The CRISPR-Cas9 system shows diverse levels of genome editing activities on eukaryotic chromatin, and high-efficiency sgRNA targets are usually desired in application. In this study, we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing. However, the strategy that increases chromatin openness by fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR-Cas9 targets in Lenti-X 293T cells. Under the enlightenment that histone acetylation increases eukaryotic chromatin accessibility, we developed a composite strategy to further improve genome editing by activating histone acetylation. We demonstrate that promoting histone acetylation using the histone acetyltransferase activator YF-2 can improve the genome editing by Cas9 and, more robustly, by the Cas9 transcriptional activator (Cas9-AD). This strategy holds great potential to enhance CRISPR-Cas9 genome editing and to enable broader CRISPR gRNA target choices for experiments in eukaryotes.

Increasing Genome Editing Efficiency of Cas9 Nucleases by the Simultaneous Use of Transcriptional Activators and Histone Acetyltransferase Activator.

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

https://www.frontiersin.org/articles/10.3389/fbioe.2022.930966/pdf

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

Transcriptional dysregulation caused by genomic and epigenetic alterations in cancer is called “transcriptional addiction”. Transcriptional addiction is an important pathogenic factor of tumor malignancy. Hepatocellular carcinoma (HCC) genomes are highly heterogeneous, with many dysregulated genes. Our study analyzed the possibility that transcriptional addiction-related genes play a significant role in HCC. All data sources for conducting this study were public cancer databases and tissue microarrays. We identified 38 transcriptional addiction genes, and most were differentially expressed genes. Among patients of different groups, there were significant differences in overall survival rates. Both nomogram and risk score were independent predictors of HCC outcomes. Transcriptional addiction gene expression characteristics determine the sensitivity of patients to immunotherapy, cisplatin, and sorafenib. Besides, HDAC2 was identified as an oncogene, and its expression was correlated with patient survival time. Our study conclusively demonstrated that transcriptional addiction is crucial in HCC. We provided biomarkers for predicting the prognosis of HCC patients, which can more precisely guide the patient’s treatment.

/pdf/profiling-and-integrated-analysis-of-transcriptional-2uqz3amh.pdf

Profiling and integrated analysis of transcriptional addiction gene expression and prognostic value in hepatocellular carcinoma

Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

/pdf/gpi-anchored-glutathione-s-transferase-as-marker-allows-2hbsmafa.pdf

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells

Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.

Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.

Histone deacetylase 2 regulates STAT1-dependent upregulation of atypical chemokine receptor 3 to induce M2 macrophage migration and immune escape in hepatocellular carcinoma.

Xiaodan Liu

Papers

Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.

Histone deacetylase 2 regulates STAT1-dependent upregulation of atypical chemokine receptor 3 to induce M2 macrophage migration and immune escape in hepatocellular carcinoma.