This work was funded in part by the Kahn Neurotechnology Development Award (to O.C.) and the National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) (R01 NS085011 to O.C.).

1. System Design and Virus Production
<ol>
	<li>Plasmid design and construction 
	NOTE: The construction of an all-in-one LV-gRNA-dCas9-DNMT3A vector is performed by using a production-and expression-optimized expression cassette published by Ortinski et al.30. The vector cassette carries a repeat of the recognition site of transcription factor Sp1 and a state-of-the-art deletion within the untranslated (U3&#39;) region of a 3&#39;-long terminal repeat (LTR...

Duke University filed a provisional patent application related to this study.

LVs have begun to emerge as the vehicle of choice for epigenome editing, especially in the context of genetic diseases, mainly due to their ability to (i) accommodate large DNA payloads and (ii) efficiently transduce a wide range of dividing and nondividing cells. The large packaging efficacy of the LVs is especially beneficial for the applications involving packaging of the CRISPR/dCas9 systems which are oversized. From this perspective, LVs represent the platform-of-choice for the delivery of all-in-one CRISPR/Cas9 sys...

Multiple epigenome-editing platforms have been recently developed to target any DNA sequences in the regions that control gene expression1,2. The created epigenome-editing tools are designed to (i) regulate transcription, (ii) alter posttranslational histone modifications, (iii) modify DNA methylation, and (iv) modulate regulatory element interactions. The approach to anchor the transcription/chromatin modifiers to a deactivated (dead) Cas9 (dCas9) raised from previously developed epigenome-editing platforms, such as zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), harboring a potent transcriptional effector domain (ED) fused to the designed DNA-binding domain (DBD)3. The outcomes of the desired phenotype such as activation or repression is defined by the effector molecule anchored to the endogenous loci (Figure 1). To create programmable transcriptional activators, dCas9/gRNA modules are linked to VP164,5,6 (Figure 1A), a viral activation domain that recruits Pol II and the general transcription machinery. The modification of this system has included VP64, a tetramer of VP16 domains, providing an even more robust activation rate5,6. The system has been successfully employed to activate coding and noncoding regions by targeting promoters and regulatory elements. Importantly, even though VP64 molecules do not directly modify the chromatin structure in the target region, it recruits chromatin modifiers which bind results in deposition of the active (euchromatin) marks, including as H3/H4 acetylation and H3-K4 di/tri-methylation5,6. In addition to VP64, the p65 subunit of the human NF-&#954;B complex has been tethered to the dCas9/gRNA module7. Interestingly, the tethering of these effectors to the regions upstream of transcription start sites (TSSs) and within promoters results in a strong gene induction. Nevertheless, VP64 and p65 effectors can also exert the activatory effects while being linked to the regions located downstream of TSSs and at distal enhancers7,8. To elicit a more robust transcriptional response, multiple dCas9-VP64 or dCas9-p65 fusions need to be recruited to a single target locus9,10. As such, the recent development of next-generation activators, which recruit multiple effector domains by a single dCas9-gRNA complex, such as SunTag, has resulted in a stronger activation capability comparing to dCas9-VP64 fusion counterparts11,12. An improved transcriptional activation has been obtained through the fusion of VP64, p65, and Rta (VPR), a transactivation domain from gamma-herpesviruses, to the C-terminus of dCas913 (Figure 1A). Similar CRISPR/dCas9 systems have been developed for target-specific repression (Figure 1B).
