Working with CRISPR in Geneious Prime

This CRISPR series shows how to find gRNA sites in Geneious Prime using Cas9 or Cpf1 PAM motifs, scored with Doench et al. 2014/2016 activity and Zhang 2013 specificity against an off-target database, or find paired sites for Cas9(D10A) nickase use. Two more videos cover preparing Illumina reads with BBDuk, then analyzing CRISPR editing results against a reference. Four videos.

Course Content

Finding CRISPR Sites

Find gRNA CRISPR sites using Annotate & Predict, specifying a PAM (3' Cas9 or 5' Cpf1) and target motif. Score activity with Doench et al. 2014/2016, and specificity against an off-target database using the Zhang 2013 method.
LENGTH 5 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.

More Geneious Academy

TUTORIALHow to Find CRISPR Sites in Geneious Prime
Find guide RNA sites in the LYP1 gene of baker's yeast, then screen the whole genome for off-target binding.
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TUTORIALHow to Analyze CRISPR Editing in Geneious Prime
Prepare sequence files, set up a CRISPR editing analysis, and interpret the results.
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VIDEO SERIESAnnotate Sequences in Geneious Prime
Annotate sequences in Geneious Prime by importing files, transferring or copying annotations between homologs, using a database, or annotating by BLAST.
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VIDEO SERIESPrimer Design in Geneious Prime
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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