oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.

Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1-60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.

Reliable multiplex generation of pooled induced pluripotent stem cells.

Reprogramming somatic cells into pluripotent stem cells (iPSCs) enables the study of systems in vitro. To increase the throughput of reprogramming, we present induction of pluripotency from pooled cells (iPPC)-an efficient, scalable, and reliable reprogramming procedure. Using our deconvolution algorithm that employs pooled sequencing of single-nucleotide polymorphisms (SNPs), we accurately estimated individual donor proportions of the pooled iPSCs. With iPPC, we concurrently reprogrammed over one hundred donor lymphoblastoid cell lines (LCLs) into iPSCs and found strong correlations of individual donors' reprogramming ability across multiple experiments. Individual donors' reprogramming ability remains consistent across both same-day replicates and multiple experimental runs, and the expression of certain immunoglobulin precursor genes may impact reprogramming ability. The pooled iPSCs were also able to differentiate into cerebral organoids. Our procedure enables a multiplex framework of using pooled libraries of donor iPSCs for downstream research and investigation of in vitro phenotypes.

Modeling of mitochondrial genetic polymorphisms reveals induction of heteroplasmy by pleiotropic disease locus 10398A>G.

Mitochondrial (MT) dysfunction has been associated with several neurodegenerative diseases including Alzheimer's disease (AD). While MT-copy number differences have been implicated in AD, the effect of MT heteroplasmy on AD has not been well characterized. Here, we analyzed over 1800 whole genome sequencing data from four AD cohorts in seven different tissue types to determine the extent of MT heteroplasmy present. While MT heteroplasmy was present throughout the entire MT genome for blood samples, we detected MT heteroplasmy only within the MT control region for brain samples. We observed that an MT variant 10398A>G (rs2853826) was significantly associated with overall MT heteroplasmy in brain tissue while also being linked with the largest number of distinct disease phenotypes of all annotated MT variants in MitoMap. Using gene-expression data from our brain samples, our modeling discovered several gene networks involved in mitochondrial respiratory chain and Complex I function associated with 10398A>G. The variant was also found to be an expression quantitative trait loci (eQTL) for the gene MT-ND3. We further characterized the effect of 10398A>G by phenotyping a population of lymphoblastoid cell-lines (LCLs) with and without the variant allele. Examination of RNA sequence data from these LCLs reveal that 10398A>G was an eQTL for MT-ND4. We also observed in LCLs that 10398A>G was significantly associated with overall MT heteroplasmy within the MT control region, confirming the initial findings observed in post-mortem brain tissue. These results provide novel evidence linking MT SNPs with MT heteroplasmy and open novel avenues for the investigation of pathomechanisms that are driven by this pleiotropic disease associated loci.