Endogenous gene repression can be achieved with engineered repressor fusions through a variety of mechanisms (Figure 1B). It has been demonstrated that CRISPR/dCas9 systems, linked to the repressor DBD (even without an effector domain/s), can efficiently silence gene expression while tethered to a promoter or upstream/downstream-TSS regions3,6,14. The effects on transcription is caused by the steric interference of transcription factor binding and RNA polymerase processing. Nevertheless, more comprehensive approaches are needed, as gene repression by steric hindrance alone is often not sufficient for robust silencing. The recent development of the next generation of silencers based on CRISPR/dCas9 systems carrying transcriptional repressor domains (TRDs), histone modifiers (H3-K9 di-/tri-methylation, H3-K27 di-/tri-methylation; H3-K36 di-/tri-methylation, H3/H4 deacetylation), and DNA (CpG) methylation led to the construction of epigenetic tools allowing more robust silencing effects4,5,15,16,17,18,19,20. It has been demonstrated that the recruitment of these epigenetic modifiers to the DNA may lead to the formation of more closed and condensed chromatin, which typically generate a more potent silencing outcome21,22. The most commonly silencing domain used with DBDs is the Kr&#252;ppel-associated box (KRAB)4,5. The recruitment of the factor has been demonstrated to correspond with chromatin changes; nevertheless, the mechanisms of these modifications are yet to be elucidated16,17,18. Recently, it has been shown that the localization of KRAB to DNA can promote the assembly of the histone methyltransferase SETDB1 and the histone deacetylation (HDAC) NuRD complexes, suggesting the possibility that these interactions mediate the formation of chromatin condensation and transcriptional silencing3,13. As an alternative approach, effector domains can be fused to DBDs to create a custom epigenetic silencing protein. This system directly catalyzes repressive DNA marks or histone modifications.
Recently, the use of synthetic CRISPR/dCas9 systems tethered to the DNMT3A enzyme has been repurposed for transcriptional deactivation. DNMT3A catalyzes DNA methylation that exerts transcriptional repression throughout the formation of heterochromatin on endogenous gene promoters and other regulatory regions (Figure 1B)18,20. McDonald et al.18 and Vojta et al.20 were the first authors to report that DNA methylation can be used for epigenome-gene silencing or repression, demonstrating that the plasmid-delivered dCas9-DNMT3A fusion system can potently enhance cytosine methylation around the TSS18,20. McDonald and coworkers demonstrated that the employment of the strategy may result in a significant reduction (about 40%) in a tumor-suppressor gene, CDKN2A mRNA levels18. Similarly, targeting the unmethylated promoter region of the BACH or IL6ST genes shows increased CpG methylation that has been correlated with a twofold reduction in the gene expression20. Our lab has recently repurposed the use of DNA methylation for attenuating the pathological outcomes of SNCA overexpression (Figure 2)23. The strategy is based on selective enhancement in DNA methylation within the SNCA intron 1 region, as it was previously reported to be hypomethylated in PD and dementia with Lewy bodies (DLB) brains24,25,26. This hypomethylation has been linked to SNCA overexpression, thus offering an attractive target for therapeutic intervention24,27,28. We recently showed a low level of DNA methylation in the SNCA intron 1 region in hiPSC-derived dopaminergic NPCs obtained from a PD patient with the SNCA triplication23. The advantage of this experimental model is that the NPCs can be robustly propagated in culture or further differentiated into mature neurons, enabling an efficient screening to identify genetic factors that mediate cellular phenotypes, including oxidative stress and apoptosis29. Furthermore, this model system enables scientists to recapitulate the developmental events that occurred prior to symptom onset in patients. In addition, hiPSC-derived NPCs represent a great tool to test the cellular and molecular pathways associated with gene expression. Importantly, hiPSC-derived NPCs combined with state-of-the-art CRISPR/Cas9-epigenome technology can greatly facilitate the development of &#8220;next-generation drugs&#8221; for many neurodegenerative diseases.
