The plasmids in this study were provided from Addgene, including p3s-Cas9-HN (addgene no.104171), pCMV_ABEmax (addgene no.112095), pCMV-AncBE4max (addgene no. 112094), pUGI-NLS (addgene no.101091), and pRG2 (Addgene no. 104174). Sequences corresponding to sgRNAs were cloned into BsaI-digested pRG2 vector (Addgene no. 104174). For this step, oligos containing the spacer sequence were annealed to form double-stranded DNA fragments with compatible overhangs and ligated using T4 ligase (Enzynomics). To construct pCMV-AncBE4max without UGI, the c-terminal part of BE4max contacting 2× UGI was digested by Cas9 and AgeI endonuclease (NEB) and Gibson cloned using NEBuilder HiFi DNA Assembly master mix (NEB). All plasmids used for transfection experiments were prepared using a NucleoBond Xtra Midi Plus EF kit (MN).
P3s cas9 hn
Manufactured by Addgene
The P3s-Cas9-HN is a laboratory equipment product. It is a Cas9 protein fused with a nuclear localization signal (NLS) and a histidine tag (HN) for purification purposes. The core function of this product is to enable CRISPR-Cas9 genome editing experiments.
Automatically generated - may contain errors
2 protocols using p3s cas9 hn
1
Plasmid Engineering for CRISPR Genome Editing
2
Engineered Cas9 Variants and sgRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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eCas9-1.1- and Cas9-HF1-encoding plasmids (p3s-eCas9-1.1, Addgene #104172; p3s-Cas9-HF1, Addgene #104173) were created via site-directed mutagenesis of a WT Cas9 construct (p3s-Cas9-HN, Addgene #104171). HH-ribozyme sgRNA constructs were cloned via ligation of annealed oligonucleotides that included a HH-ribozyme sequence and a protospacer sequence into a plasmid (pRG2, Addgene #104174) in which sgRNA expression is under the control of the U6 promoter.
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