Lentiviral CRISPR Formats
Certain cell types are difficult to transfect using lipid reagents, electroporation, or nucleofection. To enable genome editing in these cell types, Sigma has built upon capabilities within MISSION lenti-shRNA products to develop an all-in-one lenti-CRISPR vector containing both gRNA and Cas9 elements (see vector map below). A unique feature of the all-in-one vector (pLV-U6g-EPCG) is a Cas9 ORF flanked by puro and GFP elements, providing multiple options for monitoring stable cell populations which are expressing CRISPR components.
Previous genome editing workflows implementing ZFNs and TALENs primarily utilized transient modes of nuclease expression. A unique feature of lentiviral delivery is the option for highly efficient chromosomal integration of CRISPR components. This allows for longer term CRISPR expression and drug-based enrichment of CRISPR-expressing cell populations27,28. Furthermore, drug selection provides a gentler alternative for clonal isolation versus 96-well or FACS-based single cell cloning where some cell types experience significant levels of apoptosis due to rapid and extreme isolation.
A key experimental parameter to estimate when using lentivirus is multiplicity-of-infection (MOI). When comparing MOI values among different publications, protocols, datasets, etc., it is important to determine what methods were used for MOI calculation. MOI is specifically defined as the number of transducing units (TU) per cell. However, TU is broadly defined in many ways depending on vector type, including:
- Count of fluorescent cells post-transduction via FACS (for lenti-vectors expressing fluorescent proteins).
- Drug selection and counting of viable colonies (i.e. colony forming units or CFU).
- ELISA assays which measure lentivirus associated p24 core protein. Note: this can measure total p24 in the supernatant, but some methods attempt to quantitate only p24 within intact
In our experience with MISSION shRNA vectors, titers based on bulk p24 measurement (meaning free p24 + particle-associated p24) are approximately 20-fold higher than titers measured by counting of viable puro-resistant colonies. Similar comparisons with all-in-one lenti-CRISPR vectors yield a p24:CFU ratio of approximately 200 to 400 (dependent upon cell type). This reduction in CFU-based titer is most likely due to the large size of the Cas9 ORF (typically 3-4 kb). When produced via 96-well formats, Sigma’s first generation all-in-one lenti-CRISPR vector has been characterized to yield between 1×105 and 1×106 TU/ml as measured by bulk p24 (free and particle-associated). Higher titer formats are available upon custom request.
Note: Lentiviruses are quite labile. Multiple freeze-thaw cycles and prolonged exposure to ambient temperatures will decrease the lentiviral titer. Thaw lentiviral particles on ice. Keep them stored on ice when not in use.
- Seed cells into a 96-well at approximately 50% confluency (~3,000 to 5,000 cells).
- Calculate the volume (µL) needed for your target MOI based on previous experience, or first pass CFU measurements
- In a separate test tube, add polybrene to the media at a final concentration of 8 µg/m
- Add the appropriate volume of virus to the media/polybrene solution. A typical transduction has 10-15 µL of virus with the final volume of 50 ul made up of media/
- Remove media from the 96-well and add the virus/media mixture.
- Mix on a slow rocker 5-15 min then place at 37˚C for 24
- Change the media
- At 48 hours post-transduction, add puromycin to the media at a concetration determined via kill curve (see Tip No.5 below).
- Select on puromycin for the desired amount of time (typically 4 to 14 days). Less active gRNA designs may require the longer incubation times to achieve maximal
- Perform the desired phenotypic assays, genotyping assays, or single cell cloning operations.
Step 1: Pick plasmid or lentiviral format.
Single vector: U6-gRNA/EF1a-puro-2A-Cas9-2A-GFP. Expresses gRNA and Cas9 protein from single plasmid. GFP and puro selection assist in simplifying sorting and clonal selection.
Step 2: Pick gRNA sequence
Option 1: pre-designed human, mouse, or rat targets. 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.