The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive, and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell-types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species including humans. Here we describe a novel recombinant adeno-associated virus (rAAV) that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABA-ergic function in virtually any vertebrate species.

Summary Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered sub-cellular transport as well as activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates at least a subset of these changes are more broadly conserved in ALS.

SummaryMicroglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or β-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer’s disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.

To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number. STEM CELLS 2006;24:125-138

Alzheimer’s disease (AD), the most common form of dementia, is recognized as a heterogeneous disease with diverse pathophysiologic mechanisms. In this study, we interrogate the molecular heterogeneity of AD by analyzing 1543 transcriptomes across five brain regions in two AD cohorts using an integrative network approach. We identify three major molecular subtypes of AD corresponding to different combinations of multiple dysregulated pathways, such as susceptibility to tau-mediated neurodegeneration, amyloid-β neuroinflammation, synaptic signaling, immune activity, mitochondria organization, and myelination. Multiscale network analysis reveals subtype-specific drivers such as GABRB2, LRP10, MSN, PLP1, and ATP6V1A. We further demonstrate that variations between existing AD mouse models recapitulate a certain degree of subtype heterogeneity, which may partially explain why a vast majority of drugs that succeeded in specific mouse models do not align with generalized human trials across all AD subtypes. Therefore, subtyping patients with AD is a critical step toward precision medicine for this devastating disease.