Genome editing is a hugely powerful tool, which can help you to address a multitude of questions in your research. There is a range of CRISPR applications in disease and CRISPR screening is already used for identifying potential drug targets. However, it is not necessarily the best tool for the job in every situation. Below is a discussion of the main pros and cons of CRISPR-Cas9 for genome editing.
It’s Simple to Amend Your Target Region
OK, setting up the CRISPR-Cas9 genome-editing system for the first time is not simple. It takes a lot of grafting to optimize your protocol before you have any success. However, once your protocol is up and running, it is really simple to ‘chop’ and change your setup in order to target alternative genomic regions for editing. All you need to do is design and order your new guide RNAs, which can then be introduced into your up-and-running system.
The perfect example of this utility is the DepMap project, which has deleted in the region of 18,000 genes in each of 500 cancer cell lines in a high-throughput CRISPR gene dependency screen. A phenomenal amount of work, but remember that one deletion in one cell line may be all you need to get that publication or figure for your thesis.
There Are Lots of Publications Using CRISPR-Cas9 Genome Editing
Since the first papers were published on using CRISPR-Cas9 as a genome-editing tool (1, 2), the number of publications using the technology has rocketed: there are more than 23,000 articles listed in PubMed alone. Considering the publication bias towards positive results (3), this means that there are probably thousands of additional labs, projects, and scientists around the world using this system.
CRISPR-Cas9 has very quickly become a tried and tested genome-editing tool for a reason. It works. It’s a simple yet effective way to investigate the function of your gene or genetic region. A quick PubMed search can help you uncover whether or not someone else has been able to successfully genome edit your cells of interest, giving you encouragement that it is possible, as well as an experimental protocol to follow.
CRISPR-Cas9 editing is a relatively inexpensive way of deleting, silencing, or otherwise modifying a gene or region. If you’re lucky, you can pick up Cas9 and guide RNA expression vectors from a colleague or collaborator’s lab; then all you need to buy are your primers to synthesize the guide RNA vectors. The remaining preparatory steps can be performed by you in the lab, and the only other reagents that you need are those that you’ll find in any genetics lab with cell culture facilities: cloning equipment, cells, media, and transfection reagents.
Setting up from Scratch Is a Considerable Time Investment
Not all labs have an established genome-editing pipeline. If you’re in a lab without such a pipeline but have identified genome editing with CRISPR-Cas9 as the ideal technique to further your project, then chances are your PI will task you with creating and optimizing the protocol. Use these BiteSize Bio articles to help you shape your approach:
- CRISPR Genome Editing – What You Need to Know to Get Started
- Get Started in Genome Editing with CRISPR
- How to Design a CRISPR Experiment and Start Genome Editing
- How to Confirm Your CRISPR-Cas9 Genome Editing Was Successful
Optimizing a CRISPR-Cas9 protocol can be challenging and time-consuming. But with skill, luck, and perseverance, you can do it! It is an incredibly useful technique. If you can perform it for colleagues or give tutorials, it can even help boost your CV and research profile with collaborations or co-authorship on papers.
It Is Not Always Efficient
Editing efficiency can be influenced by many factors and can severely hamper your efforts in any genome-editing experiment. Editing efficiency essentially describes the percentage of cells that have been successfully edited in your culture vessel. An editing efficiency of less than 100% is by no means a disaster, but it does mean that you need to interpret your results carefully. Any subtle effect of your editing may be masked by the unedited cells within your population.
There are ways to prevent this. The most common method is to include selection markers in your Cas9 expression vector and to select the population of cells that have successfully been transfected. However, this approach has two potentially fatal caveats:
- Your cells may no longer behave ‘normally’ in the presence of toxic selection with antibiotics.
- If your cells do not readily divide or expand in culture, then the selection of a sub-population of your cells may limit the number of cells that you have to work with.
You need to be confident that low efficiency will not ruin your experiment and this should be a prominent consideration when you plan and optimize your approach.
You’ve taken all the precautions and have designed your CRISPR guide RNAs to be specific and target only the genetic region that you’re interested in. You’ve double-checked that the guide RNA sequence is unique in the genome. Cas9 should only cut at that one specific site, right? Wrong.
In theory, the CRISPR-Cas9 system is incredibly specific, but in practice, it is not. It can create mutations elsewhere in the genome, known as ‘off-target’ modifications. Off-target effects are random and can unduly influence other genes or regions of the genome. You need to factor this into the discussion of your results.
Off-target effects can be reduced by using the modified version of Cas9, known as the Cas-9 nickase, which creates a nick in only one DNA strand rather than a double-stranded break. However, you can never be 100% confident that you don’t have any off-target effects. The consequences can be potentially catastrophic.
These are the main pros and cons of CRISPR-Cas9 as a genome-editing system. Give each point due consideration before you embark on your own genome-editing adventure and you’ll maximize the chances of it working for you.
Discover more about CRISPR in the Bitesize Bio CRISPR Research Hub.
Originally published September 23, 2019. Reviewed and republished April 2021.
- Cong et al., (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science, 339(6121): 819–823. doi:10.1126/science.1231143.
- Mali et al., (2013) RNA-Guided Human Genome Engineering via Cas9. Science, 339(6121): 823–826. doi:10.1126/science.1232033.
- The importance of no evidence. (2019) Nat Hum Behav 3: 197. doi.org/10.1038/s41562-019-0569-7