The development of CRISPR/Cas9 technology has made it relatively straightforward to selectively edit genomes and has revolutionized the way in which we approach biological questions. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and in simple terms, this technique allows you to direct a nuclease to cut at a specific site in the genome of interest. The cell’s DNA repair machinery will then do the rest of the work for you. In recent years, researchers from diverse fields have plunged into the world of genome editing with CRISPR.
With this introductory article, you’ll be well on your way to starting your first gene-editing experiment. For a more in-depth look at how CRISPR gene editing works and considerations before you get started, including gRNA design, selection markers, and delivering CRISPR machinery into your cells, check out our related article “CRISPR Gene Editing: Considerations and Getting Started“.
Go Simple First
Until a few years ago, information and reagents for CRISPR/Cas9 technology were scarce. Nowadays, there is an overwhelming amount of CRISPR information, publications, and reagents. Trying to read, digest, and implement all the latest on CRISPR technology may seem like an impossible task.
If you wish to delve into the CRISPR world we recommend starting with the core publications listed at the end of this article. In addition, good online resources include genome-engineering.org and the Addgene website.
Reproduce Established CRISPR Protocols
Even though you might be tempted to go head-first into your first CRISPR project and get some exciting results, it is critical to make sure the technique works well in your hands first. The best way to do this is to start out with guide RNAs (short synthetic sequences critical for Cas9 binding) and other CRISPR reagents that have proven to be successful elsewhere, e.g. the widely used Px330 or Px458 from Feng Zhang’s group at MIT – you can order these from Addgene.
There are few widely used guide RNAs that work well in mammalian cell lines or many other systems available on Addgene‘s website. It is wise to establish your genome editing technique in vitro first in an easy-to-handle cell line such as HEK293T, before moving to different cell lines or systems. To do this, start out with the published guide RNA of the EMX1 gene. Good guides include Multiplex Genome Engineering Using CRISPR/Cas Systems (1) or Genome Engineering Using the CRISPR-Cas9 System (2).
You will find that most CRISPR publications use protocols based on generic cell lines. Even if you need to eventually use a different cell line, look into these generic protocols. They will give you the basics that you can then adapt to your specific needs. For instance, you may need to tweak the CRISPR/Cas9 construct by changing the Cas9 promoter for your cell line. If you make protocol changes, be sure to test every component of your new system in vitro to ensure that each is working correctly.
Once you get the technique up and running, you can adapt your protocol to your cells of interest.
…But Be Patient
Whatever system you are using (cell line, mouse, plant, etc.) might not work straight from the start. Bear in mind that it can take up to 6 months to get your system up and running. So be patient and don’t hesitate to go online for help and advice. There are many good online discussion forums, where you can get help to troubleshoot your CRISPR system.
And Enjoy the Editing
Once you have gained some hands-on experience with the CRISPR/Cas9 system, you will find it easy to tweak and highly efficient. So rest assured, after all the troubleshooting, there will be time to enjoy the fun and power of genome editing!
Have your own tip? Leave a comment below.
Core CRISPR Publications
- Le Cong et al. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339(6121):819-823.
- Mali et al. (2013) RNA-Guided Human Genome Engineering Via Cas9. Science 339(6121): 823-826.
- Ran FA et al. (2013) Genome Engineering Using the CRISPR-Cas9 System. Nat Protoc. 8(11):2281-308
- Wang H et al. (2013) One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-mediated Genome Engineering. Cell. 9;153(4):910-8
- Pyzocha NK et al. (2014) RNA-Guided Genome Editing of Mammalian Cells. Methods Mol Biol. 1114:269-77