Worm Cas9 Expression Vector

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system has greatly facilitated inactivation of genes in vitro and in vivo in a wide range of organisms. In this genome-editing system, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with complementary 18-22 nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome. Cas9 screens the genome and cleaves within sequences complementary to the gRNA, provided they are immediately followed by the protospacer adjacent motif (PAM) NGG. Double strand breaks are then repaired via homologous recombination or non-homologous end-joining, resulting in indels (insertion or deletion of bases in the genome) of variable length.

Utilizing the CRISPR/Cas9 system in C. elegans allows for the rapid generation of knockout lines by simply delivering either an all-in-one vector (a single vector expressing both Cas9 and gRNA) or separate vectors for driving Cas9 and gRNA expression, respectively. This vector system is the latter, containing a C. elegans codon-optimized Cas9 downstream of a synthetic intron, which enhances gene expression, and terminated by a C. elegans UTR with polyA. The promoter driving Cas9 expression can either be pasted or chosen from our database of ubiquitous (eft-3 and pie-1), tissue-specific (spe-11, myo-2, myo-3, and rab-3), or inducible promoters (hsp16-2 and hsp16-48).

This plasmid can be co-injected with a C.elegans gRNA plasmid into the distal arm of the gonad, where they can be incorporated into germ cell nuclei. Mutant progeny can then be selected, allowing generation of a stable line with heritable gene knockout. Alternatively, this plasmid can be injected alone into the gonad to produce a Cas9-expressing line.

For further information about this vector system, please refer to the papers below.

Technical simplicity: Delivery of plasmid vectors into cells by microinjection is technically straightforward and far easier than virus-based vectors which requires the packaging of live virus.

High-level expression: A synthetic promoter-proximal intron is incorporated into the vector backbone. This leads to very high expression levels of the genes carried on the vector.

Non-uniformity of gene delivery: Although high copy numbers of transgenes can be achieved, this can be non-uniform. Some cells may carry many copies while others may carry very few, or none.

PAM requirement: Cas9 must be localized by a gRNA to the target site, which is dependent on a strict requirement for a protospacer adjacent motif (PAM) of NGG, located on the immediate 3’ end of the gRNA recognition sequence. 

Promoter: The promoter driving your gene of interest is placed here.

Synthetic intron: This is placed proximal to the promoter to enhance gene expression.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest, e.g. C. elegans codon-optimized Cas9 (CeCas9), is placed here.

unc-54 3’ UTR+polyA: Myosin-4 3’ UTR with downstream polyA from C. elegans. It allows transcription termination and polyadenylation of mRNA transcribed by Pol II RNA polymerase.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.