CRISPR for neuroscientists

Kalamakis, Georgios; Platt, Randall J.

doi:10.1016/j.neuron.2023.04.021

Cited by 13 publications

(7 citation statements)

References 220 publications

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“…Here, we mirrored previous methods for a unidirectional transition of iPSC into spinal motor neurons (MNs) via the delivery of ISL1 and LHX3 transgenes into well-defined safe harbor sites (H11, ROSA26, and AAVS1) in the human iPSC genome with CRISPR-assisted gene editing techniques. With this method, we attempted to avoid poor outcomes from the interference of the local chromatin structure with the transgene expression and versa vice, and possible interruption of host gene expression by the transgene insertion [29,30]. CRISPR gene editing technology has gained popularity for the high precision rates of gene insertion into desired chromosome locus and has found a wide range of applications, including the generation of recombinant cell lines, transgenic animal models, and applications for in vivo gene therapies [30].…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function

Davis-Anderson,

Micheva-Viteva,

Solomon

et al. 2023

IJMS

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Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function

Davis-Anderson,

Micheva-Viteva,

Solomon

et al. 2023

IJMS

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“…HEK293T cells that were WT, or null for SEL1L or HRD1 were obtained from Dr. Qi and were described previously 35 , 71 – 73 . HEK293T cells that are knock-in for SEL1LΔFN2 were created in the laboratory of Dr. Qi via CRISPR-Cas9 editing as previously described 74 . Briefly, WT HEK293T cells were electroporated with Cas9 and gRNAs (gRNA1 and gRNA3; Supplementary Data 2 ) flanking the SEL1L exon 4 which encodes SEL1L FN2 domain, and an additional gRNA (gRNA2; Supplementary Data 2 ) in close proximity to gRNA1 to enhance editing efficiency.…”

Section: Methodsmentioning

confidence: 99%

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

Podolsky,

Kheyfets,

Pandey

et al. 2024

Nat Commun

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Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

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“…AAV-hypPB permits gRNA capture with sparse scRNA-seq readout. One challenge of in vivo CRISPR screens is to retain high gRNA expression for efficient gene editing as well as gRNA recovery in the sparse scRNA-seq data (Kalamakis and Platt, 2023). We next tested if the transposase system could also enhance gRNA expression levels and found that in vitro hypPB co-transfection led to a 4.2-fold increase in the gRNA expression detected (Fig.…”

Section: Transposase Hyppb Stabilizes and Enhances Transgene Expressionmentioning

confidence: 99%

Massively parallelin vivoPerturb-seq reveals cell type-specific transcriptional networks in cortical development

Zheng,

Wu,

Liu

et al. 2023

Preprint

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Systematic analysis of gene function across diverse cell types in vivo is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell's perturbation in high-throughput single-cell assays. Leveraging AAV's versatile cell type tropism and high labeling capacity, we expanded the resolution and scale of in vivo CRISPR screens: allowing phenotypic analysis at single-cell resolution across a multitude of cell types in the embryonic brain, adult brain, and peripheral nervous system. We undertook extensive tests of 86 AAV serotypes, combined with a transposon system, to substantially amplify labeling and accelerate in vivo gene delivery from weeks to days. Using this platform, we performed an in utero genetic screen as proof-of-principle and identified pleiotropic regulatory networks of Foxg1 in cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct networks essential for cell fate specification. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% (mediated by lentivirus), and achieve analysis of over 30,000 cells in one experiment, thus enabling massively parallel in vivo Perturb-seq. Compatible with various perturbation techniques (CRISPRa/i) and phenotypic measurements (single-cell or spatial multi-omics), our platform presents a flexible, modular approach to interrogate gene function across diverse cell types in vivo, connecting gene variants to their causal functions.

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CRISPR for neuroscientists

Cited by 13 publications

References 220 publications

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

Massively parallelin vivoPerturb-seq reveals cell type-specific transcriptional networks in cortical development

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