Biology subjects

Haideri, T.

Publications and source records attributed to Haideri, T..

2 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

Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells

CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity to explore the application of genome editing in human pluripotent stem cells (hPSCs). Due to the inherent simplicity of CRISPR systems, requiring a Cas protein and its corresponding single guide RNA (sgRNA), they are more widely adopted and used for diverse biomedical research than their predecessors (zinc finger nucleases and TALENs). However, a bottleneck of applying CRISPR systems in hPSCs is how to deliver CRISPR effectors easily and efficiently into hPSCs. Herein, we developed modified mRNA (modRNA) based CRIPSR systems that utilized Cas9 or base editor (ABE8e) modRNA for genome editing of hPSCs via simple lipid-based transfection. We have achieved 71.09% {+/-} 9.13% and 68.53% {+/-} 3.81% gene knockout (KO) efficiency with Cas9 modRNA and ABE8e modRNA, respectively, which is significantly higher than plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA based CRISPR methods hold great promise as the more efficient and accessible techniques for genome editing of hPSCs.

bioengineering↗