Precise gene-editing with revolutionary CRISPR-Cas9 technology holds immense potential in various life science fields. But whilst it’s a very effective tool, it does not come without its limitations.
Challenges including low editing efficiency and off-target effects can limit progress to downstream therapeutic applications.
This infographic includes a host of tips and tricks for your CRISPR-Cas9 success.
Download this infographic to discover tips on:
- Optimal gRNA design
- Selecting the most efficient delivery method for your cells
- How to troubleshoot common CRISPR-Cas9 challenges
Tips and tricks for CRISPR-Cas9 success The discovery of CRISPR-Cas9 gene editing technology completely transformed life science research in 2012 by introducing a precise and efficient “cut-and-paste” tool for inserting or removing portions of DNA. Target-specific crRNA Target genomic locus PAM gRNA tracrRNA Cas9 NGG For Research Use Only. Not for use in diagnostic procedures. © 2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL118554 0922 Learn more at thermofisher.com/genomeediting Additional CRISPR tools and resources To access additional resources like our comprehensive genome editing resource guide, validated protocols, and technical support, visit thermofisher.com/crispr101 For more quick guides with tips and tricks to popular protocols, subscribe to Connect to Science. Try this Achieve higher editing efficiency and fewer off-target effects by circumventing transcription and translation. Instead of using Cas9-expressing plasmids, opt for direct transfection of purified Cas9 protein and synthetic gRNA delivery. Low editing efficiency Off-target effects Opt for purified Cas9 protein, like Invitrogen™ TrueCut™ Cas9 Protein v2, that’s engineered for the highest editing efficiency (>90%). Minimize off-target effects by carefully designing your CRISPR gRNA and avoid homology with other regions in the genome. Use a synthetic gRNA and a high-fidelity Cas9 protein like Invitrogen™ TrueCut™ HiFi Cas9 Protein to further reduce off-target effects. Design and test 3 gRNAs for every target to help maximize editing efficiency. Leverage software tools, like the Invitrogen™ TrueDesign™ Genome Editor, to design the most optimal gRNA for your intended gene target and cell types. Use optimal gRNA design Design and test 3 guide RNAs (gRNAs) for every target to help maximize editing efficiency for a greatly increased chances of success. Select most efficient delivery method for your cells Based on your specific cell types, select the best delivery method: lipid-mediated transfection, electroporation, or viral transduction. Confirm and validate editing efficiency Confirm cleavage efficiency with a genomic cleavage detection (GCD) assay or validate edits using other sequencing methods such as next-generation sequencing and Sanger sequencing. Pro tip Troubleshooting common CRISPR-Cas9 challenges Having trouble? You’re not alone—here are a few of the most common hurdles researchers face when using CRISPR and how to handle them. It was science fiction writer Jack Williamson who first popularized the term “genetic engineering” in his 1951 novel, Dragon’s Island, two years before Watson, Crick, and Franklin revealed the double helix structure of DNA and more than 70 years before CRISPR made precise gene editing possible in 2012.
