In Vivo Testing
Regular Plasmid CRISPR Vector (Single gRNA)
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). These plasmid vectors encode a sequence-specific RNA-guided DNA nuclease (or nickase) enzyme, which can be used to edit the DNA sequence of specific user-defined sites in the genome.
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. For simplicity, our CRISPR/Cas9 Vectors are designed to efficiently express both the Cas9 nuclease (or nickase) enzyme and the guide RNA (gRNA) from a single vector.
Two variants of Cas9 enzyme are available in our CRISPR/Cas9 Expression Vectors. The standard humanized Cas9 (hCas9) variant efficiently generates double-strand breaks (DSBs) at target sites, while the “nickase” mutant form (hCas9-D10A) generates only single-stranded cuts in DNA. If hCas9-D10A nickase is used in conjunction with two gRNAs targeting the two opposite strands of a single target site, then the nickase enzyme will generate single strand cuts on both strands, resulting in DSBs 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.
Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) 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 with the CRISPR/Cas9 vector. This can result in replacement of the target genomic DNA sequence with template sequence, generating small targeted base changes, such as point mutations. Nicked genomic DNA also frequently undergoes homology-directed repair (HDR), and if exogenous template DNA is introduced into the cell along with a targeted hCas9-D10A nickase, then small base changes can be generated.
Most DNA sequence can be effectively targeted using the CRISPR/Cas9 system. However, there is a strict requirement for an NGG (sometimes NAG) sequence, known as protospacer adjacent motif (PAM), which is located on the immediate 3’ end of the gRNA recognition sequence within the target DNA.
For further information about this vector system, please refer to the papers below.
|Science 339:819-23 (2013)||Description of genome editing using the CRISPR/Cas9 system|
|Cell. 154:1380–9 (2013)||Use of Cas9 D10A double nicking for increased specificity|
|Nat. Biotech. 31:827–832 (2013)||Specificity of RNA-guided Cas9 nucleases|
Our Simple CRISPR/Cas9 Expression Vectors are designed for quickly and efficiently creating small deletions at target sites in a cellular genome. To introduce mutations at a specific target site, a gRNA is chosen which matches the target DNA sequence. Nuclease variants can be chosen to introduce DSBs (with hCas9) or single-strand cuts (with hCas9-D10A nickase).
Transient expression: Transfection of the CRISPR/Cas9 plasmid vector results in strong transient expression of the Cas9 protein and gRNA within the target cells. Without drug selection, the plasmid will be lost over time eliminating the Cas9 and gRNA from the target cells after genome editing has taken place.
Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design.
Lower specificity: Some off-target activity has been reported for the CRISPR/Cas9 system, and in general the TALEN system has lower off-target activity than CRISPR/Cas9. However, off-target effects can be significantly mitigated by using hCas9-D10A nickase in conjunction with two gRNAs (as described above).
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.
U6 Promoter: This drives high level expression of the gRNA.
Guide sequence: Specifies the target sequence of the Cas9 nuclease.
gRNA scaffold: Structural portion of the gRNA to allow complexing with Cas9.
Terminator: Terminates transcription of the gRNA.
CBh promoter: Chicken beta-actin promoter. Drives expression of Cas9 nuclease.
Nuclease: Cas9 nuclease variant chosen by user.
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.