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CRISPR Plasmids: Prime Edit

Prime editing is a “search and replace” gene editing method in which Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is fused to the C-terminus of Cas9 H840A nickase. The fusion enzyme is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). As with a typical gRNA, the pegRNA directs the nickase to the target site by homology to a genomic DNA locus. The longer pegRNA also encodes a primer binding site (PBS) and the desired edits on a RT template.

In the initial version of this tool, PE1 (depicted in the image below), the pegRNA directs the Cas9 nickase to the target sequence where it nicks the non-target strand and generates a 3’ flap. The 3’ flap binds to the primer binding site (PBS) of the pegRNA and the desired edit is incorporated into the DNA by reverse transcription. The edited DNA strand displaces the unedited 5’ flap and the resulting heteroduplex is resolved by the cell’s mismatch repair (MMR) system. Alternatively, the edited 3’ flap may be excised and the target sequence will remain unchanged but available as a substrate for another round of prime editing.

Further improvements engineered into the prime editing components help to optimize and improve the efficiency of this method. In the PE2 system, five mutations were introduced into the RT enzyme (D200N/L603W/T330P/T306K/W313F) to increase activity, enhance binding between the template and PBS, increase processivity, and improve thermostability. PE3 uses the PE2 Cas9 nickase-pentamutant RT fusion enzyme and pegRNA plus an additional simple sgRNA, which directs the Cas9 nickase to nick the unedited strand at a nearby site. The newly edited strand is then favored as the template for repair during heteroduplex resolution. The process of double nicking, however, increases indel formation slightly. Designing the sgRNA with a spacer that only binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after the edit has occurred.

Note: PE2, PE3, and PE3b systems all use the PE2 enzyme. In the PE3 and PE3b systems an sgRNA targeting the opposite strand is added.

CRISPR prime editing schematic depicts 1. Complex formation, target binding, and nicking of non-target strand, 2. Incorporation of edit into target DNA by reverse transcriptase, and 3. Cellular endonucleases and mismatch repair resolving the heteroduplex.
Browse, sort, or search the tables below for CRISPR prime editing plasmids.
Plasmids are available for expression in mammalian systems, bacteria, plants, and Drosophila.


Plasmid Gene/Insert Promoter Selectable Marker PI Publication


Plasmid Gene/Insert Promoter Selectable Marker PI Publication


Plasmid Gene/Insert Promoter Selectable Marker PI Publication


Plasmid Gene/Insert Promoter Selectable Marker PI Publication

Empty Prime Editing gRNA Vectors

A selection of empty gRNA vectors suitable for prime editing are highlighted in the table below. Use the search bar to find a gRNA vector based on expression system, promoter, the type of gRNA (e.g., pegRNA, epegRNA, nicking sgRNA), cloning enzyme, selectable marker, and whether the plasmid contains Cas9.

Plasmid Expression System Promoter Guide RNA Type Cloning Enzyme Co-expressed Cas9 Selection PI
pU6-pegRNA-GG-acceptorMammalianhU6pegRNABsaINomRFP1David Liu
QPM-sgR (pTaU3)PlantTaU3nicking sgRNAEps3I + NcoINoCaixia Gao
pYPQ141D-pegPlantOsU3pegRNANoYiping Qi
pCFD3-NSDrosophilaDrosophila U6:3pegRNABbsINoVermilionNorbert Perrimon
pCFD5-NSDrosophilaDrosophila U6:3pegRNA + nicking sgRNABbsINoVermilionNorbert Perrimon
pHSG1C3MammalianU6pegRNA + nicking sgRNABbsI for sgRNAs; BbsI + PstI for pegRNAsNoXiao Wang
pOsU3PlantOsU3pegRNABsaI + HindIIINoCaixia Gao
pPEgRNA BacteriaJ23119 (BBa_J23119)pegRNASpeI + HindIIINoTilmann Weber
pnsgRNA BacteriaJ23119 (BBa_J23119)nicking sgRNANoTilmann Weber
pPBT-peRNA_GG-PuroMammalian, piggyBachU6pegRNABsmBINoPuromycinJacob Giehm Mikkelsen
pPBT-PE2-PuroTK-pegRNA_GG Mammalian, piggyBachU6pegRNABsmBIYes (Cas9 H840A + MMLV RT)PuroTKJacob Giehm Mikkelsen
pU6-tevopreq1-GG-acceptorMammalianhU6epegRNABsaINomRFP1David Liu
pU6-tmpknot-GG-acceptorMammalianhU6epegRNABsaINomRFP1David Liu
U6-pegRNA-H1-nick sgRNA-mCherryMammalian, AAVhU6 + H1pegRNA + nicking sgRNANomCherryHyongbum Kim
pDAS12069_U6-pegRNA-mCherry MammalianhU6pegRNABpiI for pegRNA, Esp31 for PBS-RTNomCherryErvin Welker
pDAS12222_U6-pegRNA-BFP MammalianhU6pegRNABpiI for pegRNA, Esp31 for PBS-RTNoBFPErvin Welker

Do you have suggestions for other plasmids that should be added to this list?

Fill out our Suggest a Plasmid form or e-mail [email protected] to help us improve this resource!