pSLQ1658-dCas9-EGFP

CRISPR Human - Activation BRCA1

Experiment
CRISPR Human - Activation BRCA1
Product
pSLQ1658-dCas9-EGFP from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Preparation of the TET1CD- dCas9-EGFP fusion protein
The fusion protein of TET1 catalytic domain (TET1CD), and dCas9 was generated by sequentially assembling the coding sequences of the desired proteins using standard restriction enzyme digest and ligation method. The source plasmids of dCas9 (#51023 from Bo Huang and Stanley Qi lab), and TET1 (#49792 from Anjana Rao lab) were obtained from the Addgene plasmid repository (https://www.addgene.org/). In addition, a fusion protein of only dCas9-EGFP was used as a negative control to the catalytic activity of TET1. Inserts were then amplified by Polymerase Chain Reaction (PCR) from the respective source plasmids with desired restriction sites flanking on either side of the amplicons with CloneAmpHiFi PCR Premix (639298, Clontech Laboratories Inc.). The PCR reaction was carried out as specified by the manufacturer for the template DNA concentration >100 ng with 35 cycles of amplification. Detail of the PCR amplification and sequencing primers have been summarized in Table S1. The inserts were incorporated into the ‘pAAV_EF1α_WPRE_hGHpA’ backbone of mammalian expression vector (plasmid #47457, from Zhang F, Addgene). The original vector was sequentially digested with Acc651 and BamHI to incorporate TET1CD, and BamHI and XbaI to incorporate dCas9. Incorporation of TET1CD and dCas9 into the vector has replaced the CRY2PHR_NLS-VP64 of the original plasmid. The final fusion protein hence formed was in the frame of pAAV_EF1α_TET1CD-dCas9-NLS-2A-GFP_WPRE_hGHpA. Two fusion protein constructs were made by using without or with a long linker sequence between the TET1CD and dCas9 functional domains and defined respectively as TDE-I or TDE-II constructs. PCR amplified inserts and the vector template were digested with restriction endonucleases followed by gel purification using the QIAEX II gel extraction kit (20021, QIAGEN). Purified vector and inserts thus made were ligated along with requisite amount of T4 DNA ligase buffer and enzyme system (M0202S, New England Biolabs) and kept at room temperature for 15 min. The ligated product was then transformed into the stellar competent cells (PT5056-2, Clonetech Laboratories Inc.) and plated out on an Ampicillin (Amp) supplemented LB agar plate. Suitable clones were propagated in LB-Amp media and the plasmids were extracted with QIAprep Spin Miniprep Kit (27104, QIAGEN). The full length nucleotide sequence of the fusion proteins can be found in the Supplementary Information (Supplementary Sequence-1 and 2). The fusion protein was sequenced against a panel of primers as summarized in Table S1. In addition, we have generated fusion protein of dCas9 with inactive TET1CD for the negative control experiments. The preparation of inactive TET1CD-dCas9 fusion protein, PCR primers (Table S2), and the full nucleotide sequence (Supplementary Sequence-3) of the fusion protein can be found in the Supplementary Information.

Detection of TET1CD-dCas9 fusion protein
The total protein was extracted from the cells, individually or co-transfected with TDE plasmids and sgRNAs respectively, and the excitation spectra of the EGFP fluorescence of the fusion proteins were recorded using the fluorometer (Cary Eclipse, Agilent Technologies). Expression of the fusion proteins in HeLa cells were confirmed with western blot. For performing the western blot, cells were rinsed twice with ice-cold PBS followed by lysis with M-PER mammalian protein extraction reagent supplemented with Halt protease inhibitor cocktail (Thermo Scientific). The concentration of the extracted proteins was then determined with Coomassie Plus (Bradford assay kit; Pierce). Twenty micrograms of the extracted proteins were loaded per lane onto a 4% to 15% polyacrylamide gel (Bio-rad, USA) for electrophoresis and electro-transferred to a nitrocellulose membrane (Biorad). The membrane was then blocked in 5% nonfat dry milk in (TBS-T) Tris-buffered-saline-Tween buffer (10mM Tris (pH 8.0), 140 mMNaCl, 0.1% Tween 20) overnight at 4°C, followed by incubation with rabbit polyclonal anti-EGFP antibody (ab290; Abcam) at 1:1000 dilution overnight in 5% milk-TBS-T. After washing with fresh TBS-T for three times, the membrane was incubated with rabbit secondary antibody conjugated to AlexaFluor 680 (ab150077; Thermo-Scientific) at 1:2000 dilutions in the blocking buffer for 1 h at room temperature. The membrane was re-washed with TBS-T for three times and scanned using a Li-Cor Odyssey scanner (CLx) system.

Designing of the sgRNA
Four sgRNA strings were designed as PCR cassettes (501 bp) with U6 promoter (Life Technologies, USA), which bind to four different specific sequences in the BRCA1 promoter. For subsequent uses, the sgRNAs were amplified with a set of primer, which are as follows: Forward: 5′-GGCCTATTGGTTAAAAAATGAGCTG-3′ and Reverse: 5′-GAGCGGATAACAATTTCACACAGGA-3′. PCR amplicons of the sgRNA cassettes were then purified through gel-extraction (20021, QIAGEN). Full length sequence of the sgRNA PCR cassettes and their corresponding binding sites has been described in the Supplementary Information (Supplementary Sequence-4-9).

Cell culture and transfection
HeLa or MCF7 cells were seeded at the density of 0.7 × 106 and cultured at 37°C with 5% CO2 in presence of DMEM/F-12 supplemented with 10% FBS, 1% glutamax and 1% penicillin-streptomycin (Life Technologies). Cells were co-transfected with TET1-dCas9 plasmid plus sgRNA strings using Lipofectamine LTX (Life Technologies) system according to the manufacturer's instruction. Briefly, 70-80% confluent cells in 35 mm plates were co-transfected with 2.5 μg of TET1-dCas9 plasmid and 1 μg of sgRNA strings. Cells individually transfected with dCas9 plasmids were considered as the negative control. Transfection efficiency of the plasmids was assessed with microscopic analyses. The fluorescence intensities of transfected cells were examined under fluorescence microscope (EVOS FL cell imaging system, Life Technologies), 24 h after transfection to assess the expression of the EGFP fluorescence from the TDE fusion proteins. During imaging, cells were incubated with HBSS buffer (pH adjusted to 7.4 with 2 M NaOH).

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Discussion

Discussion

1 year ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pSLQ1658-dCas9-EGFP below.

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