Plasmid based CRISPR: mESCs / Cell Lines

All-in-one Cas9-reporter Vectors for High Efficiency Single Cell Cloning

To expedite cell engineering workflows, Sigma offers a CRISPR/Cas subcloning service which delivers targeted CRISPR/Cas plasmids with the following features:

  • A single vector format including the Cas9 protein expression cassette and gRNA. This maximizes the chances of successful cellular delivery of all necessary CRISPR/Cas
  • GFP (or RFP) is co-expressed from the same mRNA as the Cas9 protein via a 2A peptide linkage, enabling tracking of transfection efficiency and enrichment of genome editing activity in cell populations via fluorescence activated cell sorting (FACS).
  • The human U6 promoter is used to drive gRNA expression, while CMV promoter drives expression of Cas9 and GFP or RFP proteins. The human U6 promoter was chosen since it has proven to be superior to H1 promoter in previous RNAi applications.
  • A T7 promoter sequence is localized immediately upstream of Cas9 cDNA sequence, allowing for in vitro Cas9-GFP mRNA synthesis, if desired.
 All-in-one plasmid maps and features.

The Cas9 protein implemented in these vectors contains both HNH and RuvC activities enabling the creation of double strand breaks. Cas9 is linked to EVROGENTM TagGFP2 or TagRFP fluorescent proteins. TagGFP2 is the improved variant of TagGFP, a mutant of the Aequorea macrodactyla GFP-likeprotein.19,20 TagGFP2 possesses bright green fluorescence with excitation/emission maxima at 483 and 506 nm, respectively. TagRFP is a monomeric red (orange) fluorescent protein generated from the wild-type RFP from sea anemone Entacmaea quadricolor.21 It possesses bright fluorescence with excitation/emission maxima at 555 and 584 nm, respectively. In both GFP and RFP vectors, the 2A-FP encoding sequence is flanked by two Hpa I restriction sites, which allows removal or replacement of the 2A-FP element. The XbaI site can be used to linearize the vector for production of Cas9-FP mRNA via in vitro transcription using T7 RNA polymerase.

one-plasmid

 

Step 1: Pick GFP or RFP : With GFP: U6-gRNA/CMV-Cas9-GFP

: With RFP: U6-gRNA/CMV-Cas9-RFP

Step 2: Pick gRNA sequence

Option 1: pre-designed human, mouse, or rat targets. http://crispr.sigmainformatics.com/CrisprSearch.php

Note: The TargetID number is critical to ordering pre-designed CRISPRs. It is like the TRC clone number for ordering shRNA.

Option 2: custom targets or organisms, typically used for specific applications like point mutations, SNPs, etc. The researcher will fill out a Custom CRISPR Application form and will work with our bioinformatics team to identify the best gRNA sequence for their project. This order must be placed using a quote.

Dual vector system (plasmid)

Cas9 and gRNA are expressed on separate vectors. Researcher needs to order Cas9 AND gRNA (two items).

Step 1 : Pick Cas9 Plasmid.

The Cas9 expression plasmids use the CMV promoter for strong transient expression of Cas9. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9 expression plasmids can be linearized using XbaI for T7-based mRNA production.

Option 1: No color vector = pCMV-Cas9: CAS9P-1EA

Option 1

Option 2:  GFP vector = pCMV-CG: CAS9GFPP-1EA

Option 2

Option 3: RFP vector = pCMV-RG: CAS9RFPP-1EA

Option 3

Step 2: Pick gRNA

gRNA sequences can be pre-designed (i.e. Target ID HS000040875) or custom

gRNA

Triple vector system (CRISPR Nickase + gRNA1 + gRNA2)

Recent evidence indicates off-targeting by CRISPR endonucleases is a significant concern for applications requiring high specificity.9,10 To address this problem, Sigma has developed paired nickase technology to expand CRISPR DNA recognition tracts to lengths similar to those of ZFNs and TALENs. While we have found that both D10A and H840A-type Cas9 mutations can result in active paired nickases, our experience suggests that paired nickases based on the Cas9-D10A mutant (i.e. RuvC minus) are most reliable. These results have been independently confirmed.11-13 Our development work also suggests that for both D10A and H840A-type Cas9 mutants, the critical factor in producing active paired nickases is the positioning of gRNAs in a 5’-to-5’ orientation. A quick way to verify the correct 5’-to-5’ orientation is to ensure that the PAMs of chosen paired gRNA sites, as viewed on the sense strand, are in a 5’-CCNN19-20….N19-20NGG-3’ format (see Figure 4). Our data indicate that 5’- to-5’ PAM spacings ranging from 30 to 150 bp can result in paired nickase activity useful for genome editing workflows (i.e. indel rates > 1%).

Three separate vectors expressing Nickase, gRNA1, and gRNA2. Researcher needs to order CRISPR Nickase and two paired gRNAs.

Step 1: Pick Cas9 Nickase.

The Cas9-D10A expression plasmids use the CMV promoter for strong transient expression of Cas9. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9- D10A expression plasmids can be linearized using XbaI for T7-based mRNA production.

Option 1: No color vector = pCMV-Cas9-D10A

Option 2:  GFP vector = pCMV-D10A-G

Option 3: RFP vector = pCMV-D10A-R

P2 F8

Step 2: Pick paired gRNAs

For optimal Cas9-D10A paired nickase functionality, gRNAs should be designed in a 5’-to-5’ orientation with PAM spacing between 30 and 150 bp.

To simplify the use of paired nickases, Sigma has on-line tools which allow access to a library of pre-designed paired nickases. Please check Sigma’s on-line CRISPR product offerings for the latest design sets and custom CRISPR and donor design services. We recommend testing 3-4 sets of paired nickases to maximize chances of finding an active pair.

For plasmid-based delivery of paired CRISPR nickases, we recommend the use of a three plasmid system comprised of the Cas9-D10A expression vector and two U6-driven gRNA vectors. The reason for this is two-fold:

  1. In our experience, the three plasmid system has worked more reliably than single plasmid formats at producing highly active paired
  2. The three plasmid system allows for flexible titration of gRNA and Cas9-D10A plasmids to optimize genome editing rates. For example, if one gRNA in a paired nickase has low activity, but a valuable location, you can independently increase the plasmid transfection levels of that gRNA in attempts to boost activity.

For initial experiments, we recommend:

  • ≥ 1.0 μg/μL and ≤ 5 μL of Cas9 or Cas9-D10A plasmids (per 5 to 1 million cells nucleofected)
  • ≥ 1.0 μg/μL and ≤ 5 μL of U6-gRNA plasmids (per 5 to 1 million cells nucleofected)