A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Vijayraghavan, Sriram; Kantor, Boris

doi:10.3791/56915

Cited by 17 publications

(21 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lentiviral particles were generated by the Duke University Viral Vector Core Facility as previously described (Vijayraghavan & Kantor, ). HeLa cells were grown in growth media supplemented with 8 μg/ml hexadimethrine bromide, and infected at a 10:1 multiplicity of infection.…”

Section: Methodsmentioning

confidence: 99%

The MyMOMA domain of MYO19 encodes for distinct Miro‐dependent and Miro‐independent mechanisms of interaction with mitochondrial membranes

et al. 2019

View full text Add to dashboard Cite

MYO19 interacts with mitochondria through a C‐terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity‐capture databases, we have identified a number of putative MYO19‐interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr‐Miro2 in combination with GFP‐tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19‐GFP to mitochondria. Coexpression of MYO19898‐970‐GFP with mchr‐Miro2 enhanced MYO19898‐970‐GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898‐970‐GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization‐activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane‐insertion motif but lacking the Miro2‐interacting region displayed slow exchange kinetics. MYO19898‐970‐GFP, which does not include the membrane‐insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well‐conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898‐970‐GFP localization in cells ectopically expressing mchr‐Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898‐970‐GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane‐inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin‐ and microtubule‐based mitochondrial activities.

show abstract

Section: Methodsmentioning

confidence: 99%

The MyMOMA domain of MYO19 encodes for distinct Miro‐dependent and Miro‐independent mechanisms of interaction with mitochondrial membranes

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Lentiviral vectors were generated using the transient transfection protocol, as described previously. 64 Briefly, 15 mg vector plasmid, 10 mg psPAX2 packaging plasmid (Addgene 12260 generated in Dr. Didier Trono's lab, EPFL, Switzerland), 5 mg pMD2.G envelope plasmid (Addgene 12259, generated in Dr. Trono's lab), and 2.5 mg pRSV-Rev plasmid (Addgene 12253, generated in Dr. Trono's lab) were transfected into 293T cells. Vector particles were collected from filtered conditioned medium at 72 hr post-transfection.…”

Section: Vector Productionmentioning

confidence: 99%

“…Vector particles were collected from filtered conditioned medium at 72 hr post-transfection. The particles were purified using the sucrose-gradient method 64 and concentrated >250-fold by ultracentrifugation (2 hr at 20,000 rpm).…”

Section: Vector Productionmentioning

confidence: 99%

See 1 more Smart Citation

Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

et al. 2018

Self Cite

View full text Add to dashboard Cite

Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson's disease (PD), while normal physiological levels of SNCA are needed to maintain neuronal function. We ought to develop new therapeutic strategies targeting the regulation of SNCA expression. DNA methylation at SNCA intron 1 regulates SNCA transcription, and PD brains showed differential methylation levels compared to controls. Thus, DNA methylation at SNCA intron 1 is an attractive target for fine-tuned downregulation of SNCA levels. Here we developed a system, comprising an all-in-one lentiviral vector, for targeted DNA methylation editing within intron 1. The system is based on CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A). Applying the system to human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons from a PD patient with the SNCA triplication resulted in fine downregulation of SNCA mRNA and protein mediated by targeted DNA methylation at intron 1. Furthermore, the reduction in SNCA levels by the guide RNA (gRNA)-dCas9-DMNT3A system rescued disease-related cellular phenotype characteristics of the SNCA triplication hiPSC-derived dopaminergic neurons, e.g., mitochondrial ROS production and cellular viability. We established that DNA hypermethylation at SNCA intron 1 allows an effective and sufficient tight downregulation of SNCA expression levels, suggesting the potential of this target sequence combined with the CRISPR-dCas9 technology as a novel epigenetic-based therapeutic approach for PD.

show abstract

“…Lentiviral particles were generated by the Duke University Viral Vector Core Facility as previously described [Vijayraghavan and Kantor 2017]. HeLa cells were grown in growth media supplemented with 8µg/ml hexadimethrine bromide, and infected at a 10:1 multiplicity of infection.…”

Section: Establishment Of Cell Lines Stably Expressing Myo19 824-970 mentioning

confidence: 99%

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

Bocanegra

Fujita

Melton

et al. 2019

Preprint

View full text Add to dashboard Cite

for critical reading and discussions related to this manuscript. Hypotheses and experiments in this study were conceived by OAQ. Experiments were performed by JLB, BMF, NRM, JMC, ELS, TYT, and OAQ.Data analysis and manuscript preparation were completed by JLB, BMF, NRM, JMC, TYT, MBM, and OAQ. Except for the proteomics analysis, all experiments for the initial submission were completed during the 10-week 2018 summer research session at University of Richmond. We would also like to that Edward Salmon for his example and inspiration.3 Abstract MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA).The specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using new promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to further explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Co-expression of MYO19 898-970 -GFP with mchr-Miro2 enhanced MYO19 898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19 898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence (PARF) analysis. MyMOMA constructs containing a putative membrane insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19 898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that the MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19 898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19 898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin-and microtubulebased mitochondrial activities.

show abstract

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Cited by 17 publications

References 71 publications

The MyMOMA domain of MYO19 encodes for distinct Miro‐dependent and Miro‐independent mechanisms of interaction with mitochondrial membranes

The MyMOMA domain of MYO19 encodes for distinct Miro‐dependent and Miro‐independent mechanisms of interaction with mitochondrial membranes

Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

Contact Info

Product

Resources

About