Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells
Huaigeng Xu, Yuto Kita, Uikyu Bang, Peter Gee, Akitsu Hotta
Abstract
Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).
Topics & Concepts
CRISPRGenome editingInduced pluripotent stem cellCas9Computational biologyRibonucleoproteinGuide RNABiologyCell biologyGeneticsEmbryonic stem cellRNAGeneCRISPR and Genetic EngineeringPluripotent Stem Cells ResearchNeuroscience and Neural Engineering