title: Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders.
abstract: The recent identification of multiple new genetic causes of neurological disorders highlights the need for model systems that give experimental access to the underlying biology. In particular, the ability to couple disease-causing mutations with human neuronal differentiation systems would be beneficial. Gene targeting is a well-known approach for dissecting gene function, but low rates of homologous recombination in somatic cells (including neuronal cells) have traditionally impeded the development of robust cellular models of neurological disorders. Recently, however, CRISPR/Cas9 gene editing technologies have expanded the number of systems within which gene targeting is possible. Here we adopt as a model system LUHMES cells, a commercially available diploid human female mesencephalic cell line that differentiates into homogeneous mature neurons in 1-2 weeks. We describe optimised methods for transfection and selection of neuronal progenitor cells carrying targeted genomic alterations using CRISPR/Cas9 technology. By targeting the endogenous X-linkedMECP2locus, we introduced four independent missense mutations that cause the autism spectrum disorder Rett syndrome and observed the desired genetic structure in 3-26% of selected clones, including gene targeting of the inactive X chromosome. Similar efficiencies were achieved by introducing neurodevelopmental disorder-causing mutations at the autosomalEEF1A2locus on chromosome 20. Our results indicate that efficiency of genetic "knock-in" is determined by the location of the mutation within the donor DNA molecule. Furthermore, we successfully introduced an mCherry tag at theMECP2locus to yield a fusion protein, demonstrating that larger insertions are also straightforward in this system. We suggest that our optimised methods for altering the genome of LUHMES cells make them an attractive model for the study of neurogenetic disorders.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Ruth R
authors.identifier: 0000-0002-0284-8756
authors.initials: RR
authors.lastname: Shah
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Justyna
authors.initials: J
authors.lastname: Cholewa-Waclaw
authors.affiliation: Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
authors.forename: Faith C J
authors.initials: FCJ
authors.lastname: Davies
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Paton
authors.affiliation: Centre de Recherche, Institut Curie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 3215, Institut National de la Santé et de la Recherche Médicale U934, Paris, France.
authors.forename: Ronan
authors.initials: R
authors.lastname: Chaligne
authors.affiliation: Centre de Recherche, Institut Curie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 3215, Institut National de la Santé et de la Recherche Médicale U934, Paris, France.
authors.forename: Edith
authors.initials: E
authors.lastname: Heard
authors.affiliation: Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Abbott
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Adrian P
authors.initials: AP
authors.lastname: Bird
keywords: CRISPR; Cas9; EEF1A2; LUHMES; MeCP2; human; neurological; neurons