Multiple lines of evidence implicate dysregulated microglia-mediated synaptic pruning in the pathophysiology of schizophrenia. In vitro human cellular studies represent a promising means of pursuing ...this hypothesis, complementing efforts with animal models and postmortem human data while addressing their limitations. The challenges in culturing homogeneous populations of cells derived from postmortem or surgical biopsy brain material from patients, and their limited availability, has led to a focus on differentiation of induced pluripotent stem cells. These methods too have limitations, in that they disrupt the epigenome and can demonstrate line-to-line variability due in part to extended time in culture, partial reprogramming, and/or residual epigenetic memory from the cell source, yielding large technical artifacts. Yet another strategy uses direct transdifferentiation of peripheral mononuclear blood cells, or umbilical cord blood cells, to microglia-like cells. Any of these approaches can be paired with patient-derived synaptosomes from differentiated neurons as a simpler alternative to co-culture. Patient-derived microglia models may facilitate identification of novel modulators of synaptic pruning and identification of biomarkers that may allow more targeted early interventions.
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases
has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)
. To address this limitation, we engineered an enhanced ...Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants
to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with ...frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.
Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation ...domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
The CYFIP1 gene, located in the neurodevelopmental risk locus 15q11.2, is highly expressed in microglia, but its role in human microglial function as it relates to neurodevelopment is not well ...understood.
We generated multiple CRISPR (clustered regularly interspaced short palindromic repeat) knockouts of CYFIP1 in patient-derived models of microglia to characterize function and phenotype. Using microglia-like cells reprogrammed from peripheral blood mononuclear cells, we quantified phagocytosis of synaptosomes (isolated and purified synaptic vesicles) from human induced pluripotent stem cell (iPSC)–derived neuronal cultures as an in vitro model of synaptic pruning. We repeated these analyses in human iPSC-derived microglia-like cells derived from 3 isogenic wild-type/knockout line pairs derived from 2 donors and further characterized microglial development and function through morphology and motility.
CYFIP1 knockout using orthogonal CRISPR constructs in multiple patient-derived cell lines was associated with a statistically significant decrease in synaptic vesicle phagocytosis in microglia-like cell models derived from both peripheral blood mononuclear cells and iPSCs. Morphology was also shifted toward a more ramified profile, and motility was significantly reduced. However, iPSC-CYFIP1 knockout lines retained the ability to differentiate to functional microglia.
The changes in microglial phenotype and function due to the loss of function of CYFIP1 observed in this study implicate a potential impact on processes such as synaptic pruning that may contribute to CYFIP1-related neurodevelopmental disorders. Investigating risk genes in a range of central nervous system cell types, not solely neurons, may be required to fully understand the way in which common and rare variants intersect to yield neuropsychiatric disorders.
Abstract Background The SARS-CoV-2 virus activates maternal and placental immune responses. Such activation in the setting of other infections during pregnancy is known to impact fetal brain ...development. The effects of maternal immune activation on neurodevelopment are mediated at least in part by fetal brain microglia. However, microglia are inaccessible for direct analysis, and there are no validated non-invasive surrogate models to evaluate in utero microglial priming and function. We have previously demonstrated shared transcriptional programs between microglia and Hofbauer cells (HBCs, or fetal placental macrophages) in mouse models. Methods and results We assessed the impact of maternal SARS-CoV-2 on HBCs isolated from 24 term placentas ( N = 10 SARS-CoV-2 positive cases, 14 negative controls). Using single-cell RNA-sequencing, we demonstrated that HBC subpopulations exhibit distinct cellular programs, with specific subpopulations differentially impacted by SARS-CoV-2. Assessment of differentially expressed genes implied impaired phagocytosis, a key function of both HBCs and microglia, in some subclusters. Leveraging previously validated models of microglial synaptic pruning, we showed that HBCs isolated from placentas of SARS-CoV-2 positive pregnancies can be transdifferentiated into microglia-like cells (HBC-iMGs), with impaired synaptic pruning behavior compared to HBC models from negative controls. Conclusion These findings suggest that HBCs isolated at birth can be used to create personalized cellular models of offspring microglial programming.
Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting ...aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.
Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory ...exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.
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