CRISPR/Cas9 Plasmid Reagents for sgRNA Expression & Gene Editing

CRISPR Vector Specifications

SpCas9 Vectors

Cas9 endonucleases derived from the type II CRISPR systems in S. pyogenes (SpCas9) were the first Cas9 enzymes developed for mammalian genome editing. When combined with guide RNA (gRNA) sequences, these enzymes create site-specific double strand breaks (DSBs) in the genome. The CRISPR/Cas9 system accelerated genome editing for its ease of use, specificity, and high efficiency. GenScript is pleased to offer Broad Institute-validated WT SpCas9 constructs for gene editing in mammalian cells. Constructs are available either as all-in-one or dual vector systems, and can be used for non-viral, lenti-viral or adeno-associated virus (AAV) transfection. The lenti-vectors are compatible with 2nd and 3rd generation lentiviral packaging 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.

Transcription Activation (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

• 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.
• 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.
• 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.

CRISPR Handbooks and Protocols

CRISPR Handbook

CRISPR Plasmid Questions & Answers

• What are your CRISPR plasmid delivery specifications? Read More »

Deliverables:

• 4 μg of lyophilized plasmid (1 μg for low-copy plasmid) *
• Electronic vector map

*Check plasmid copy

QC:

• Sequence chromatograms encompassing your custom insert
• Quality assurance certificate

• How many gRNA sequences are needed for targeted knock-out? Read More »

A minimum of 3 gRNA sequences are recommended to ensure knock-out and experimental accuracy. Independently obtained knock-out mutants provide redundancy to safeguard against any hidden off-target effects.

• How should gRNA sequences be designed? Read More »

Designing your gRNA sequences involves 4 steps:

1. Determining the target gene locus.
2. Finding suitable sequences for Cas9 targeting.
3. Checking the potential for off-target binding.
4. Selecting gRNAs sequences that lie within your preferred binding region.

GenScript\'s gRNA database and online design tool will take out much of the guesswork when you\'re choosing gRNA sequences, by providing off-target scores and chromosomal location.

• Do I need an all-in-one or dual vector system? Read More »

All-in-one vector systems have two main advantages:

• Cells only need to be transfected once.
• gRNA/Cas9 expression is driven in an ideal 1:1 ratio.

Dual vectors, where Cas9 and gRNA are expressed independently on separate constructs, are more suitable if you plan to express multiple gRNAs for multiplex targeting.  For these applications, Cas9 should first be stably expressed in the cell line, after which the cells can be transfected with different gRNA vectors to generate a cell pool.

• When are lentiviral or adeno-associated viral (AAV) vectors necessary? Read More »

Vector selection for CRISPR gene editing should consider both application and cell type.

• In most easy-to-transfect cell lines non- viral vectors can work well.  
• Lentiviral transfection is typically necessary in cells with low transient transfection efficiency, such as primary cell cultures or hard-to-transfect cell lines. 
• AAV vectors have low immunogenicity and are preferred for in vivo gene delivery. Since the cargo limit of AAV vectors is generally smaller than other vectors (<5 kb), packaging the SpCas9 gene into these vectors can be challenging.  The Staphylococcus aureus Cas9 orthologue (SaCas9) is smaller than SpCas9 and is the preferred Cas9 variant for AAV vectors.

• When should I use SpCas9 nickase vectors? Read More »

SpCas9 nickase vectors are advantageous to use in experiments which are more sensitive to off-target editing. However, it is important to remember, that two gRNAs will need to be designed to target both forward and reverse strands. These gRNAs must be oriented so that PAM sites are distal to each other. gRNA sequences should be offset with windows of up to 100bp between them.

• How are gRNA sequences designed for the transcription activation (SAM) system?
  Read More »

For robust SAM transcription activation, gRNAs must target the first 200 bp upstream of the transcription start site (TSS). To decrease the degree of transcription activation, design gRNAs that target the SAM complex to greater distances upstream of the TSS. To repress transcription, design gRNAs that target SAM to +50 relative to the TSS, which will effectively block the TSS.

GenScript maintains a genome-wide SAM gRNA database which contains 6 SAM gRNAs designed to activate each coding region of the human genome.  For other species, our scientists offer complimentary SAM gRNA design help.  Request custom SAM gRNA design here.