Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9

Methods : a Companion to Methods in Enzymology
Saniye YumluRalf Kühn

Abstract

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.

Citations

Apr 8, 2018·Applied Microbiology and Biotechnology·Maciej GrzybekPaweł Lisowski
Nov 6, 2018·Human Genetics·Svetlana A SmirnikhinaAlexander V Lavrov
Jan 19, 2020·Disease Models & Mechanisms·Viola Volpato, Caleb Webber
Apr 23, 2020·Current Psychiatry Reports·Debamitra DasDimitrios Avramopoulos
Jul 22, 2019·International Journal of Molecular Sciences·Serge M RozovElena V Deineko
Sep 22, 2017·JCI Insight·Viktoriia KyrychenkoEric N Olson
Dec 30, 2020·Stem Cell Research·Johanna SiehlerIngo Burtscher
Mar 23, 2021·Biotechnology Progress·Michael VitelliMelih Tamer
Aug 19, 2021·The CRISPR Journal·Alireza ShahryariHeiko Lickert
Dec 22, 2021·The EMBO Journal·Hector Flores-RomeroAna J Garcia-Saez

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