A Dual-Targeting Delivery System for Effective Genome Editing and In Situ Detecting Related Protein Expression in Edited Cells

One of critical steps in genome editing by CRISPR-Cas9 is to deliver the CRISPR-Cas9 system into targeted cells. In this study, we developed a dual-targeting delivery system based on polymer/inorganic hybrid nanoparticles to realize highly efficient genome editing in targeted tumor cells as well as in situ detection on the related protein expression in edited cells. The CRISPR-Cas9 plasmid for CDK11 knockout was encapsulated in the core of the delivery system composed of protamine sulfate, calcium carbonate, and calcium phosphate by coprecipitation, and functional derivatives of carboxymethyl chitosan (biotinylated carboxymethyl chitosan with biotin ligands and aptamer-incorporated carboxymethyl chitosan with AS1411 ligands) were decorated on the nanovector surface by electrostatic interactions to form the dual-targeting delivery system. On the basis of the tumor cell targeting capability of biotin and AS1411 ligands as well as the nuclear targeting of AS1411, the dual-targeting system can deliver the CRISPR-Cas9 plasmid into the nuclei of tumor cells to realize highly efficient genome editing, resulting in a dramatic decrease (>90%) in CDK11 protein together with the significant downregulation of other proteins involved in tumor development, including an ∼90% decrease in MMP-9, >40% decrease in VEGF, and ∼70% decrease in survivin. Using the same vector, molecular beacons can be easily delivered to edited cell nuclei to in situ detect the mRNA level of related proteins (p53 and survivin as typical examples) and mRNA distribution in subcellular organelles. Our strategy can realize effective genome editing and in situ detection on related protein expression simultaneously

High ABCG4 Expression Is Associated with Poor Prognosis in Non-Small-Cell Lung Cancer Patients Treated with Cisplatin-Based Chemotherapy

<div><p>ATP-binding cassette (ABC) transporters are associated with poor response to chemotherapy, and confer a poor prognosis in various malignancies. However, the association between the expression of the ABC sub-family G member 4 (ABCG4) and prognosis in patients with non-small-cell lung cancer (NSCLC) remains unclear. NSCLC tissue samples (n = 140) and normal lung tissue samples (n = 90) were resected from patients with stage II to IV NSCLC between May 2004 and May 2009. ABCG4 mRNA and protein expressions were detected by RT-PCR, western blot, and immunohistochemistry. Patients received four cycles of cisplatin-based post-surgery chemotherapy and were followed up until May 31^st, 2014. ABCG4 positivity rate was higher in NSCLC than in normal lung tissues (48.6% <i>vs</i>. 0%, <i>P</i><0.001) and ABCG4 expression was significantly associated with poor differentiation, higher tumor node metastasis (TNM) stage, and adenocarcinoma histological type (all <i>P</i><0.001). Univariate (HR = 2.284, 95%CI: 1.570–3.324, <i>P</i><0.001) and multivariate (HR = 2.236, 95%CI: 1.505–3.321, <i>P</i><0.001) analyses showed that ABCG4 expression was an independent factor associated with a poor prognosis in NSCLC. Patients with ABCG4-positive NSCLC had shorter median survival than ABCG4-negative NSCLC (20.1 <i>vs</i>. 43.2 months, <i>P</i><0.001). The prognostic significance of ABCG4 expression was apparent in stages III and IV NSCLC. In conclusion, high ABCG4 expression was associated with a poor prognosis in patients with NSCLC treated with cisplatin-based chemotherapy.</p></div

Correlation between ABCG4 expression status and prognosis of NSCLC patients treated with cisplatin-based chemotherapy.

<p>(A) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients based on ABCG4 expression (negative <i>vs</i>. positive). (B) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients based on tumor node metastasis (TNM) stage (II <i>vs</i>. III <i>vs</i>. IV). (C) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients based on differentiation (poorly <i>vs</i>. moderately/well). (D) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients with TNM stage II based on ABCG4 expression (negative <i>vs</i>. positive). (E) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients with TNM stage III based on ABCG4 expression (negative <i>vs</i>. positive). (F) Kaplan-Meier curves were plotted to determine cumulative survival rate of NSCLC patients with TNM stage IV based on ABCG4 expression (negative <i>vs</i>. positive). Negative:-; Positive: +, ++ and +++.</p

ABCG4 protein expression in NSCLC and normal lung tissues was detected by immunohistochemistry.

<p>(A) Normal lung tissue; (B) Negative (-) staining of ABCG4 in NSCLC tissue; (C) Weakly positive (+) staining of ABCG4 in NSCLC tissue; (D) Moderately positive (++) staining of ABCG4 in NSCLC tissue; (E) Strongly positive (+++) staining of ABCG4 in NSCLC tissue; (F) Immunohistochemistry showing ABCG4-negative staining in NSCLC tissues (++) using ABCG4 blocking peptide. Magnification: 200×.</p

ATP-binding cassette sub-family G member 4 (ABCG4) mRNA expression was detected in non-small-cell lung cancer (NSCLC) (n = 14) and normal lung (n = 2) tissues by reverse transcription-polymerase chain reaction (RT-PCR).

<p>β-actin was used as an internal reference. M: marker. (<b>A)</b> Lane 1: positive control (A549-ABCG4). Lanes 2–3: normal lung tissues (NORM) (n = 2); <b>(B)</b> NSCLC tissues (NSCLC) (n = 14, lanes 1–14).</p