Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering

Moyer, Tyler C.; Holland, Andrew J.

doi:10.1016/bs.mcb.2015.03.017

Cited by 29 publications

(28 citation statements)

References 29 publications

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“…PLK4 is the major kinase for the initiation of centriole assembly 37 , 38 . Expression of the phosphodegron-removed mutant PLK4 Δ24 could be induced by doxycycline 37 , 39 – 41 . The PLK4 Δ24 -expressing cells were synchronized at G1/S and released into a fresh medium with or without MG132 for 8 h. At the time of thymidine release, doxycycline was treated to induce centriole assembly by PLK Δ24 .…”

Section: Resultsmentioning

confidence: 99%

Caspase-mediated cleavage of the centrosomal proteins during apoptosis

Seo

Rhee

2018

Cell Death Dis

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The centrosome is the major microtubule-organizing center and plays important roles in intracellular transport, cellular morphology, and motility. In mitotic cells, centrosomes function as spindle poles to pull a set of chromosomes into daughter cells. In quiescent cells, primary cilia are originated from the centrosomes. Given its involvement in various cellular processes, it is little surprising that the organelle would also participate in apoptotic events. However, it remains elusive how the centrosome changes in structure and organization during apoptosis. Apoptosis, a programmed cell death, is required for homeostatic tissue maintenance, embryonic development, stress responses, etc. Activation of caspases generates a cascade of apoptotic pathways, explaining much of what happens during apoptosis. Here, we report the proteolytic cleavage of selected centrosomal proteins in apoptotic cells. SAS-6, a cartwheel component of centrioles, was specifically cleaved at the border of the coiled-coil domain and the disordered C-terminus. Pericentrin, a scaffold of pericentriolar material, was also cleaved during apoptosis. These cleavages were efficiently blocked by the caspase inhibitors. We propose that the caspase-dependent proteolysis of the centrosomal proteins may destabilize the configuration of a centrosome. Loss of centrosomes may be required for the formation of apoptotic microtubule networks, which are essential for apoptotic fragmentation. This work demonstrates the first centrosomal targets by caspases during apoptosis.

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Section: Resultsmentioning

confidence: 99%

Caspase-mediated cleavage of the centrosomal proteins during apoptosis

Seo

Rhee

2018

Cell Death Dis

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“…Top and bottom oligos were obtained for each guide (Table 1) Guides were cloned into a Cas9 containing plasmid (PX459) Addgene #62988, (Cambridge, MA) following methods previously outlined (Moyer & Holland, 2015). The search tool was used to select guides close to the C-terminus of the protein of interest (~100 nt surrounding and including the stop codon).…”

Section: Methodsmentioning

confidence: 99%

“…Guides were cloned into a Cas9 containing plasmid (PX459) Addgene #62988, (Cambridge, MA) following methods previously outlined (Moyer & Holland, 2015). Briefly, top and bottom oligos were annealed and then phosphorylated by T4 PNK (NEB, Ipswich, MA).…”

Section: Crispr Gene Editingmentioning

confidence: 99%

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Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

Cytoskeleton

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The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

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“…We performed genome editing as described previously, with slight modifications [46]. Guide RNAs against target genomic sequences from human or mouse PCYT1A (RefSeq NC_000003.11 or NC_000082.5, respectively) were designed using the CRISPR MIT portal [47] (S1 Table), modified into oligonucleotides for cloning purposes, and cloned according to the submitter’s laboratory protocol into the pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene, Cat#62988), a gift from Feng Zhang’s laboratory [48].…”

Section: Methodsmentioning

confidence: 99%

Loss of function variants inPCYT1Acausing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation

Jurgens

Chen

Sobreira

et al. 2019

Preprint

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46Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal 47 recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding 48 the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-49 limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in 50 understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we 51 generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited 52 PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured 53 skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in 54 CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of 55 wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response 56 to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts 57 (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers 58 or sizes as compared to controls. Lipid droplet phenotypes in PCYT1A-null HEK293 cells were 59 rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His human PCYT1A cDNAs. 60 While both edited cellular models had normal morphology and proliferation rates compared to 61 unedited controls, Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte 62 differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-63 200 lipid levels in PCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as 64 compared to wild-type controls. The specific lipids altered and extent of change varied by cell 65 type. Importantly, both PCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines 66 had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels 67 of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting 4 68 compensatory PC production through increased LPC remodeling by LPCAT or decreased 69 conversion of PC to LPC by phospholipase A 2 . Our results show that all tested PCYT1A alleles 70 associated with SMD-CRD are hypomorphic and suggest involvement of PCYT1A in chondrocyte 71 differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation. 72 Author Summary 73 Rare genetic disorders can reveal the function of genes on an organismal scale. When 74 normal gene activity is lost, patients can experience a range of symptoms, often dependent on the 75 residual activity of the encoded protein. Rare variants in the gene PCYT1A can cause multiple 76 inherited disorders, including a disorder of the skeleton and the retina characterized by short 77 stature, bone abnormalities, and blindness. PCYT1A is required for normal cellular function, 78 particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. 79 T...

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Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering

Cited by 29 publications

References 29 publications

Caspase-mediated cleavage of the centrosomal proteins during apoptosis

Caspase-mediated cleavage of the centrosomal proteins during apoptosis

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Loss of function variants inPCYT1Acausing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation

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