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Genome Editing08:31

Genome Editing

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage,...
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Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing09:03

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells...
4.6K
Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation09:51

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.
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Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development09:37

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive β-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease...
13.5K
Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information09:37

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing...
10.3K
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model...
35.5K