Stanley Qi, Postdoctoral Faculty Sponsor
Enhanced Cas12a multi-gene regulation using a CRISPR array separator.
The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
View details for DOI 10.7554/eLife.66406
View details for PubMedID 34499031
The spatial RNA integrity number assay for in situ evaluation of transcriptome quality.
2021; 4 (1): 57
The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.
View details for DOI 10.1038/s42003-020-01573-1
View details for PubMedID 33420318
A Widespread Neurogenic Potential of Neocortical Astrocytes Is Induced by Injury
CELL STEM CELL
2020; 27 (4): 605-+
Parenchymal astrocytes have emerged as a potential reservoir for new neurons in non-neurogenic brain regions. It is currently unclear how astrocyte neurogenesis is controlled molecularly. Here we show that Notch signaling-deficient astrocytes can generate new neurons after injury. Using single-cell RNA sequencing, we found that, when Notch signaling is blocked, astrocytes transition to a neural stem cell-like state. However, only after injury do a few of these primed astrocytes unfold a neurogenic program, including a self-amplifying progenitor-like state. Further, reconstruction of the trajectories of individual cells allowed us to uncouple astrocyte neurogenesis from reactive gliosis, which occur along independent branches. Finally, we show that cortical neurogenesis molecularly recapitulates canonical subventricular zone neurogenesis with remarkable fidelity. Our study supports a widespread potential of parenchymal astrocytes to function as dormant neural stem cells.
View details for DOI 10.1016/j.stem.2020.07.006
View details for Web of Science ID 000577245400014
View details for PubMedID 32758425
View details for PubMedCentralID PMC7534841
Activation of a neural stem cell transcriptional program in parenchymal astrocytes.
Adult neural stem cells, located in discrete brain regions, generate new neurons throughout life. These stem cells are specialized astrocytes, but astrocytes in other brain regions do not generate neurons under physiological conditions. After stroke, however, striatal astrocytes undergo neurogenesis in mice, triggered by decreased Notch signaling. We used single-cell RNA sequencing to characterize neurogenesis by Notch-depleted striatal astrocytes in vivo. Striatal astrocytes were located upstream of neural stem cells in the neuronal lineage. As astrocytes initiated neurogenesis, they became transcriptionally very similar to subventricular zone stem cells, progressing through a near-identical neurogenic program. Surprisingly, in the non-neurogenic cortex, Notch-depleted astrocytes also initiated neurogenesis. Yet, these cortical astrocytes, and many striatal ones, stalled before entering transit-amplifying divisions. Infusion of epidermal growth factor enabled stalled striatal astrocytes to resume neurogenesis. We conclude that parenchymal astrocytes are latent neural stem cells and that targeted interventions can guide them through their neuronal differentiation.
View details for DOI 10.7554/eLife.59733
View details for PubMedID 32744501
Blocking Notch-Signaling Increases Neurogenesis in the Striatum after Stroke
2020; 9 (7)
Stroke triggers neurogenesis in the striatum in mice, with new neurons deriving in part from the nearby subventricular zone and in part from parenchymal astrocytes. The initiation of neurogenesis by astrocytes within the striatum is triggered by reduced Notch-signaling, and blocking this signaling pathway by deletion of the gene encoding the obligate Notch coactivator Rbpj is sufficient to activate neurogenesis by striatal astrocytes in the absence of an injury. Here we report that blocking Notch-signaling in stroke increases the neurogenic response to stroke 3.5-fold in mice. Deletion of Rbpj results in the recruitment of a larger number of parenchymal astrocytes to neurogenesis and over larger areas of the striatum. These data suggest inhibition of Notch-signaling as a potential translational strategy to promote neuronal regeneration after stroke.
View details for DOI 10.3390/cells9071732
View details for Web of Science ID 000554092400001
View details for PubMedID 32698472
Visualization and analysis of gene expression in tissue sections by spatial transcriptomics
2016; 353 (6294): 78-82
Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.
View details for DOI 10.1126/science.aaf2403
View details for Web of Science ID 000378816200040
View details for PubMedID 27365449
Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions.
Development (Cambridge, England)
2016; 143 (7): 1075-86
In a few regions of the adult brain, specialized astrocytes act as neural stem cells capable of sustaining life-long neurogenesis. In other, typically non-neurogenic regions, some astrocytes have an intrinsic capacity to produce neurons when provoked by particular conditions but do not use this ability to replace neurons completely after injury or disease. Why do astrocytes display regional differences and why do they not use their neurogenic capacity for brain repair to a greater extent? In this Review, we discuss the neurogenic potential of astrocytes in different brain regions and ask what stimulates this potential in some regions but not in others. We discuss the transcriptional networks and environmental cues that govern cell identity, and consider how the activation of neurogenic properties in astrocytes can be understood as the de-repression of a latent neurogenic transcriptional program.
View details for DOI 10.1242/dev.133975
View details for PubMedID 27048686
A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse.
Science (New York, N.Y.)
2014; 346 (6206): 237-41
Neurogenesis is restricted in the adult mammalian brain; most neurons are neither exchanged during normal life nor replaced in pathological situations. We report that stroke elicits a latent neurogenic program in striatal astrocytes in mice. Notch1 signaling is reduced in astrocytes after stroke, and attenuated Notch1 signaling is necessary for neurogenesis by striatal astrocytes. Blocking Notch signaling triggers astrocytes in the striatum and the medial cortex to enter a neurogenic program, even in the absence of stroke, resulting in 850 ± 210 (mean ± SEM) new neurons in a mouse striatum. Thus, under Notch signaling regulation, astrocytes in the adult mouse brain parenchyma carry a latent neurogenic program that may potentially be useful for neuronal replacement strategies.
View details for DOI 10.1126/science.346.6206.237
View details for PubMedID 25301628