Biology subjects

Lian, X. L.

Publications and source records attributed to Lian, X. L..

3 recordsLinked to original sources

bioRxiv2023

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Precise insertion of a fluorescent protein into a lineage-specific gene in human pluripotent stem cells (hPSCs) presents challenges due to the low knockin efficiency and difficulties in selecting the correctly targeted cells. Here we introduce the ModRNA-based Activation for Gene Insertion and Knockin (MAGIK) approach to enhance knockin efficacy in hPSCs. MAGIK operates in two steps: first, it employs a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug-selection cassette) to significantly enhance efficiency; second, a dCas9 activator modRNA and a dgRNA are used to temporarily activate the successfully targeted gene, allowing for live cell sorting without single cell cloning. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single cell colony screening, expediting precise genetic integration. We have demonstrated that MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5 and PDX1, across multiple hPSC lines, showcasing its robustness. This innovative MAGIK approach streamlines the process and provides a promising solution for targeted genetic modifications in hPSCs.

bioengineering↗

bioRxiv2022

STAMP-Based Digital CRISPR-Cas13a (STAMP-dCRISPR) for Amplification-Free Quantification of HIV-1 Plasma Viral Load

The development of new nucleic acid techniques to quantify HIV RNA in plasma is critical for identifying the disease progression and monitoring the effectiveness of antiretroviral therapy. While RT-qPCR has been the gold standard for HIV viral load quantification, digital assays could provide an alternative calibration-free absolute quantification method. Here, we report the development of a self-digitalization through automated membrane-based partitioning (STAMP) technique to digitalize the CRISPR-Cas13 assay (dCRISPR) for amplification-free and absolute quantification of HIV-1 viral RNAs. The analytical performances of STAMP-dCRISPR were evaluated with synthetic HIV-1 RNA, and it was found samples spanning 4 orders of dynamic range between 100 aM to 1 pM can be quantified as fast as 30 min. We also examined the overall assay from RNA extraction to STAMP-dCRISPR quantification with spiked plasma samples. The overall assay showed a resolution of 42 aM at a 90% confidence level. Finally, a total of 20 clinical plasma samples from patients were evaluated with STAMP-dCRISPR. The obtained results agreed well with the RT-qPCR. Our result demonstrates a new type of easy-to-use, scalable, and highly specific digital platform that would offer a simple and accessible platform for amplification-free quantification of viral RNAs, which could be exploited for the quantitative determination of viral load for an array of infectious diseases.

bioengineering↗

bioRxiv2021

Direct induction of hemogenic endothelial progenitors from hPSCs by defined factors revealed by single-cell transcriptome analysis

Transcription factors (TFs) play critical roles in stem cell maintenance and differentiation. Using single cell RNA sequencing, we investigated TFs expressed in hemogenic endothelial (HE) progenitors differentiated from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the HE population. To test whether overexpression of these factors increases HE differentiation efficiency, we established inducible hPSC lines and found only SOX17 improved differentiation. Temporal expression analysis further revealed SOX17 was turned on immediately before VE-Cadherin, indicating SOX17 may be a causative factor for HE differentiation. Upon SOX17 knockdown via CRISPR-Cas13d, HE differentiation was significantly abrogated. Strikingly, we discovered SOX17 overexpression alone is sufficient to generate more than 50% CD34+VE-cadherin+CD73- cells that could be directed to hematopoietic progenitors, which emerged via an endothelial-to-hematopoietic transition and significantly upregulated definitive hematopoietic transcriptional programs. Functional assays showed that these progenitors can differentiate into blood cells from multiple lineages. Our analyses reveal an uncharacterized function of SOX17 in directing hPSCs differentiation towards HE cells. Significance StatementHemogenic endothelial (HE) cells have been generated from human pluripotent stem cells (hPSCs) to study blood development. However, their full transcriptomic characterization and key genes involving in directing HE differentiation is unclear. Utilizing single cell RNA-seq analysis, we find that SOX17 is solely expressed in HE cells and is also required for HE differentiation. Strikingly, we find that overexpression of SOX17 alone is sufficient to program hPSCs into CD34+VE-cadherin+CD73-HE cells, which could further differentiate into blood progenitors. Our research reveals that SOX17 is sufficient to direct hPSCs differentiation to HE cells. ClassificationPhysical Sciences/Engineering; Biological Sciences/Cell Biology.

bioengineering↗