dCas9 plasmid

CRISPR Human - Repression BRCA1

CRISPR Human - Repression BRCA1
dCas9 plasmid from Addgene

Protocol tips

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate

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.

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3 years 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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Manufacturer protocol

Download the product protocol from Addgene for dCas9 plasmid below.

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