CRISPR Plasmids: Drosophila

The following CRISPR plasmids have been designed for use in Drosophila and other insects.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Nick

CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).

Prime Edit

Cas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). The pegRNA directs the nickase to the target site by homology to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template.

Activate

Catalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator to your specific locus.

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.

	gRNA Plasmid	Promoter	Cloning Enzyme(s)	Delivery	Resistance	Co-expressed Cas9	Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
	pCFD1-dU6:1gRNA	dU6:1	BbsI	Injection or in vitro transcription	Virmilion	none, need Cas9 plasmid	Bullock and Port
	pCFD2-dU6:2gRNA	dU6:2	BbsI	Injection or in vitro transcription	Virmilion	none, need Cas9 plasmid	Bullock and Port
	pCFD3-dU6:3gRNA	dU6:3	BbsI	Injection or in vitro transcription	Virmilion	none, need Cas9 plasmid	Bullock and Port
	pCFD4-U6:1_U6:3tandemgRNAs	dU6:1 and dU6:3	BbsI	Injection or in vitro transcription	Virmilion	none, need Cas9 plasmid	Bullock and Port
	pAc-sgRNA-Cas9	dU6	BspQI	Transfection	Puromycin	yes, cut	Ji-Long Liu
	U6-BbsI-crRNA	dU6	BbsI	Transfection		none, need Cas9 plasmid	O'Connor-Giles, Harrison, Wildonger
	pU6-BbsI-chiRNA	dU6	BbsI	Transfection		none, need Cas9 plasmid	O'Connor-Giles, Harrison, Wildonger