Designing and Generating CRISPR-Cas Mutants

We are using the double-nickase mutation strategy. This involves the generation of two CRISPR-Cas plasmids per mutation, each of which nicks a different strand, spaced apart by about 40 nt. To generate a point mutant, you can also include a piece of DNA containing the mutation and allow for homologous recombination.

• Determine where you want to target the gene and copy that DNA sequence. It needs to be the genomic DNA from that particular species that is targeted, so if your region of interest spans several introns, you need to copy the genomic DNA not the mRNA sequence.
• Paste this sequence into the CRISPR tool at http://crispr.mit.edu/ and select your target species. When complete select Double Nickase Design
• Based on where you want to cut the DNA select the two guide DNA sequences.
• Print out, or sketch all of the targeting information into your notes.

You will need to generate two nickase plasmids using the pX335 backbone. Below is a schematic of the cloning taken from http://www.addgene.org/42335/

Desigining and Ordering Primers

• For each guide DNA sequence you need to order a pair of primers to make this gDNA sequence.
• To make the forward primer copy the sequence but remove the NGG site at the end. Add CACC to the 5' end of it to generate the appropriate overhang.
• To make the reverse primer reverse transcribe the copied sequence (removing the NGG and the end, and not including the CACC at the other end). Add AAAC to the 5' end of that reverse transcribed sequence to make the appropriate overhang.
• Name the primers as follows: mm-Pygm-gDNA-62-FWD where mm = species; Pygm = your gene name, 62 = where this nickase cuts, and FWD or REV is whether the primer is forward or reverse.

Cloning

• First digest pX335 vector by adding to a PCR tube:
  • 1 ug of pX335,
  • 1uL of 10X NEB Buffer 2.1
  • 1 uL of CIAP
  • 1 uL of BbsI
  • water up to 10 uL
  • Digest for 1h in, gel purify the fragment from an agarose gel.
• Next prepare the insert by annealing and phosphorylating the primers in a PCR tube:
  • 1 uL of a 100 uM stock of each oligo with 4.5 uL of water
  • 2.5 uL of ligase buffer
  • 1 uL of T4 PNK
  • Incubate at 37C for 30 mins then 95C for 5 mins to heat inactivate PNK then ramp down to 25C at 5C/min to allow the oligos to anneal
• Combine the ligation mixture in an eppendorf tube:
  • 5 ng of vector (~10 pmoles of vector)
  • 3 uL of annealed insert (~30 pmoles of Insert) or water as a blank
  • 2.5 uL of 4X ligation buffer
  • 1 uL of T4 DNA Ligase
  • Water to 10 uL
• Incubate for 10 min at RT
• Transform into competent cells (see Transformation of Bacteria)