Worm Single-gRNA 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 18-22nt 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 single gRNA driven by a C. elegans U6 promoter. This plasmid can be co-injected with a C. elegance Cas9 plasmid into the distal arm of the gonad of C. elegans, where they can be incorporated into germ cell nuclei. Alternatively, the gRNA plasmid can be injected into the gonad of a Cas9-expressing worm. Mutant progeny can then be selected, allowing generation of a stable line with heritable gene knockout.

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.

Flexibility: Our regular plasmid gRNA expression vector can be used in conjunction with a variety of Cas9 variants, depending upon the user’s experimental goal.

Non-uniformity of gene delivery: Regular plasmid vectors mostly remain as episomal DNA, without genome integration. If a drug resistance cassette or fluorescent marker is incorporated into the plasmid, cells that have stably integrated the plasmid can be derived by drug selection after extended culture.

PAM requirement: CRISPR/Cas9 based targeting 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. 

CeU6: C. elegans U6 small nuclear RNA promoter. It drives strong expression levels of small RNAs.

gRNA: Guide RNA compatible with the Cas9 variant being used.

Terminator: Pol lll transcription terminator. It allows transcription termination of small RNA transcribed by Pol lll 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.