To reduce pathological levels of SNCA expression, we recently developed a lentivirus-based system carrying a dCas9-DNMT3A fusion protein and gRNA to specifically target CpG methylation within the SNCA intron 1 (Figure 2A)23. This protocol will describe lentiviral vector (LV) design and production in detail. LVs represent an effective means of delivering CRISPR/dCas9 components for several reasons, namely (i) their capacity to carry bulky DNA inserts, (ii) a high efficiency of transducing a broad range of cells, including both dividing and nondividing cells30, and (iii) their ability to induce minimal cytotoxic and immunogenic responses. Recently, we applied the LV system to hiPSC-derived dopaminergic neurons from a patient with the triplication of the SNCA locus and demonstrated the therapeutic potential of LVs for the delivery of epigenome-editing methylation tools23 (Figure 2B). Indeed, an LV-gRNA/dCas9-DNMT3A system causes a significant increase in DNA methylation at the SNCA intron 1 region. This increase corresponds with the reduction in the levels of SNCA mRNA and protein23. Moreover, SNCA downregulation rescues PD-related phenotypes in the SNCA triplication/hiPSC-derived dopaminergic neurons (e.g., mitochondrial ROS production and cell viability)23. Importantly, we demonstrated that the reduction in SNCA expression by the LV-gRNA-dCas9-DMNT3A system is capable of reversing the phenotypes which are characteristic for hiPSC-derived dopaminergic neurons from a PD patient who carried the SNCA triplication, such as mitochondrial ROS production and cell viability23. The goal of this protocol is 1) to outline the protocol of production and concentration of an optimized LV platform for generating high-tittered viral preparations and 2) to describe the differentiation of hiPSCs into NPCs patterned to become mature dopaminergic neurons31,32 and the characterization of the methylation levels of the targeted region within SNCA intron 1.
Lentiviral platforms have a major advantage over the most popular vector platform, namely adeno-associated vectors (AAVs), which is the former&#8217;s ability to accommodate larger genetic inserts33,34. AAVs can be generated at significantly higher yields but possess a low packaging capacity (&#60;4.8 kb), compromising their use for delivering all-in-one CRISPR/Cas9 systems. Thus, it seems that the LVs would be the platform-of-choice in the applications involved in the delivery of CRISPR/dCas9 tools. Therefore, the protocol outlined here will be a valuable tool for researchers desiring to effectively deliver epigenome-editing components to the cells and organs. The protocol further outlines the strategy to increase the production and expression capabilities of the vectors via a modification in cis of the elements within the vector expression cassette30,35. The strategy is based on the novel system developed and studied in our lab and highlights its ability to produce viral particles in the range of 1010 viral units (VU)/mL30,35.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN-80 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A99839</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;M filter unit, 1L</td>
			<td>Corning</td>
			<td>430513</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-&#956;m filter unit, 500mL</td>
			<td>Corning</td>
			<td>430773</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm TC-Treated Cell Culture dishes with 20 mm Grid</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 800</td>
			<td>StemCell Technologies</td>
			<td>34811</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 25R tabletop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369434</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS</td>
			<td>Becton Dickinson</td>
			<td>338960</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical bottom ultracentrifugation tubes</td>
			<td>Seton Scientific</td>
			<td>5067</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube adapters</td>
			<td>Seton Scientific</td>
			<td>PN 4230</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Cell Imaging Slides</td>
			<td>Eppendorf</td>
			<td>30742060</td>
			<td></td>
		</tr>
		<tr>
			<td>High-binding 96-well plates</td>
			<td>Corning</td>
			<td>3366</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DM IRB2</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (50)</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Reversible Strainer</td>
			<td>StemCell Technologies</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>SW32Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>369650</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR&#174; Disposable Serological Pipets, Glass, Nonpyrogenic</td>
			<td>VWR</td>
			<td>93000-694</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR&#174; Vacuum Filtration Systems</td>
			<td>VWR</td>
			<td>89220-694</td>
			<td></td>
		</tr>
		<tr>
			<td>xMark&#8482; Microplate Absorbance plate reader</td>
