CRISPR Cas Plasmids

SpCas9 Nickase Vectors

While the CRISPR/Cas9 technology is still more specific when compared to other popular gene editing strategies, off-targeting concerns are still a reality. In an effort to improve specificity, the endonuclease activity of Cas9 was modified. WT Cas9 has two catalytic domains, RuvC and HNH, and mutations to catalytic residues within these domains (specifically, D10A in RuvC and H840A in HNH) cause Cas9 to create single strand nicks as opposed to double strand breaks (Ran et al, 2013). This Cas9 enzyme with nickase activity, or Cas9n, is guided by guide RNAs (gRNA) to opposite sides of the target genomic DNA. Cells will preferentially repair these SSBs by HDR rather than NHEJ. By proceeding through an HDR mechanism, the frequency of unwanted indel mutations from off-target DSBs is minimized. GenScript is pleased to offer Broad-validated nickase vectors for gene editing in mammalian cells types.

SaCas9 Vectors

The Cas9 orthologue derived from Staphylococcus aureus, or SaCas9, has similar efficiency to SpCas9; however, SaCas9 is approximately 1 kb shorter. The primary advantage of SaCas9 is adeno-associated virus (AAV) packaging: the cargo size of AAV is approximately 4.5kb, and consequently packaging SpCas9 into this vector can be challenging (Ran et al, 2015). The relatively smaller size of SaCas9 makes CRISPR gene editing with AAV vectors possible. Considering the lower immunogenicity of these constructs, SaCas9 is therefore more suited for in vivo editing applications, such as for therapeutics.

SAM Vectors

CRISPR/Cas9 Synergistic Activation Mediator (SAM) is a protein complex engineered to enable robust transcriptional activation of endogenous genes – either a single gene at a time, or up to 10 genes simultaneously in the same cell. SAM takes advantage of the specificity and ease of reprogramming of Cas9 nucleases, which are targeted to a specific locus in the endogenous genome by guide RNA. Through a license with the Broad Institute*, GenScript offers validated SAM gRNA sequences to target any coding region in the human genome, as well as complimentary design of SAM gRNA for any other species. SAM guide RNA sequences are custom-synthesized and cloned into efficient lentiviral vectors, and accompanied by the Cas9-VP64 and MS2-P65-HSF1 components that form the three-part SAM complex.

The SAM complex consists of three components

1.A nucleolytically inactive Cas9-VP64 fusion: dCas9 is used to ensure that no strand breaks are introduced into endogenous genome; VP64 is a transcription activation domain that acts synergistically with p65 and HSF1 to enhance transcription.

2.An sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop: the sgRNA should be designed to target the first 200 bp upstream of the transcription start site in order to target the SAM complex for ideal transcription activation. While sgRNA normally binds to Cas9, the MS2 RNA aptamers are required to allow the third member of the SAM complex to bind to the Cas9-sgRNA complex.

3.The MS2-P65-HSF1 activation helper protein: this contains two transcription activation domains, P65 and HSF1, that synergize with VP64 to robustly activate transcription of downstream coding regions. The MS2 domain allows his helper protein to bind to the sgRNA-dCas9 complex.