Patient-derived hiPSCs corresponding to the non-variant MYH7 (WT Ib) and heterozygous MYH7 E848G variant were previously generated [6 (link)]. A total of 1000 k pelleted hiPSCs were mixed with 1 μL 10 μM SP-dCas9-VPR (Addgene, 63798, Watertown, MA, USA), 9 μL Buffer R2 (STEMCELL Technologies, 100-0691, Cambridge, MA, USA), 1 μL 30 μM of gRNA (Table 2 ), and 1.5 μg of pJet-MYH7-EGFP-PGK-PuroR plasmid (Supplementary File S1). For TP53 ablation, pJet plasmid was omitted. Cells were electroporated at 1400 V for 20 ms with a 10 μL tip using the Neon Transfection System (Thermo Fisher, MPK5000, Waltham, MA, USA). Transfected cells were plated in mTeSR+ without penicillin/streptomycin, and supplemented with CloneR2 (STEMCELL Technologies, 100-0691, Cambridge, MA, USA) and 10 μM ROCK inhibitor on plates coated with 80 μg/mL Matrigel. Cells were fed every other day with mTeSR+ with 0.175 μg/mL puromycin dihydrochloride (Thermo Fisher, A1113803, Waltham, MA, USA) and replated at 88 cells/cm2 in a 10 cm plate coated with 80 μg/mL Matrigel for colony picking. Clones were replated in 96-well plates for expansion and genomic DNA harvesting.
Sp dcas9 vpr
Manufactured by Addgene
Sourced in United States
The SP-dCas9-VPR is a gene activation tool that can be used to upregulate target gene expression. It consists of a catalytically inactive Cas9 (dCas9) fused to the VPR transcriptional activation domain. This system can be programmed with guide RNAs to bind to specific DNA sequences and induce transcriptional activation at those loci.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin dihydrochloride, by Thermo Fisher Scientific (2 mentions)
Buffer r2, by STEMCELL (2 mentions)
Cloner2, by STEMCELL (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Cas9m4 vp64, by Addgene (2 mentions)
Lenticas9 blast, by Addgene (1 mentions)
Pmcb306, by Addgene (1 mentions)
Sgrna, by Addgene (1 mentions)
Pcdna dcas9 p300 core, by Addgene (1 mentions)
Geneticin selective antibiotic g418, by Thermo Fisher Scientific (1 mentions)
Pspcas9 2a puro px459, by Addgene (1 mentions)
4d nucleofector kit, by Lonza (1 mentions)
Pspcas9 bb 2a gfp, by Addgene (1 mentions)
Pspcas9 bb 2a puro, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Stellaris fish probe designer, by Biosearch Technologies (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Plv hu6 sgrna hubc dcas9 krab t2a puro, by Addgene (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
16 protocols using sp dcas9 vpr
1
Generation of MYH7 variant hiPSCs
2
CRISPR-Cas9 Genome Editing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BACs were purchased from Thermo Fisher Scientific (Waltham, MA) in the case of BAC CTD-2166E9, and from the BACPAC Resources Center (Children’s Hospital Oakland Research Institute, CA) in the case of all other BACs. BACs were purified from E. coli using the Nucleobond Xtra BAC Maxi Kit (Macherey-Nagel, Duren, Germany). SP-dCAS9-VPR (Addgene ID: 63798) was provided by the Qi lab [19 (link)], and the sgRNA-encoding plasmid along with the Cas9 plasmid, pMCB306 (Addgene ID: 89360) and lentiCas9-Blast (Addgene ID: 52962) respectively, were gifts from Michael Bassik [30 (link),31 (link)]. Guides were cloned into the pMCB306 BlpI/BstXI sites using annealed oligos with the appropriate sticky ends (S1 File ).
3
CRISPR Plasmid Cloning and Assembly
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids encoding SpCas9 (Plasmid #41815), sgRNA (#47108) and SpdCas9-VPR (#63798) were obtained from Addgene. The backbone for the targeting vectors was synthesized by IDT as gene blocks and cloned into a pCDNA3.1 plasmid. The oligonucleotides used to create the guide sequences were obtained from IDT, hybridized, phosphorylated and cloned in the sgRNA vector using BbsI (15 (link),34 (link)). The target sequences are provided in Supplementary Table S1 .