			<td>Bio-Rad</td>
			<td>1681150</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human embryonic kidney 293T (HEK 293T) cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>StemCell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>StemCell Technologies</td>
			<td>7010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FOXA2 Antibody</td>
			<td>Abcam</td>
			<td>Ab60721</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nestin Antibody</td>
			<td>Abcam</td>
			<td>Ab18102</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution, 100X</td>
			<td>Sigma Aldrich</td>
			<td>A5955-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BES</td>
			<td>Sigma Aldrich</td>
			<td>B9879 - BES</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin Fraction V (7.5% solution)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>StemCell Technologies</td>
			<td>72052</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>Corning</td>
			<td>08-774-552</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Lonza</td>
			<td>12-719</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose media</td>
			<td>Gibco</td>
			<td>11965</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>EpiTect PCR Control DNA Set</td>
			<td>Qiagen</td>
			<td>596945</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ DNA Methylation Kit</td>
			<td>Zymo Research</td>
			<td>D5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1800-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15750078</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>stemCell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant bFGF</td>
			<td>StemCell Technologies</td>
			<td>78003</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant&#160;EGF</td>
			<td>StemCell Technologies</td>
			<td>78006</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant Shh (C24II)</td>
			<td>StemCell Technologies</td>
			<td>78065</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acid (NEAA)</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark PCR Kit</td>
			<td>Qiagen</td>
			<td>978703</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>StemCell Technologies</td>
			<td>72232</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff&#160;Neural Induction Medium</td>
			<td>StemCell Technologies</td>
			<td>5835</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Medium</td>
			<td>StemCell Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Assay FOXA2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs00232764</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Assay GAPDH</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs99999905</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Assay Nestin</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs04187831</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Assay OCT4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs04260367</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Assay PPIA</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs99999904</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>StemCell Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>p24 ELISA reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>3537</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit horseradish peroxidase IgG</td>
			<td>Sigma Aldrich</td>
			<td>12-348</td>
			<td>Working concentration 1:1500</td>
		</tr>
		<tr>
			<td>Goat serum, Sterile, 10mL</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr>
			<td>HIV-1 standards</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP968F</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal mouse serum, Sterile, 500mL</td>
			<td>Equitech-Bio</td>
			<td>SM30-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP451T</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB peroxidase substrate</td>
			<td>KPL</td>
			<td>5120-0076</td>
			<td>Working concentration 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>52961</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsrGI</td>
			<td>New England Biolabs</td>
			<td>R0575S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England Biolabs</td>
			<td>R0195S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R0547S</td>
			<td></td>
		</tr>
		<tr>
			<td>SphI</td>
			<td>New England Biolabs</td>
			<td>R0182S</td>
			<td></td>
		</tr>
	</tbody>
</table>

lentiviral vector platform for the efficient delivery of epigenome-editing tools into human induced pluripotent stem cell-derived disease models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Validation of the production titers of the LV-dCas9-DNMT3A-GFP/Puro vectors compared to the naive GFP counterpart
We performed p24gag ELISA to compare between physical titers of LV-dCas9-DNMT3A-GFP/Puro with the naive GFP/Puro counterparts. Representative results, presented in Figure 5A, demonstrate that physical yields of the vectors, generated using the protocol herein ...

