CRISPR Plasmids: C. elegans
The following CRISPR plasmids have been designed for use in C. elegans.
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.
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.
|Delivery||Resistance||Co-expressed Cas9||Depositing lab|
|pMB70||cU6||BsaI||Transfection||none, need Cas9 plasmid||Boxem|
|pMB60||T7||BsaI||In vitro transcription||none, need Cas9 plasmid||Boxem|
|PU6::klp-12_sgRNA||cU6||PCR (see paper)||Transfection||none, need Cas9 plasmid||Calarco|
|pDD162 (Peft-3::Cas9 + Empty sgRNA)||R07E5.16 U6||yes, cut||Goldstein|
|DR274||T7||BsaI||In vitro transcription||none, need Cas9 plasmid||Joung|
|SP6-sgRNA-scaffold||SP6||AflII||In vitro transcription||none, need Cas9 plasmid||Sternberg|
|L4440_BioBrick-sgRNA||T7||BbsI, BsaI||Ingestion by C. elegans||none, need Cas9 plasmid||Ristow|