4
Plasmid-Based Lentiviral Vector Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the plasmids (Gagpol MLV, GagMLV-Cas9, VSV-G) and gRNA-expressing plasmids to produce the NBs were described previously25 (link),26 (link) and are available at Addgene (https://www.addgene.org ). SP-dCas9-VPR was a gift from George Church (Addgene, plasmid no. 63798). Lenti CRISPR was a gift from F. Zhang (Addgene, plasmid no. 49535). The GagMLV-CAS9 fusion was constructed by sequential insertions of PCR-amplified fragments in a eukaryotic expression plasmid harboring the human cytomegalovirus (CMV) early promoter, the rabbit β-globin intron, and polyadenylation signals. The MA-CA-NC sequence from Friend murine leukemia virus (accession no. M93134 ) was fused to the MA/p12 protease-cleavage site (9 aa) and the Flag-nls-spCas9 amplified from pLenti CRISPR. The BaEVRless envelope glycoproteins were described previously28 (link) and can be obtained under material and transfer agreement from the corresponding author (E.V.). The BaEVRless envelope glycoprotein is from the baboon endogenous retrovirus and its R-peptide was removed to improve LV pseudotyping as described. Both envelope glycoproteins (VSV-G and BAEV) were expressed in the phCMV-G expression plasmid.48 (link) The HIV-SFFV-eGFP vector and the HIV CMV-eGFP LV encoding plasmids to produce the GFP murine and human organoid cell line were described previously.31 (link),49 (link)
5
Generating CRISPRa Cell Line using CRISPR/Cas9-NHEJ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate the CRISPRa cell line, the CRISPR/Cas9 and nonhomologous end joining (NHEJ) pathway (CRISPR/Cas9-NHEJ)-mediated genome editing strategy was applied [34 (link)]. First, the CRISPR/Cas9 plasmids, pSpCas9 2A-Puro (PX459) (a gift from Feng Zhang, Addgene plasmid #62988) targeting the 3′ region of chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (GAPDH #1) and targeting the common region of the three CRISPRa vectors (CRISPRa #1), Cas9m4-VP64 (Addgene plasmid #47316) [35 (link)], SP-dCas9-VPR (Addgene plasmid #63798) [13 (link)], or pcDNA-dCas9-p300 Core (Addgene plasmid #61357) were constructed [35 (link),36 (link)]. The 2.5 × 105 DF-1 cells were then transfected with 1 µg of GAPDH #1, 1 µg of CRISPRa #1, and 2 µg of each CRISPRa vector using Lipofectamine 3000 according to the manufacturer’s instructions. The transfected cells were treated with Geneticin Selective Antibiotic (G418, 300 µg/mL) (Thermo Fisher Scientific, Waltham, MA, USA, 24–48 h post transfection, and the drug selection was maintained to establish cell lines for at least 2 weeks. The gRNA and oligo sequences used in all-in-one CRISPR/Cas9 vector construction are listed in Table S1 .
6
Generating isogenic hiPSC lines with MYH7 variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patient-derived hiPSCs corresponding with non-variant MYH7 (WT Ib) and heterozygous MYH7E848G/+ variant were previously generated [6 (link)]. 1000k pelleted hiPSCs were mixed with 1 μL 10 μM SP-dCas9-VPR (Addgene, 63798), 9 μL Buffer R2 (STEMCELL Technologies, 100-0691), 1 μL 30 μM of gRNA (Table 2 ), and 1.5 μg of pJet-MYH7-EGFP-PGK-PuroR plasmid (Supp. File 1). For TP53 ablation, pJet plasmid was omitted. Cells were electroporated at 1400 V for 20 ms with a 10 μL tip using the Neon Transfection System (Thermo Fisher, MPK5000). Transfected cells were plated in mTeSR+ without penicillin/streptomycin supplemented with CloneR2 (STEMCELL Technologies, 100-0691) and 10 μM ROCK inhibitor on plates coated with 80 μg/mL Matrigel. Cells were fed every other day with mTeSR+ with 0.175 μg/mL puromycin dihydrochloride (Thermo Fisher, A1113803) and replated at 88 cells/cm2 in a 10 cm plate coated with 80 μg/mL Matrigel for colony picking. Clones were replated in 96 well plates for expansion and genomic DNA harvesting.