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Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Lentiviral vector platform have a major advantage over the most popular vector platform, specifically because of the ability of lentiviral vectors to accommodate larger genetic insert. Thus, it seems that the lentiviral vector would be the platform of choice in the application involve the delivery of CRISPR/Cas tools. This technique imitate the natural regulatory mechanism of alpha-synuclein expression which when impaired are associated with disease.It allows fine-tuning of the expression level versus robust changes in gene expression. I would strongly recommend validating this or similar approaches using multiple guide RNAs for screening. There is great variability in guide RNA potency.The only way to determine it is there robust and comprehensive screening and validation. The developed system can be easily adapted to other model systems such as animal models including rodent and nonhuman primate. These follow up experiment could serve as a proof of concept for future clinical studies.Demonstrating the procedure will be Dr.Ekaterina Ilich, a senior staff member in my lab, focusing on execution of molecular cloning procedure for creating Lentiviral Vector Plasmids related to this project. She will also demonstrate the key steps involved in Lentiviral Vector production. Also demonstrating the procedure will be Dr.Lidia Tagliafierro, a postdoc from my lab focusing on the differentiation of human iPS cells into pathological relevant cells.She will demonstrate the key steps into the establishment of assays to evaluate the methylation profiles of synuclein intron1. Ahila Sriskanda, a staff member in my lab focusing on the culture of human iPS cell-derived neural progenitor cells. To begin, develop the DNMT3A catalytic domain from the d-Cas9-DNMT3A-GFP by amplifying the DNMT3A portion of the vector.After digesting the DNMT3A fragment using a BamH1 restriction enzyme. Cloat it into the BamH1 site of the modified PBK-301 vector carrying d-Cas9 and verified by direct Sanger sequencing, to replace the pure Meissen reporter gene and the resulting plasmid with GFP digest d-Cas9-DNMT3A-P2A pure Meissen and plasmid with FSC 1. Purify the vector fragment using a gel purification method.Prepare the insert by digesting PBK-201 with FSC-1. Clone the FSC 1 fragment into the vector to create d-Cas9-DNMT3A-P2A-GFP. Coat 15 centimeter plates with 0.2%gelatin for the transfection and wait for 10 minutes.Then aspirate the gelatin and add 22.5 milliliters of high glucose medium and 2.5 milliliters of previously cultured HEK-293T cell suspension. Incubate the plates at 37 degrees Celsius with 5%CO2 until reaching 70 to 80%confluence. To prepare the plasmid mix sufficient for 115 centimeter plate use the four plasmids described in the manuscript.Add 312.5 micro liters of one molar calcium chloride to the same 15 milliliter tube with the plasmids and bring the final volume to 1.25 milliliters using sterile double distilled water. gently add 1.25 milliliters of two X BBM solution drop wise to the tube while vortexing. Incubate for 30 minutes at room temperature.Aspirate the medium from the cells and add 22.5 milliliters of freshly prepared high glucose DMEM without serum. Add 2.5 milliliters of the transfection mixture drop wise to each plate. Swirl the plates and incubate at 37 degrees Celsius with 5%CO2 for two to three hours after incubation add 2.5 milliliters of serum per plate to achieve 10%dilution and incubate overnight at 37 degrees Celsius with 5%CO2.To harvest the virus collect the supernatant from all the transfected cells and pull them in 50 milliliter conical tubes. Centrifuge at 400 to 450 g for 10 minutes at room temperature and then filter the supernatant through 0.45 micrometer vacuum filter unit. Next create a sucrose gradient by preparing conical ultra centrifugation tubes.Carefully add the filtered supernatant to the gradient by equally distributing the viral supernatant among each ultra centrifugation tube filling at least three fourths of each tube. Centrifuge the samples at 70000 g for two hours at 17 degrees Celsius. Gently collect 30%to 60%sucrose fractions into clean tubes add cold 1X PBS up to 100 milliliters of total volume and pipette multiple times to mix.Carefully add four milliliters of 20%sucrose in 1X PBS to each tube to stratify the viral preparation continue by pipetting approximately 20 to 25 milliliters of the viral solution per each tube. Carefully balance the tubes and then centrifuge at 70000 g for two hours at 17 degrees Celsius. Carefully aspirate the remaining liquid to completely remove all the liquid leaving the virus containing pellets which are barely visible as small translucent spots.After emptying the supernatant invert the tubes on paper towels to allow the remaining liquid to drain. Add 70 micro liters of 1X PBS to the first tube and thoroughly pipette to re-suspend