Worm gRNA and Cas9 Coexpression 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 former, allowing users to edit the genome using a single plasmid containing both a gRNA and a C. elegans codon-optimized Cas9 (CeCas9). This plasmid can be injected into the distal arm of the gonad of C. elegans, where it is incorporated into germ cell nuclei. Mutant progeny can then be selected, allowing generation of a stable line with heritable gene knockout.

To achieve effective gene editing capability with the CRISPR/Cas9 system in C. elegans, VectorBuilder has developed the gRNA and Cas9 coexpression vector. In this vector, gRNA transcription is under the control of a C. elegans U6 promoter. Downstream of the gRNA terminator, CeCas9 is driven by the strong C. elegans promoter eft-3 and terminated by a C. elegans UTR with polyA.

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.

Heritability of mutation: Incorporation of the CRISPR/Cas9 components into germ cells allows generation of a stable mutant line.

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: 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.

eft-3: C. elegans ef1ɑ promoter. It drives strong expression levels in somatic cells.

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

CeCas9: CRISPR-associated endonuclease that cuts DNA at a location specified by gRNA. This S. pyogenes Cas9 is codon-optimized for C. elegans and contains multiple synthetic introns, a carboxy terminal nuclear localization signal, and an HA tag.

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.