CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) nuclease expression vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN).

Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 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-mediated cleavage of the DNA target site ultimately results in a double-strand break (DSB) which can then be repaired by either of the two following repair pathways – the non-homologous end joining (NHEJ) pathway or the homology directed repair (HDR) pathway. Cellular repair of DSBs by NHEJ is more common and usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSBs on both the host DNA and an exogenous DNA template can result in repair that incorporates the exogenous DNA, generating either small targeted base changes such as point mutations or large sequence alterations such as fragment knockin.

Our gene targeting donor vectors are highly efficient vehicles for delivering exogenous donor templates to achieve targeted insertion of reporters, fluorescent tags, or other desired sequences at genomic sites of interest. The homology-independent targeted integration (HITI) donor vector utilizes the NHEJ mechanism to achieve higher integration efficiency in both dividing and non-dividing cells. The vector is designed to contain the desired insertion sequence flanked by upstream and downstream target sequences that match the cut sites in the host genome. These sequences must contain both the target sequence for the gRNA and the PAM sequence for Cas9 recognition.

When designing HITI donor vectors for NHEJ-mediated integration, it is important to ensure that DSBs will not be introduced following insertion, for instance by excluding or inactivating any PAM sequences in the desired insertion sequence. 

Since undesirable off-target effects are a major drawback associated with CRISPR genome targeting, careful designing of the target-site specific gRNA sequences with minimal off-target scores is critical. Additionally, using dual gRNA sequences to target the host DNA and “bait” sequences on the donor vector minimize off-target integration.

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

Our gene targeting HITI donor vectors are designed to achieve highly efficient and specific NHEJ-mediated insertion of reporters, fluorescent tags or other desired sequence at genomic target sites of interest. The donor vector is designed with the desired donor sequence flanked by upstream and downstream sequences that are targeted by Cas9/gRNA to facilitate efficient insertion following DSBs at both the genomic and donor vector target sites.

Site-specific changes: Delivering exogenous repair templates in the form of gene targeting donor vectors enables NHEJ-mediated introduction of sequence changes at the genomic target sites of interest.

Higher efficiency integration: NHEJ repair occurs during all phases of the cell cycle, facilitating integration in both dividing and non-dividing cells. While NHEJ is more efficient than the HDR repair mechanism, many cells will perform repair without integration. Therefore target cells must be carefully screened.

Technical simplicity: Our gene targeting donor vectors can be delivered to mammalian cells by conventional transfection along with the target site specific gRNA sequences and the Cas9 protein for NHEJ-mediated genome editing. Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

PAM requirement: CRISPR/Cas9 target sites must contain an NGG sequence, known as PAM, located on the immediate 3’ end of the gRNA recognition sequence. Of note, it is important to ensure that DSBs will not be introduced following insertion, for instance by excluding or inactivating any PAM sequences in the desired insertion sequence. 

CRISPR Target Sequence 1: The first CRISPR target sequence is cloned here.

Donor Sequence/Cassette: User-selected insertion sequence to be knocked in at the genomic target site of interest.

CRISPR Target Sequence 2: The second CRISPR target sequence is cloned here.

MC1 Promoter: Polyoma virus enhancer fused to herpes simplex virus thymidine kinase promoter. It drives the ubiquitous expression of the downstream marker gene when added.

Marker: Diphtheria toxin A gene. Allows negative selection of cells by inducing cell apoptosis by inhibiting EF-2 synthesis.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

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.