the pellet. Transfer the suspension to the next tube and repeat with pipetting and transferring until all pellets are re-suspended.Add an additional 50 micro liters of 1X PBS to the first tube and mix to wash it. Then transfer the suspension to the second tube to wash and continue with washing and transferring as previously, yielding approximately 120 micro liters of the slightly milky final suspension. Centrifuge at 10000 g for 60 seconds to obtain a clear suspension transfer the supernatant to a new tube and make five micro liter aliquot and store them at negative 80 degrees Celsius place a Cryovial with MD NPC into 37 degrees Celsius heat block for two minutes and transfer the thawed cells to a 15 milliliter conical tube with 10 milliliters of warm DMEM-F12.Centrifuge at 300 g for five minutes. Aspirate the supernatant and re-suspend the cells in two milliliters of pre warmed complete N2B2 medium. Add two milliliters of N2B27 to the cell suspension and plate two milliliters of cells in each well of the previously prepared BMM coded six well plate.Incubate the cells at 37 degrees Celsius with 5%CO2 overnight. When MD NPCs are at 70%confluence, transduce them by adding two micro liters of lentivirus guide RNA d-Cas9-DNMT3A vectors and gently swirl the plate. After incubating the cells at the same conditions as previously for 16 hours replace the N2B27 medium 48 hours after the transduction.Add N2B27 with five micrograms per milliliter of pure Meissen to obtain the stable MD NPC lines, grow the cells for three weeks in N2B27 plus pure Meissen to characterize the methylation profile of SNCA intron 1. First extract DNA from each stable transducer cell line using a DNA extraction kit, then use 800 nanograms of DNA to perform a bisulfide conversion with a commercially available kit and then a the bisulfide converted DNA to 20 nanograms per micro liter. For the pyro-sequencing analysis prepare the PCR master mix in a nucleus free tube transfer the reaction plate to a thermo-cycler and start the PCR.Visualize the amplicons using two micro liters of PCR product with a 30 and bromide staining following Agarose gel electrophoresis. For pyro-sequencing assays used mixtures of un-methylated and methylated by sulfide converted DNAs and pyro-sequencing reagents as described in the manuscript. Calculate the methylation values for each CPG site using pyro-sequencing software physical yields of Lentivirus d-Cas9-DMMT3A-GFP vector and the Naive GFP vector generated using this protocol are comparable.This suggests that the utility of the optimized vector backbone combined with the optimized production protocol results in high yields tighter of the CRISPR-dCas9 vectors transduction rates of the Lentivirus d-Cas9-DNMT3 GFP vector was four times lower than that of the Naive GFP vector suggesting that the packaging efficiency of the CRISPR d-Cas9-RNA is reduced. Transduction rates of the Naive PURO vector were five fold higher compared to the d-Cas9-DNMT3A vector. These results were further confirmed by using the Naive GFP and d-Cas9-DNMT3A-GFP.The EB based protocol described here allows the differentiation of MD NPCs. hiPSCs expressed pleasure potency marker Oct4 while the MD NPC is expressed both Nestin and FoxA2. Seven pyro-sequencing assays were designed and validated for the quantification of the methylation status in the SNCA intron 1.All 7 assays were validated and showed linear correlation, using the validated assays it was possible to determine the methylation levels at the 23 CPGs in the SNCA intron 1 The most important thing to remember than attending this procedure is to work with cells that show proper growth and health to ensure consistency across experiments. The production of lentil vectors highlighted in this presentation can be easily adopted to production of integrals deficient anti-viruses it's also can be easily scaled up to generate larger quantities or more concentrated vectors. This technique can pave the way to explore the pathogenic impact of genes de-regulation on age related nor the generative disorders including Alzheimer Disease and related dementias.The advantage of lentil vectors develop here is that it's not hazard or not infectious lentil vectors have only minimal effect on cell cycle they're safe and reliable delivery platform for gene therapy applications.

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JoVE Journal

Genetics

9.3K Görüntüleme.  Duke University Medical Center. Lentiviral vektör platformu en popüler vektör platformu üzerinde büyük bir avantaja sahip, özellikle lentiviral vektörlerin yeteneği nedeniyle daha büyük genetik eklemek karşılamak için. Böylece, bu lentiviral vektör uygulamada tercih edilen platform crispr / Cas araçlarının teslim dahil olacağını görünüyor. Bu teknik, alfa-sinüklein ekspresyonunun doğal düzenleyici mekanizmasını taklit eder.Gen ekspresyonundaki sağlam değişikliklere karşı ifade düze...

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