CRISPR Plasmids: Prime Edit

Prime editing is a “search and replace” gene editing method in which a reverse transcriptase (RT) is fused to the C terminus of Cas9 H840A nickase. The fusion enzyme is capable of installing targeted insertions, deletions, and point mutations using a prime editing guide RNA (pegRNA). As with a typical gRNA, the pegRNA is designed with a spacer that binds to a specific genomic DNA locus and directs the nickase to the target site. The longer pegRNA also encodes a primer binding site (PBS) and the desired edits on an RT template.

During prime editing, 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 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, if the edited 3’ flap is excised, the target sequence remains unchanged and available as a substrate for another round.

Figure 1: Overview of prime editing. Created with BioRender.com.

The original prime editing enzyme is named PE1. PE1 and most other prime editors use the Moloney murine leukemia virus (M-MLV) reverse transcriptase. Engineering of the PE enzyme and related components have helped to improve the efficiency of editing, including exploring new sources for the reverse transcriptase. Many PE enzymes and related tools are available:

• PE2 - introduction of five mutations in RT enzyme
• PE3 - PE2 plus additional sgRNA
• PE4 - PE2 plus MLH1 (a protein component of MMR) mutant to inhibit MMR
• PE5 - PE3 plus MLH1 mutant to inhibit MMR
• PEmax - optimized PE2 enzyme containing RT optimized for human codons, additional nuclear localization signals, and two mutations in Cas9 to enhance nuclease activity; can be used in PE2–PE5 systems
• PE6 - small prime editors with optimized RT and/or Cas9 domains
• PE7 - addition of RNA-binding exonuclease protection factor La to PEmax to enhance pegRNA stability
• epegRNA - additional protection added to 3’ tail of pegRNA to prevent RNA degradation

For more in-depth information on these PE tools, check out our prime editing blog post. Need help designing a pegRNA? Try the DeepPrime design tool from Hyongbum Kim's lab.

Browse, sort, or search the tables below for CRISPR prime editing plasmids.
Plasmids are available for expression in mammalian systems, bacteria, plants, and Drosophila.

Mammalian

Bacteria

Plant

Drosophila

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-acceptor	Mammalian	hU6	pegRNA	BsaI	No	mRFP1	David Liu
	QPM-sgR (pTaU3)	Plant	TaU3	nicking sgRNA	Eps3I + NcoI	No		Caixia Gao
	pYPQ141D-peg	Plant	OsU3	pegRNA		No		Yiping Qi
	pCFD3-NS	Drosophila	Drosophila U6:3	pegRNA	BbsI	No	Vermilion	Norbert Perrimon
	pCFD5-NS	Drosophila	Drosophila U6:3	pegRNA + nicking sgRNA	BbsI	No	Vermilion	Norbert Perrimon
	pHSG1C3	Mammalian	U6	pegRNA + nicking sgRNA	BbsI for sgRNAs; BbsI + PstI for pegRNAs	No		Xiao Wang
	pOsU3	Plant	OsU3	pegRNA	BsaI + HindIII	No		Caixia Gao
	pPEgRNA	Bacteria	J23119 (BBa_J23119)	pegRNA	SpeI + HindIII	No		Tilmann Weber
	pnsgRNA	Bacteria	J23119 (BBa_J23119)	nicking sgRNA		No		Tilmann Weber
	pPBT-peRNA_GG-Puro	Mammalian, piggyBac	hU6	pegRNA	BsmBI	No	Puromycin	Jacob Giehm Mikkelsen
	pPBT-PE2-PuroTK-pegRNA_GG	Mammalian, piggyBac	hU6	pegRNA	BsmBI	Yes (Cas9 H840A + MMLV RT)	PuroTK	Jacob Giehm Mikkelsen
	pU6-tevopreq1-GG-acceptor	Mammalian	hU6	epegRNA	BsaI	No	mRFP1	David Liu
	pU6-tmpknot-GG-acceptor	Mammalian	hU6	epegRNA	BsaI	No	mRFP1	David Liu
	U6-pegRNA-H1-nick sgRNA-mCherry	Mammalian, AAV	hU6 + H1	pegRNA + nicking sgRNA		No	mCherry	Hyongbum Kim
	pDAS12069_U6-pegRNA-mCherry	Mammalian	hU6	pegRNA	BpiI for pegRNA, Esp31 for PBS-RT	No	mCherry	Ervin Welker
	pDAS12222_U6-pegRNA-BFP	Mammalian	hU6	pegRNA	BpiI for pegRNA, Esp31 for PBS-RT	No	BFP	Ervin Welker