CRISPR – Tips

Storage and Stability

Store CRISPR plasmid at –20°C immediately upon arrival. Store CRISPR RNA and mRNA at –80°C immediately upon arrival. Please avoid repeated freeze thawing of the plasmid or RNA. The reagents can be stored at –20°C or –80°C for up to 12 months.

Practice aseptic technique to avoid DNase contamination of the components. Keep reagent vials and sample tubes closed when not in use.

Note: Unless specified via custom requests, Sigma CRISPR plasmid products are delivered as mini-prep aliquots, which may not be suitable for transfection into particular cell types. For best results, we advise maxi-prepping plasmids using endotoxin-free DNA purification kits prior to transfection.

CRISPR design and specificity

CRISPR endonucleases have shown wide variation in their activity, even among multiple CRISPRs designed within close genomic proximity.2 For this reason, we highly recommend that you test 3 to 4 CRISPR nucleases (or paired nickases) that target different DNA sequences. Since size of the DNA target site for CRISPR systems is significantly smaller than that required for CompoZrTM ZFNs (e.g., 30–36 bp), care must be taken during design to ensure minimal off-target breaks elsewhere in the genome. Recent evidence indicates off-targeting by CRISPR endonucleases is a significant concern and some guidance for avoiding off-target activity is beginning to develop.9,10 If you feel your particular application requires enhanced specificity, paired CRISPR nickases may be used (Figures 2 and 4). Many on-line tools are available for CRISPR design; however, Sigma has applied its core capabilities in specific ZFN design to the CRISPR/Cas system to create an in silico collection of genome-wide CRISPR target sequences for both single site and paired nickase applications. Please check Sigma’s on-line CRISPR product offerings for the latest CRISPR design sets and custom design services for ZFNs, CRISPR, and related donor DNAs.

Delivery of CRISPR plasmids for cell culture applications

Note: Unless specified via custom requests, Sigma CRISPR plasmid products are delivered as mini-prep aliquots, which may not be suitable for transfection into particular cell types. For best results, we advise maxi-prepping plasmids using endotoxin-free DNA purification kits prior to transfection.

Previous experience with many ZFN projects has shown nucleofection is a robust delivery method for a wide variety of cell types. For initial experiments in human cells, we advise nucleofecting maxi-prepped CRISPR plasmid DNA into well-validated cell types such as K562 or U2OS to assess double strand break activity or donor integration levels. These cell lines have been shown to respond with high levels of homologous recombination22, making them ideal for testing compatibility of ZFN and CRISPR nucleases with newly designed donor constructs before moving to a more challenging or unknown cell type. For mouse and rat cell culture testing, neuro2A and C6 are suitable cell types for initial CRISPR experiments. A good starting point for dosage experiments is:

  • 2–8 μg of CRISPR plasmid (for single palsmid Cas9-FP linked vectors)
  • ≥ 1.0 μg/μL and ≤ 5 μL of Cas9 or Cas9-D10A plasmids combined with ≥ 1.0 μg/μL and ≤ 5 μL of U6- gRNA pla

(All dosages above assume 0.5 to 1 million cells nucleofected)

Since Sigma’s single vector format contains a GFP (or RFP)-linked expression cassette for Cas9, transfected cells can be subsequently inspected by microscopy or FACS to monitor transfection efficiency (Figure 5) prior to performing genotyping assays or single cell cloning efforts.

Monitoring CRISPR double strand break activity

Several published protocols exist for monitoring double strand break (DSB) activity. The most rapid, flexible, and economical assay is the mismatch cleavage assay, which can detect the variety of insertions and deletions (indels) generated by NHEJ activity in eukaryotic cells. The most common protocols use the CEL-I enzyme (a.k.a. Surveyor nuclease) and T7 endonuclease I (T7EI). If sufficient bioinformatics and deep sequencing resources are available at your institution, deep sequencing can also be used to detect and quantitate indel activity in CRISPR-treated cell populations.

If, for a particularly favored CRISPR design, you cannot detect DSB activity despite many attempts, consider enriching the cell population for Cas9-FP expression as described in the next section.

 Single cell cloning and genotyping

Since some Sigma CRISPR plasmids implement an FP-linked Cas9 expression cassette, fluorescence-activated cell sorting (FACS) can be used to isolate cell populations with significantly increased frequencies of Cas9- induced modifications. This FACS-enrichment approach is particularly useful in scenarios where delivery efficiencies and/or Cas9 expression levels are low or undetectable. Furthermore, the FP co-expression approach has advantages over surrogate reporters including:

  1. Cas9-FP linked expression does not require the extra step of cloning of artificial target sites into surrogate reporter
  2. A reduction in the total amount of transfected dsDNA. In many cases, certain cell types are sensitive to high amounts of dsDNA, and adding additional dsDNA mass in the form of a surrogate reporter plasmid to existing

CRISPR and donor vector constructs can result in cell toxicity.

  1. The option to use “dsDNA free” approaches by implementing Cas9-FP mRNA with in vitro transcribed gRNA, and single stranded oligos (ssODNs).

The Cas9 and FP coding regions are linked by a small sequence encoding a 2A peptide. The 2A peptide is a ‘‘self-cleaving’’ peptide, which allows production of two individual proteins from one transcript and utilizes “ribosomal skipping” rather than proteolytic cleavage mechanism to generate two individual proteins.

Cells can be harvested for FACS according to commonly used protocols. We suggest cells be sorted into fractions with low, medium, and high FP expression levels. Cell populations with the highest FP expression have been shown to enrich genome edits with the extent of the “high” fraction ranging from 1–30% of the total cell population. The optimal size of the “high” and “medium” populations may vary, depending on genomic loci, cell types, delivery methods, and other experimental variables. A good start is to divide the cell population into three low, medium, and high fractions each of which comprises 1–20% of the total cell population