CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-0716-2301-5_6 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Optimizing approaches for targeted integration of transgenic cassettes by integrase-mediated cassette exchange in mouse and human stem cells.
Stem Cells
January 2025
Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
To enable robust expression of transgenes in stem cells, recombinase-mediated cassette exchange at safe harbor loci is frequently adopted. The choice of recombinase enzyme is a critical parameter to ensure maximum efficiency and accuracy of the integration event. We have explored the serine recombinase family of site-specific integrases and have directly compared the efficiency of PhiC31, W-beta, and Bxb1 integrase for targeted transgene integration at the Gt(ROSA)26Sor locus in mouse embryonic stem cells.
View Article and Find Full Text PDFGeneration of an inducible dCas9-SAM human PSC line for endogenous gene activation.
Front Cell Dev Biol
November 2024
Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain.
The CRISPR/Cas9 system has transformed genome editing by enabling precise modifications for diverse applications. Recent advancements, including base editing and prime editing, have expanded its utility beyond conventional gene knock-out and knock-in strategies. Additionally, several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains have been developed to modulate endogenous gene expression when directed to their regulatory regions by specific single-guide RNAs.
View Article and Find Full Text PDFMultiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1.
Bio Protoc
November 2024
Department of Cell and Developmental Biology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from ) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs.
View Article and Find Full Text PDFCIRCLE-Seq for Interrogation of Off-Target Gene Editing.
J Vis Exp
November 2024
Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus; Gates Institute, University of Colorado School of Medicine, Anschutz Medical Campus;
Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing.
View Article and Find Full Text PDF-targeted, stable expression of ChR2 in human brain organoids for consistent optogenetic control.
Bioeng Transl Med
November 2024
School of Biological Sciences, College of Natural Sciences, Seoul National University Seoul Republic of Korea.
Self-organizing brain organoids provide a promising tool for studying human development and disease. Here we created human forebrain organoids with stable and homogeneous expression of channelrhodopsin-2 (ChR2) by generating safe harbor locus-targeted, ChR2 knocked-in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2-expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non-virus mediated, spatiotemporal optogenetic control of neural activities.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!