14.11 CRISPR site finder

The CRISPR system is an RNA-guided endonuclease technology for gene editing. This system uses a guide RNA (gRNA) of around 20 bp, next to an enzyme-specific PAM (Protospacer Adjacent Motif), to direct the enzyme complex to the cleavage site. The Find CRISPR Sites tool searches for gRNA (“CRISPR”) sites in your selected sequences, and scores them based on on-target sequence features and off-target interactions.

Find CRISPR Sites can be run on any number of sequences and sequence list documents, including on selections within the documents. For best performance the target sequence for each document should be limited to 1000 bp.

To use the tool, select your target sequences and click Find CRISPR Sites in the Cloning menu. Check Anywhere in sequence or Selected region depending on whether you are using a selection or not, and choose the PAM Site Location (see below). Enter the Target and PAM Site motifs you want to search for in the CRISPR site panel (for syntax help, hit the help button to the top right of the panel). You can then select your scoring and pairing options and hit OK. Any CRISPR sites found will be annotated back on to your original sequences, with the associated scores listed in the label on the CRISPR annotation.

    PAM Site Location
    Activity Scoring
    Specificity Scoring
    Pair CRISPR sites
    Results output
PAM Site Location

Different CRISPR enzymes recognize different PAM site motifs. The CRISPR-Cas9 family recognizes PAM Sites on the 3’ end of the guide sequence, but the more recently discovered CRISPR-Cpf1 enzymes (Zhang et al. (2016)) recognize a 5’ PAM site. Change the PAM Site location option to match the orientation of the enzyme you are using. Note that activity (on-target) scoring methods for Cpf1 sites are not currently available. If you have a particular scoring method you would like to use for Cpf1, please contact Support.

Activity Scoring

Activity, or on-target scoring looks at the sequence features of the CRISPR site itself, and compares them to an experimentally determined model. The model then scores the site based on its predicted level of activity.

Geneious Prime offers activity scoring for Cas9 CRISPR sites using the method from Doench et al. (2014). This method analyzes the one- and two-base features of the gRNA, as well as the GC content, to generate the score. Scores are between 0 and 1, with a higher score denoting higher expected activity. Note that ambiguous bases will not contribute to this score.

Specificity Scoring

Use these options to score your CRISPR sites based on how unique they are and how likely they are to cause off-target effects. Scores are between 0 and 100, with a higher score denoting better specificity and less off-target activity.

When finding CRISPR sites within a Selected region of your sequence, Geneious will check for off-target interactions in the unselected regions of your sequence. If Score against an off-target database is selected, Geneious will also search sequence documents in the selected folder for off-target interactions. While doing so, Geneious will skip any sequences in the off-target database that are exact duplicates of the target sequence, and report the intervals it skipped at the end of the operation.

Use Maximum mismatches allowed against off-targets and Maximum mismatches allowed to be indels to tailor how similar off-target sites must be to the CRISPR site before they contribute to the score.

If multiple sequences are given as the targets, the option to Score each sequence against all other selected sequences is available. This checks for off-target interactions between the target sequences and incorporates them into the off-target scores.

The scoring algorithm for CRISPR sites with 3’ PAMs was proposed by Zhang et al. (2013). Mismatched bases between the CRISPR site and off-target sites have different weightings in the final score, which are experimentally determined and based on their position. Geneious also recognizes a 10 bp seed region immediately next to the PAM, which can tolerate a maximum of 2 mismatches (Cho et al. (2014)).

For CRISPR sites with 5’ PAMs, a modified version of the Zhang et al. (2013) algorithm is used. Geneious recognizes a 6 bp seed region next to the PAM, which tolerates up to 2 mismatches and has a high mismatch weighting in the score. From position 7 to 18, mismatches have an average weighting, and after position 18, mismatches do not contribute to the Specificity score. These weightings were not experimentally determined and were chosen to give a similar score spread to the Zhang et al. (2013) method. See Kim et al. (2016) and Kleinstiver et al. (2016) for details on the seed, trunk, and non-weighted regions used.

Scoring against an off-target database will significantly increase the time taken for the operation to complete.

Pair CRISPR sites

There is a mutant variety of the CRISPR-Cas9 enzyme known as Cas9 D10 Nickase. It induces a single stranded break at the target instead of the usual double stranded break. This enzyme can be used with a pair of CRISPR sites on complementary strands to induce a double stranded break with sticky ends. This method also minimises off-target effects because any that occur are single-stranded, and therefore repaired by the cell with high fidelity. This process is described by Zhang et al. (2013).

Select Pair CRISPR sites to only return guides that are within range of another guide on the opposite strand. You can specify the Maximum overlap of paired sites and the Maximum allowed space between the paired sites, which are measured from the 5’ end of the CRISPR sites. Optimal CRISPR pairs will be linked when they are annotated onto your target sequence.

Results output

CRISPR sites are returned as annotations on your original sequences. Hovering the mouse over one of these annotations will bring up a tooltip about the CRISPR site: its Target Sequence, PAM (Protospacer Adjacent Motif), and any scores that were calculated for the CRISPR site.

If the CRISPR sites were scored through off-target analysis, the five most similar off-target sites are also included in the annotation. Mismatches between the CRISPR site and its off-target sites are highlighted in red, and insertions in the off-target site are red and underlined.

CRISPR site annotations can also be colored by their scores. Choose the score to color by using the Color CRISPR sites by option. You can change which score is used for coloring later by selecting Color by / heatmap in the annotation Track options. The colors move from green, for good scores, through yellow, and down into red, for poor scores.