Working with CRISPR in Geneious Prime

This CRISPR series shows how to find gRNA sites in Geneious Prime by selecting a PAM and target motif. Score each guide for activity with Doench et al. 2014, 2016 or 2022, and for specificity with the MIT and CFD algorithms against an off-target database. Further videos cover reviewing off-target sites, paired sites for Cas9(D10A) nickase use, read trimming with BBDuk, and editing analysis.

Course Content

Finding CRISPR Sites

Find gRNA sites with Annotate & Predict, then score each guide for activity (Doench et al. 2014, 2016 or 2022) and specificity (MIT and CFD) against an off-target database. Filter out low-scoring guides and compare the rest in the Annotations table.
LENGTH 5 minutes

Review Off-target CRISPR Sites

Geneious Prime lists up to 100 off-target sites per guide RNA. Review them in the CRISPR Off-Targets viewer: sort by CFD or MIT rank, check for mismatches in the seed region, and see whether a site falls within a coding sequence.
LENGTH 3 Minutes

Finding Paired CRISPR Sites

Find paired CRISPR sites for Cas9(D10A) nickase use by ticking Pair CRISPR Sites in the Find CRISPR Sites tool, and set the maximum overlap and spacing between sites. Each pair gets a combined score averaging both sites’ specificity.
LENGTH 3 minutes

Merging Paired Reads

Install the BBDuk Trimmer plugin, trim adapters and low-quality bases, discard reads that are too short, then merge paired reads into single sequences for CRISPR editing analysis.
LENGTH 2 minutes

Analyze CRISPR Editing Results

Analyze unaligned, merged Illumina reads against a short reference spanning the CRISPR cleavage site. Set the variant search window, minimum variant frequency, and error threshold, then review no-variant versus knockout percentages.
LENGTH 3 minutes

Recommended Resources

How do I set the off-target database for CRISPR?

Learn to search for off-target binding sites against a database of sequences.

Manual for CRISPR site finder

The Find CRISPR Sites tool searches for gRNA sites in your selected sequences.

Manual for CRISPR

A guide to using the CRISPR tools in Geneious Prime.

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Design primers in Geneious Prime, import existing sets, test them against a target, and add 5' extensions for cloning or Gateway applications.
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