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.

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