PEmax Enzyme

Description

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 enzyme^3-4.  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 protein^3-4 containing a nuclear localization signal (NLS) at the C-terminal for targeting to the nucleus. This enzyme is designed to perform CRISPR prime-editing^3-4. 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 MgCl_2, 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

References

• Jinek, M. et al. A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
• Mali, P. et al. RNA-Guided Human Genome Engineering via Cas9. Science 339, 823–826 (2013).
• Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652.e29 (2021).
• Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
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