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CRISPR Plasmids: C. elegans


The following CRISPR plasmids have been designed for use in C. elegans.

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.

Plasmid Gene/Insert Promoter PI Publication

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)
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

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

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