Cloning custom sgRNAs into CRISPR lentiviral vectors
Introduction
Materials
Procedure
- Digest and dephosphorylate 5µg of the lentiviral CRISPR plasmid with BsmBI for 30 min at 37°C.
A A | B B | C C | |
1 1 | Amount | Unit | |
2 2 | Lentiviral vector concentration | 1 | ug/uL |
3 3 | Lentiviral vector | 5 | ug |
4 4 | Lentiviral vector | 5 | uL |
5 5 | FastDigest BsmBI | 3 | uL |
6 6 | FastAP | 3 | uL |
7 7 | 10X FastDigest Buffer | 6 | uL |
8 8 | 100 mM DTT | 0.6 | uL |
9 9 | ddH2O | 42.4 | uL |
10 10 | Total | 60 | uL |
- Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB.
CRITICALIf BsmBI digested, a ~2kb filler piece should be present on the gel. Only gel purify the larger band. Leave the 2kb band.
- Phosphorylate and anneal each pair of oligos:
A A | B B | |
1 1 | Number of pairs | 4 |
A A | B B | C C | |
1 1 | Amount per rxn (ul) | Master Mix (ul) | |
2 2 | 10X T4 Ligation Buffer | 1 | 4 |
3 3 | ddH2O | 6.5 | 26 |
4 4 | T4 PNK | 0.5 | 2 |
5 5 | Master Mix Total | 8 | 32 |
6 6 | Oligo 1 (100 uM) | 1 | |
7 7 | Oligo 2 (100 uM) | 1 | |
8 8 | Reaction Total | 10 |
Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).
Put the phosphorylation/annealing reaction in a thermocycler using the following parameters:
A A | B B | |
1 1 | 37C | 30 min |
2 2 | 95C | 5 min and then ramp down to 25C at 5C/min |
- Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.
- Set up ligation reaction and incubate at room temperature for 10 min:
A A | B B | C C | D D | |
1 1 | Amount | Unit | Master Mix (uL) | |
2 2 | BsmBI digested plasmid concentration | 50 | ng/uL | |
3 3 | BsmBI digested plasmid | 50 | ng | |
4 4 | BsmBI digested plasmid | 1 | uL | 4 |
5 5 | 2X Quick Ligase Buffer | 5 | uL | 20 |
6 6 | ddH2O | 4 | uL | 16 |
7 7 | Master Mix Total | 10 | uL | 40 |
8 8 | Quick Ligase | 1 | uL | |
9 9 | Reaction Total | 11 | uL |
Also perform a negative control ligation (vector-only with water in place of oligos) and transformation.
- Transformation into Stbl3 bacteria.
CRITICALLentiviral transfer plasmids contain Long-Terminal Repeats (LTRs) and must be transformed into recombination-deficient bacteria. We use homemade Stbl3 (propagated from Invitrogen C7373-03) and get excellent plasmid yields. Although other RecA strains may work, we have found the most consistent transformations and yields using Stbl3.
Lentiviral CRISPR/Cas can infect a broad variety of mammalian cells by co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA (sgRNA) to facilitate genome editing (Shalem, Sanjana, et al., Science 2014). Protocols for cloning into the lentiviral transfer plasmid and general considerations for producing lentivirus are described below. Separate protocols are available for amplifying the genome-scale CRISPR knock-out (GeCKO) libraries. This protocol is for creating individual lentiviral CRISPR plasmids targeting a single genomic locus. For further information on which lentiviral vector to use or how to design chimeric guide RNAs, see information from the Zhang Lab. Also see the GeCKO website and the CRISPR forum for any help needed.