrated that recruitment of LEF1 onto the above binding site on the ISL1 promoter was augmented on day 3 in cardiac differentiation (arrows, bottom); however, this recruitment was significantly prevented by LGR5 deletion. **p < 0.01. (D, E, F, H, and I) Error bars represent SEM. See also Figures S6 and S7.

Article Snippet: The following sequences were selected as two single guide-RNAs (sgRNA) which target the first exon of LGR5 gene locus and have minimal off-target activity, using the CHOPCHOP software (http://chopchop.cbu.uib.no/): sgRNA1 (genomic location: Chr12, 71,440,110-71,440,091), 5’-CAGGAGCACACCGAGCCGGGAGG-3’ and sgRNA2 (genomic location: Chr12, 71,440,308- 71,440,289), 5’- GTGGGGAAGTACTTACAGGTAGG-3’ (Figure 7A). sgRNAs were cloned into a bicistronic expression vector pX330 expressing S. pyogenes Cas9 (Addgene, #42230), following previously published protocols (Cong et al., 2013).

Techniques: CRISPR, Transfection, Clone Assay, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Comparison, Quantitative RT-PCR, Gene Expression, Binding Assay, Sequencing, Chromatin Immunoprecipitation