Before you begin
Learn how to get started and sign up with Benchling here. Start this worksheet using your free academic account in order to get the most out of this worksheet. DNA sequences can be copied directly into your Benchling account. For notebook entries, you can manually copy & paste the content into a new blank entry.
Content and materials for this module were co-developed with Dr. Philip Leftwich, Biology Lecturer at the University of East Anglia (Norwich, UK)
CRISPR which stands for “Clustered Regularly Interspaced Short Palindromic Repeats” is a family of DNA sequences that are found in prokaryotic organisms such as archaea and bacteria and have been linked to innate immunity in these systems against predators such as bacteriophages. CRISPR sequences function in conjunction with Cas proteins (the most notable member being Cas9) to protect the organism by recognizing and destroying DNA from the invading phage. CRISPR-Cas9 has been repurposed and engineered by scientists from its native context to be a robust and effective method for gene-editing.
In this worksheet, you'll explore how to use Benchling to find a gene you want to target and perform CRISPR analysis on the target sequence including scoring on-and-off target guide RNAs and exporting guide RNA sequences.
Students should have a basic understanding of enzymes like nucleases and comprehension of the structure and function of DNA and RNA. They should also be aware of how genes can be edited in an organism and understand various applications for modifying a gene with CRISPR.
By the end of this worksheet, you will be able to:
Identify and obtain genes of interest for modification
Design CRISPR guide RNAs for gene/genome-editing
Score and export gRNA sequences into lab notebooks
The following exercise uses Benchling to create a knockout of the STE12 gene found in Saccharomyces cerevisiae, a common yeast for baking, winemaking, and brewing. You will import the STE12 gene from an external database into Benchling and design and analyze CRISPR guide RNAs that will target and inactivate this gene.
Import the STE12 gene into Benchling
From the global "Create" button, choose "DNA Sequence" -> "Import DNA Sequences" and navigate to the "Search External Databases" tab.
Type in "STE12" and hit Search and ensure the following parameters below are changed to the following:
Genome = "R64-1-1 (sacCer3, Saccharomyces cerevisiae"
Transcript = "None"
Import As = “STE12”
Save to = Select an appropriate Project or Folder to save this sequence
Click “Import” to save and open the STE12 gene.
Design and analyze guide RNAs for STE12
Benchling’s CRISPR tool will design guide RNAs that disrupt the target gene and will score them based on the cleavage efficiency for the desired sequence (On-target) and the cleavage efficiency for undesired sequences in the organism’s genome (Off-target).
Click on the "CRISPR" icon on the right sidebar, and click on the Design and Analyze Guides button.
A window titled, "Design CRISPR Guides: Guide Parameters" should be open and ensure that the following fields have the correct information below and then click Finish.
Go to your Sequence Map and click on the annotation for STE12 to autofill the target gene regions (Target Region = "275288" to "275307")
Now click "Create" and guide RNA sequences will be generated and scored.
You can organize guide RNAs by clicking on the headers such as: "Position", "On-Target Score", and "Off-Target Score".
Sort these by "On-Target Score" and click on the checkbox for each guide RNA to visualize them in the DNA sequence.
Save your guide RNA analysis results as "STE12 gRNAs" to view it again later.
Use this guide RNA example to answer the questions below:
1. Where does the Cas9 nuclease typically cut in the sequence relative to the PAM?
2. Is it preferable to have a higher or lower “On-Target Score”?
3. Is it preferable for your experiment to have a higher or lower “Off-Target Score”?
Try to answer this question on your own and check the "Solution" at the bottom.
Can you import ADE2, another gene in Saccharomyces cerevisiae, and design guide RNAs for it?
Note: You should use the same steps as before with STE12 and reuse identical settings for gene import and CRISPR design.
Export guide RNAs for STE12 into a Notebook Entry
You can also you can export all generated guide RNAs and related information into a related lab notebook entry or an external spreadsheet for additional analyses.
Re-open your saved CRISPR analysis to view all of the guide RNAs you've designed.
Click on the Export button and select "Export All" to temporarily save this information in a.TSV format. (Alternatively, if there is only a subset you'd like to export, just select those respective checkboxes and choose "Export Selected".)
Open a new Notebook Entry and insert a table with six column headers. Paste this information to transfer your guide RNA analysis into a Benchling table.
You can now export this table as .CSV or even paste information into an external spreadsheet such as Excel, Google Sheets, and/or Apple Numbers.
Cas9 typically cuts 3 bp upstream from the start of the PAM sequence.
A higher "On-target Score" is indicative of your target sequence being cleaved more efficiently and therefore is more preferable.
A higher "Off-target Score" is indicative of your non-target sequences being cleaved less efficiently and therefore is more preferable.
Congrats! You've finished the learning module: CRISPR Knockouts.