Gene Targeting Donor Vector

Overview

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 can be repaired by homology-directed repair (HDR), using exogenous donor DNA template, which is co-introduced into cells with the CRISPR/Cas9 components. This can result in replacement of the target genomic DNA sequence with sequence from the donor 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 donor vector is designed to contain the desired insertion sequence flanked by upstream and downstream homology arms (homologous sequences upstream and downstream of the target site of interest). Efficient HDR targeting also requires the DSB introduced by Cas9 to be located within a proximity of the target site of insertion, ideally within 10-15 bp of the homologous arms. Additionally, when designing donor vectors for HDR, it is critical to either exclude or inactivate any PAM sequences in the repair template when present, to prevent Cas9 from disrupting the donor template or the edited genomic locus after it has been edited by HDR.

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, off-target effects can be further minimized by using the mutant nickase form (hCas9-D10A) of the standard humanized hCas9 which generates single-stranded cuts in DNA instead of DSBs. If hCas9-D10A nickase is used in conjunction with two gRNAs targeting the two opposite strands of a single target site, it will generate single strand cuts on both strands, resulting in a DSB at the target site. This approach generally reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated. Nicked genomic DNA also frequently undergoes HDR, and if exogenous template DNA in the form of a donor vector is introduced into the cell along with a targeted hCas9-D10A nickase, then desired sequence changes in the genome can be generated using this approach as well.

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

References Topic
Science. 339:819 (2013) Description of genome editing using the CRISPR/Cas9 system
Biotechniques. 59:201 (2015) CRISPR/HDR-mediated knockin of large DNA fragments
Front Genet. 9:691 (2019) Methodologies for improving HDR efficiency
Cell. 154:1380 (2013) Use of Cas9 D10A double nicking for increased specificity

Highlights

Our gene targeting donor vectors are designed to achieve highly efficient HDR-mediated insertion of reporters, fluorescent tags or other desired sequence at genomic target sites of interest. The donor vector is designed with the desired insertion sequence flanked by target site specific upstream and downstream homologous sequences to facilitate efficient recombination following CRISPR generated DSBs at the genomic target sites. The lengths of the homologous arms are adjusted depending upon the size of the desired edit, with longer insertions requiring longer arms.

Advantages

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

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 HDR-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.

Disadvantages

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.

Low efficiency: Upon Cas9-induced cleavage of DNA target sites, HDR-mediated repair of the cleaved sites occurs at a much lower frequency than compared to NHEJ-mediated repair. As a result, CRISPR/Cas9 targeting in the presence of an exogenous donor template will give rise to a mixed population of cells, some repaired by the NHEJ pathway while others repaired by the HDR pathway. Therefore, careful screening of the resultant cell population is essential to isolate clones containing the desired HDR-edited sequence. For obtaining cells with homologous alleles altered in the same way, additional round(s) of knockin screen are often needed.

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. It is critical to either exclude or inactivate any PAM sequences in the donor vector when present, to prevent Cas9 from disrupting the donor template or the edited genomic locus after it has been edited by HDR.

Key components

5’-Homology Arm: Homologous sequence immediately upstream of the target site of insertion. The length of the homology arms depends on the length of the sequence to be inserted, with longer insertions requiring longer arms.

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

3’-Homology Arm: Homologous sequence immediately downstream of the target site of insertion. The length of the homology arms depends on the length of the sequence to be inserted, with longer insertions requiring longer arms.

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.

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