7
CRISPR-mediated regulation of ELF5 and EHF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single guide RNAs (sgRNAs) targeting ELF5 and EHF promoters were designed within 1 kb 5’ to the transcriptional start sites (TSS). Two to four sgRNAs were designed to target DHS cores at chr11p13 using Benchling 27 (Table S6 ) and were chosen based on the highest on‐target and lowest off‐target scores. sgRNAs were cloned into the pSPgRNA plasmid (Addgene #47108) and nucleofected with the SP‐dCas9‐VPR plasmid (Addgene #63798) into either 16HBE14o‐ or A549 cells with Lonza 4D‐Nucleofector kits (P3 Primary Cell V4XP‐3032 for 16HBE14o‐ and SE Cell Line V4XC‐1032 for A549). For each experiment, pSPgRNA alone was nucleofected with SP‐dCas9‐VPR and used as a negative control. Using protocols described above, RNA was extracted 48 hours post‐nucleofection and reverse transcription quantitative polymerase chain reaction (RT‐qPCR) assays were performed. Data were normalized individually to the empty vector control.
8
CRISPR Knockout and Activation of BRCA1P1
9
Antisense lncRNA Functional Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequences of antisense lncRNAs (RP1-261G23.7, VEGF-AS1 and EST AV731492, VEGF-AS2, Supplementary Materials Table S1 ) were ordered as gBlocks from Integrated DNA Technologies (IDT, Coralville, IA, USA) and subcloned into pcDNA3.1 (+) (Thermo Fisher Scientific, Waltham, MA, USA) using EcoRV (NEB) and XbaI (NEB) restriction sites. pcDNA3.1-GFP was used as a control. SgRNAs targeting VEGF-AS2 putative promoter were designed (Supplementary Materials Table S2 ), ordered from IDT and subcloned into pcDNA3.1-H1sgRNA described in [39 (link)]. SgRNA targeting alpha-1 antitrypsin (target site does not have a PAM sequence) was used as a control (Supplementary Materials Table S2 ). SP-dCas9-VPR was obtained from Addgene (Addgene plasmid #63798) [19 (link)]. Antisense PTOs and antisense oligonucleotides with 3′-Biotin modification targeting VEGF-AS1 and VEGF-AS2 were designed with Sfold software or Biosearch Technologies’ Stellaris FISH Probe Designer, respectively, and ordered from IDT (Supplementary Materials Table S2 ). In PTO experiments, miRN367 targeted [40 (link),41 (link)] antisense PTO was used as a control (Supplementary Materials Table S2 ).
10
Generation of Lentiviral Constructs for CRISPR Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The third-generation lentiviral transfer plasmid pLV_dCas9-VPR_sgRNA was cloned by replacing the KRAB domain from pLV_hU6-sgRNA_hUbC-dCas9-KRAB-T2A-Puro (Addgene plasmid 71236, a gift from Charles Gersbach)98 (link) with the VPR domain from SP-dCas9-VPR (Addgene plasmid 63798, a gift from George Church).51 (link) For each sgRNA, sense and anti-sense custom DNA oligonucleotides (Integrated DNA Technologies) containing the recognition sequence were annealed and ligated into pLV_dCas9-VPR_sgRNA using T4 DNA ligase (Promega, Madison, WI), as described previously.99 (link) pLV_dCas9-VPR_sgRNA with no inserted sgRNA recognition sequence was used as a control. The third-generation lentiviral packaging plasmids pMDLg/pRRE (Addgene plasmid 12251) and pRSV-Rev (Addgene plasmid 12253) and envelope plasmid pMD2.G (Addgene plasmid 12259; all gifts from Didier Trono) were used for lentiviral production.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!