IG® PEmax enzyme is the purified recombinant Streptococcus pyogenes Cas9 nickase mutant (H840A)-MMLV-Reverse Transcriptase fusion protein containing a NLS.
PE pegRNA Synthesis Kit Also Available
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids1. The CRISPR system consists of a short non-coding guide RNA (sgRNA) made up of a target complementary CRISPR RNA (crRNA) and an auxiliary transactivating crRNA (tracrRNA)2. Prime-editing is gaining popularity as a precision gene-editing technique. Prime-editing enzymes are a fusion of the Cas9 nickase mutant (H840A) with a modified Murine Moloney Leukemia Virus-Reverse Transcriptase (MMLV-RT). One of the resulting optimized prime-editing fusions is known as PEmax enzyme3-4. This prime-editing enzyme has enabled precise gene-editing without the need for double-stranded DNA breaks (DSBs) or donor DNA templates. PEmax requires a unique long gRNA known as “pegRNA” shown schematically in Figure 1.
Intact Genomics (IG®) PEmax enzyme is the purified recombinant Streptococcus pyogenes Cas9 nickase mutant (H840A)-Murine Moloney Leukemia Virus-Reverse Transcriptase (MMLV-RT) fusion protein3-4 containing a nuclear localization signal (NLS) at the C-terminal for targeting to the nucleus. This enzyme is designed to perform CRISPR prime-editing3-4. The physical purity of this enzyme is ≥85% as assessed by SDS-PAGE with Coomassie® blue staining.
Product Source
E. coli BL21 (DE3) strain expressing a recombinant PEmax gene from Streptococcus pyogenes with custom protein purification tags.
Components and Storage
PEmax Enzyme Kit contains the items below. Store all components at -20°C.
- PEmax enzyme
- 5x PEmax Nicking Buffer: 20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5 @ 25 °C
Quality Control Assays
PEmax enzyme is free from detectable RNase, Endonuclease (nicking) and non-specific DNase activities.
Functional Testing
We have demonstrated that PEmax has specific nicking activity in the presence of an in vitro transcription-synthesized pegRNA guide molecule (Fig.2) and in vitro RT activity (not shown).
Enzyme Dilution/Storage Buffer (Option): 50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 ºC
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