pcw cas9 tet on vector  (Addgene inc)

Journal: Life Medicine

Article Title: A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator of human naïve pluripotency and totipotency

doi: 10.1093/lifemedi/lnad037

Figure Lengend Snippet: Figure 1. CRISPR kinome KO screen identified novel targets that increase human EPS cells’ competence of acquiring authentic human naïve state. (A) Schematics of CRISPR knockout screen with kinome library under naïve or extended pluripotent conditions. (B) Plasmid design of iCas9 expression with BCL2 and Neomycin resistance. iCas9, induced Cas9. (C) Quantitative polymerase chain reaction (q-PCR) analysis of the expression of iCas9 in hEPSC-iCas9-hBCL2-Neo cell line upon Dox treatment. N = 2 biological replicates. Error bar indicates standard error of the mean (SEM). (D) The distribution diagram of the initial sgRNA library read counts frequency. (E) Representative flow cytometry sorting of SUSD2 positive cells during kinome KO screen under XF-LCDM condition. Control hEPSCs without SUSD2-phycoerythin (PE) staining (left), control hEPSCs with SUSD2-PE staining (middle), and kinome KO EPSCs with SUSD2-PE staining (right) are presented. The percentage of SUSD2-PE positive cells is shown. This experiment was repeated at least three times. (F) Kinases targeted by positively enriched sgRNAs in cells at passage 6 under naïve pluripotent condition 5i/LA (left) and PXGL (right). Top enriched genes and commonly enriched genes under two conditions are shown. (G) Kinases targeted by positively enriched sgRNAs in sorted SUSD2 positve cells at passage 3 under XF-LCDM condition. Top enriched genes are shown.

Article Snippet: Plasmid design and construction For plasmid PB-iCas9-hBCL2-CMV-Rtta-Neo, the Cas9 sequence was obtained from pAAVS1-tet-iCas9-BFP2 vector (addgene#125519).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Control, Staining

Journal: Life Medicine

Article Title: A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator of human naïve pluripotency and totipotency

doi: 10.1093/lifemedi/lnad037

Figure Lengend Snippet: Figure 5. Graphic summary. We performed a comprehensive CRISPR/Cas9-based kinome knockout screen with different strategies on hEPSCs and identified new kinases and related signaling pathways that regulate human naïve pluripotency. Chemical validation of these newly identified kinases revealed that inhibition of ErbB signaling not only promoted the induction of human naïve pluripotency but also human totipotency in vitro.

Article Snippet: Plasmid design and construction For plasmid PB-iCas9-hBCL2-CMV-Rtta-Neo, the Cas9 sequence was obtained from pAAVS1-tet-iCas9-BFP2 vector (addgene#125519).

Techniques: CRISPR, Knock-Out, Protein-Protein interactions, Biomarker Discovery, Inhibition, In Vitro