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.
