Pros and cons of CRISPR. Pros: its simplicity once established, widespread use in research demonstrated by numerous publications, and relatively low cost. Cons: the significant time and effort required to set up and optimize a CRISPR system, issues with editing efficiency, and the risk of off-target effects that might alter unintended parts of the genome.

Genome editing is a hugely powerful tool that can help you 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.

The Pros

It’s Simple to Amend Your Target Region

OK, setting up the CRISPR-Cas9 genome-editing system for the first time is not simple. Optimizing your protocol takes a lot of grafting 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 somewhere 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, 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.

It’s Cheap

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.

The only other reagents you need are those you’ll find in any genetics lab with cell culture facilities: cloning equipment, cells, media, and transfection reagents.

The Cons

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:

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’s Not Always Efficient

The editing efficiency can be influenced by many factors, severely hampering 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:

  1. Your cells may no longer behave ‘normally’ in the presence of toxic selection with antibiotics.
  2. 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 must be confident that low efficiency will not ruin your experiment, and this should be a prominent consideration when you plan and optimize your approach.

Off-Target Effects

You’ve taken all the precautions and designed your CRISPR guide RNAs to be specific and target only the genetic region 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’s 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.

The Pros and Cons of CRISPR Summarized

These are the main pros and cons of CRISPR-Cas9 as a genome-editing system.

Its ability to target and modify specific genetic sequences makes it invaluable for research and therapeutic applications.

However, challenges such as the complexity of initial setup, variable editing efficiencies, and potential off-target effects must be carefully considered before you embark on your own genome-editing adventure.

Discover more about CRISPR in the Bitesize Bio CRISPR Research Hub.

And because there’s so much to learn, get your free gene editing 101 eBook to get up to speed today!

Gene editing 101 eBook for readers to download to learn more about the pros and cons of CRISPR

FAQs

Q: How does CRISPR-Cas9 compare with other genome editing technologies like TALENs and ZFNs?

A: Comparing CRISPR-Cas9 with other genome editing technologies, such as TALENs (Transcription Activator-Like Effector Nucleases) and ZFNs (Zinc Finger Nucleases), reveals why CRISPR has become more popular.

While all three allow for targeted genetic modifications, CRISPR-Cas9 is generally faster, more cost-effective, and capable of editing multiple genes simultaneously, which is more difficult with TALENs and ZFNs. However, TALENs and ZFNs often have lower off-target effects compared to CRISPR, making them preferable in situations where precision is more critical than efficiency.

Q: What are the latest advancements in improving the accuracy and efficiency of CRISPR-Cas9?

A: Researchers have developed high-fidelity versions of Cas9 that reduce off-target effects. Additionally, new techniques like prime editing and base editing offer more precise editing capabilities by enabling direct, single-nucleotide alterations without creating double-strand breaks, thus minimizing unintended consequences.

These innovations continue to refine the CRISPR toolkit, making it not only more reliable but also expanding its potential applications in medicine and biotechnology. [4]


Originally published September 23, 2019. Reviewed and updated April 2021 and May 2024.

References

  1. Cong L, et al. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339(6121): 819–23
  2. Mali P, et al. (2013) RNA-Guided Human Genome Engineering via Cas9. Science 339(6121):823–26
  3. The importance of no evidence. (2019) Nat Hum Behav 3:197
  4. Hillary VE, Ceasar SA. (2023). A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering. Molecular Biotechnology 65(3):311–25

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