Designing and Generating CRISPR-Cas Mutants

Overview

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.

Designing the Targeting Strategy

• 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. In other words, if you can design your CRISPR pairs to be within one exon that is best. In that case submit the exon to the CRISPR tool. If your target site is close to an exon boundary then you need to submit the genomic sequence around that exon. You do not want your CRISPR pairs to span an intron.
• 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. Name those sequences based on the nucleotide it cuts after (see cuts after position in the CRISPR tool output). Use the actual nucleotide from the reference mRNA sequence, not the nucleotide based on the arbitrary region that you pasted into the CRISPR tool.
• Print out, or sketch all of the targeting information into your notes.

Generating the CRISPR-Cas Plasmids

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/

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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 (or use a frozen, pre-digested vector):
  • 2 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, using the QIAEX II Kit and check the concentration by nanodrop.
• Next prepare the insert by annealing and phosphorylating the primers in a PCR tube:
  • 4.5 uL of water
  • 1 uL of a 100 uM stock of each oligo
  • 2 uL of 5X 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, leave at room temperature
  • Dilute the annealed oligos 250X in water (2 uL + 498 uL of water)
• Combine the ligation mixture in an eppendorf tube:
  • 50 ng of vector
  • 3 uL of annealed insert or water as a blank
  • 2 uL of 5X ligation buffer
  • 1 uL of T4 DNA Ligase
  • Water to 10 uL
• Incubate for 10 min at RT
• Transform 2uL of this into competent cells (see Transformation of Bacteria), plating the entire transformation.