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-linked MECP2 locus, 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 autosomal EEF1A2 locus 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 the MECP2 locus 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.

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic

“…The procedure for culture and differentiation of the LUHMES cell line was previously described (18). To create two independent MECP2 knock-out lines, we used CRISPR-mediated gene disruption (30). To generate MeCP2 knock-downs, several shRNAs against MeCP2 were designed using Sigma-Aldrich Mission shRNA online software.…”

Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins

“…During this study we also established a new model for the study of T. gondii in human neurons differentiated from neuronal precursor LUHMES cells. Although for many hosts culturing is straightforward and which can differentiate into mature dopamine-like human neurons within one week (Lotharius et al, 2002;Scholz et al, 2011;Shah et al, 2016). This 360 LUHMES neuronal model could facilitate the study of fundamental questions regarding the unique interaction of T. gondii with neurons, and especially human neurons.…”

“…Lund Human Mesencephalic (LUHMES) cells were differentiated in vitro to morphologically and biochemically mature dopamine-like neurons (Lotharius et al, 2002;Scholz et al, 2011;Shah et al, 2016) and infected with the T. gondii lines GRA16-HA, GRA16-MeCP2 and GRA16-TFEB. All tested lines displayed clear secretion, export and accumulation of the tagged 245 protein in the nuclei of the neurons.…”

“…To test the level of MeCP2 rescue achieved by the transgenic T. gondii, we quantified MeCP2 delivery in MeCP2-deficient neurons. MeCP2 knock-out (MECP2-KO) LUHMES cells (Shah et al, 2016) were differentiated into mature human neurons and infected with GRA16-MeCP2 T. gondii. Nuclear levels of MeCP2 in 295 infected and non-infected neurons were quantified by immunofluorescence, and relative protein levels were calculated by comparison to endogenous MeCP2 in wild-type (WT) neurons.…”

“…Culturing and differentiation of LUHMES cells were carried according to Shah et al (Shah et al, 2016). Undifferentiated and differentiated LUHMES cells were grown in 75cm 2 flasks coated overnight with fibronectin+Poly-L-ornithine (1 μg/μl fibronectin and 43μg/ml PLO 550 in sterile distilled water).…”