CRISPR Human - Activation MIAT

CRISPR Human - Activation MIAT
M-ST1n-VP64 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

Vectors used and designed
Activation domains were cloned using a combination of Gibson and Gateway assembly or Golden Gate assembly methods. For experiments involving multiple activation domains, ADs were separated by short glycine-serine linkers. Activator sequences are listed in the Supplementary Data (vectors to be deposited in Addgene). All SP-dCas9 plasmids were based on Cas9m4-VP64 (Addgene #47319)6, ST1-dCas9 plasmids were based on M-ST1n-VP64 (Addgene #48675)15. Sequences for gRNAs are listed in the supplementary information. gRNAs for endogenous human gene activation were selected to bind between 1 and 1000 bp upstream of the transcriptional start site (TSS). gRNAs for iPSC differentiation to iNeurons, targeting NGN2 and NEUROD1, were selected to bind between 1 and 2000 base pairs upstream of the transcriptional start site. All human gRNAs were expressed from either cloned plasmids (Addgene #41817)5 or integrated into the genome through lentiviral delivery (plasmid SB700). Guide RNA sequences are listed within the Supplementary Data. Reporter targeting gRNAs were previously described (Addgene #48671 and #48672)6.

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4 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 M-ST1n-VP64 below.

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