CRISPR and the CRISPR Associated system (Cas) are powerful gene editing technologies. Originally identified and characterized in bacteria, the endogenous CRISPR systems act as an RNA-based defense mechanism against invading phage DNA.
CRISPR cas9 gene editing was adapted for genome editing in 2013 and has since been exploited for its ability to generate targeted double-stranded DNA breaks, which has revolutionized molecular biology protocols.1,2
This guide covers the basics of CRISPR experimental design and will prepare you to embark upon your own genome editing experiment.
The Basics of Commercial CRISPR Explained:
Endogenous CRISPR systems fall into three categories – type I, II and III. You can read more about these types in Makarova et al.3 Commercial CRISPR genome editing tools are adapted and simplified from endogenous type II systems and have the following components:
- Cas9: An endonuclease that induces a double-strand break in genomic DNA, allowing the removal of genes or DNA sequences, or the integration of foreign DNA at specific sites.
- Guide RNA (gRNA): This RNA has a scaffold sequence, which is needed for Cas9 binding, and a 20-nucleotide user-defined spacer or target DNA sequence.
- Homologous Recombination (HR) template (optional): This is a piece of DNA that contains the mutation(s) that you would like to introduce flanked by regions of homology.
How Does CRISPR/Cas9 Gene Editing Work?
When gRNA and Cas9 are expressed together in a cell, a gRNA:Cas9 complex is recruited to the target DNA sequence, which is located immediately upstream of a motif called a protospacer adjacent motif (PAM).4 The PAM motif targeted by most commercial Cas9 enzymes is NGG (any nucleotide followed by two guanines).
Binding of the gRNA to target DNA occurs via complementary base-pairing between the genomic target sequence and the 20-nucleotide spacer on the gRNA. The Cas9 in the gRNA:Cas9 complex then cuts the genomic DNA, inducing a double-stranded break after the PAM sequence. Crucially, Cas9 cannot digest DNA unless bound to the gRNA, thus providing specificity to the system.
The editing process is completed by repairing the break using the endogenous Non-Homologous End Joining (NHEJ) pathway. While this DNA repair system is the most efficient repair pathway, it is error prone, sometimes permitting small insertions or deletions, which can result in frameshifts and reduced protein production. An alternative option is to exploit the endogenous Homology Directed Repair (HDR) system by providing the HR template, as mentioned above. This is used when introducing targeted mutations.
Target Sequence and Guide RNA Considerations:
1. Before designing your gRNA, determine the exact sequence of your target DNA. Differences between your gRNA and target DNA can reduce efficiency. Therefore, check for any species- or cell-specific polymorphisms before you start.
1. Next, identify all PAM sequences (NGG) within your region of interest. Once identified, you can hone in on the best potential target region.
2. Design the gRNA and HR template (if using) based on the location of the PAM. The gRNA must match the target DNA. However, equally as important, the gRNA should not match any other “off target” genomic sites.
a. Check libraries/Use online tools
a.i. Before you start designing your own gRNA, make sure to check validated gRNA libraries such as GenScript’s gRNA library.
a.ii. If you don’t find what you need in existing libraries you can use a software tool, such as Desktop Genetics, GenScript, Geneious, Benchling or CRISPR Design (MIT) to aid your design. These tools can identify target sequences with PAM sequences, and rank the results by their specificity to the target region (i.e., their on- and off-target potential), potentially saving you a lot of hassle later.
b. Size is an important consideration:
b.i. For small changes of 100 bp), then the HR template requires longer homology arms (approximately 800 nucleotides long).
b.ii. After designing your templates clone them into donor vectors.
Transfection & Screening of CRISPR Constructs
Once you have designed and cloned the gRNA and HR templates, you cotransfect the Cas9 plasmid and your gRNA and HR donor vectors into the chosen cell line. Lipid transfection, electroporation or microinjection are all suitable transfection methods.
After transfection, screen for your desired genetic changes. You can do this via Restriction Fragment Length Polymorphisms assays (RFLP), Tracking of Indels by Decomposition (TIDE Analysis), Next-Generation Sequencing, or Fluorescence-Activated Cell Sorting (FACS). The optimal screening method depends on the nature of the modifications you made and your cell line. For example, researchers often introduce silent novel restriction sites into the HR template to allow for easy recombination screening with RFLP.
Optimizing recombination levels may take some trial and error. Choose a robust cell line (e.g., HEK) for troubleshooting. Once your experiment is up and running, you can move onto more expensive and less robust cell lines, if necessary.
Bear in mind that immortalized cell lines are not only cheaper than primary cells, but also have recombination pathways that are often less stringent. Therefore, you should ideally achieve a high level of recombination efficiency before moving to primary lines.
How to Increase your Chances of Successful CRISPR Genome Editing:
In the end, the efficiency of your CRISPR genome editing experiment is part plan, part luck. The interaction between the system components and Cas9 is still not well understood. Fortunately, there are a few ways you can increase your odds:
- Check your HR template for Cas9/PAM sites. These sites can lead to template degradation.
- Screen for PAM sites and remove them by making silent mutations. And remember, NGG to NAG doesn’t work, as NAG is a cryptic PAM site.
- Only use high quality intact DNA free of RNA and other contaminants. The market boasts a range of spin column-based kits for high quality DNA isolation.
If you have done everything right but are still experiencing low efficiency, then it is time to experiment. You may have better luck using sense and anti-sense templates. Others have reported better efficiency with asymmetrical arms.5 Be prepared to design a few setups – the efficiencies of overlapping designs can vary widely – and be ready to experiment to find the best design for your experiments. For more information about CRISPR/Cas9 gene editing, check out this free CRISPR handbook.
1. Cong L, Ann Ran F, Cox D, Lin S, Barretto R, Habib N, et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819-23. doi: 10.1126/science.1231143.
2. Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J, et al. (2013). RNA-guided human genome engineering via Cas9. Science 339:823-6. doi: 10.1126/science.1232033.
3. Markova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. (2011) Evolution and Classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467-77. doi: 10.1038/nrmicro2577.
4. Karvelis T, Gasiunas G, Siksnys 2. (2017) Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: a brief overview. Methods S1046-2023(16)30304-8. doi: 10.1016/j.ymeth.2017.03.006.
5. Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339-44. doi: 10.1038/nbt.3481.Image Credit: U.S. Department of Agriculture