How Does CRISPR Compare with Other Gene-Editing Methods?

In 2013, a programmable nuclease repurposed from an innate bacterial adaptive immune system was first shown to successfully edit genomic sequences in eukaryotic cells—and so started a revolution in gene editing. The new programmable nuclease was CRISPR-Cas9, which has since exploded into the biological space and been rapidly adopted as the go-to gene-editing tool.¹ Researchers can now make targeted and deliberate changes to DNA sequences relatively easily, which is having a tremendous impact on our understanding of basic biological processes and mechanisms of disease, helping to identify novel drug targets and advance gene therapy development.

But sometimes forgotten in the midst of the CRISPR furor are the gene-editing methods that came before, such as naturally occurring meganucleases, and the sequence-specific engineered zinc finger nucleases (ZFN) or TALENS. This article looks at these alternative gene-editing tools and how they compare to CRISPR.

Gene editing: Harnessing cellular DNA repair pathways

The underlying principle of gene editing is to introduce a double-strand break (DSB) into the DNA at the site of interest, and then rely on endogenous DNA repair pathways to elicit the required change. Due to its error-prone nature, the non-homologous end joining (NHEJ) pathway often causes frameshift mutations with the introduction of insertions or deletions (indels) at the target site, and so is useful for causing gene knockout. Alternatively, in the presence of a donor template sequence, a DSB can trigger homologous repair (HR), allowing an exogenous piece of DNA to be integrated at the target site, thereby facilitating a sequence change, or gene knock-in. Therefore, with the simple introduction of a DSB at the target site, the DNA sequence can be manipulated as desired—either deleting the gene of interest to uncover the function of a particular gene or allowing for targeted repair or replacement of specific sequences.

Meganucleases: The original editor

Early gene-editing efforts leveraged naturally occurring nucleases called meganucleases to introduce a DSB. Meganucleases are derived from microbial mobile genetic elements and are perhaps the most specific of the technologies, recognizing a 12–40 bp sequence and only able to tolerate minor variations at the recognition site. But this is both a strength and weakness—this high specificity means off-target effects are very low, but their use as a gene-editing tool relies on finding the right one for your chosen site—and there is a limited range to choose from.

Programmable editing: Zinc finger nucleases

Scientists then turned to protein engineering, and by using a customizable, sequence-specific DNA binding domain fused to a non-specific DNA cleavage molecule, created a nuclease that could theoretically be programmed to target any site in the genome. The first such programmable nuclease to be developed was the ZFN, which contain the zinc finger motif, one of the most common DNA binding domains in eukaryotic cells.² Zinc fingers are typically around 30 amino acid modules and recognize regions of 3–6 nucleotides, so by linking several zinc finger domains with a highly conserved linker region, synthetic proteins can be engineered to recognize longer DNA sequences. These DNA binding assemblies are combined with a Fok1 restriction endonuclease—as dimerization of Fok1 is required to cleave DNA, gene editing relies on a pair of ZFN to recognize sequences on opposite strands of DNA. However, zinc finger motifs arranged in an array can influence the specificity of the neighboring fingers, which can introduce design restrictions.

Improved resolution with TALENs

For many years, ZFNs were the only programmable site-specific nuclease available—until the discovery of a simple TALE DNA binding domain from the plant bacteria Xanthamonas. The TALE motif consists of 33–35 amino acid module that targets a single nucleotide—TALENs (or transcription activator-like effector nucleases) are modular molecules consisting of several TALE motifs in the required combination to target the sequence of interest.³ Like ZFNs, TALENs are fusion proteins, relying on the Fok1 endonuclease to introduce a DSB at the target site but compared to ZFNs, are cheaper and produce faster results—they are more flexible to design as individual TALEs do not affect binding of adjacent domains. However, as with ZFNs they still require a degree of expertise in protein engineering, although this was largely overcome with the development of freely available design tools. TALENs can be difficult to clone due to their large size—but despite these challenges, it is safe to assume that scientists would have continued to use and optimize these technologies, and highly likely that TALENs in particular would have found wider adoption, were it not for CRISPR.

Enter CRISPR: The next phase in gene editing

The CRISPR system (which stands for clustered regularly interspaced short palindromic repeats) was originally part of a microbial immune system used to defend against foreign DNA. It has since been repurposed into a simple but very powerful gene-editing tool—consisting of just a short RNA molecule (single-guide RNA, or sgRNA) and Cas9 endonuclease. The Cas9 protein recognizes the 3’-NGG-5’ PAM site (protospacer adjacent motif), which occurs frequently throughout the genome—if the sequence adjacent to the PAM site shares homology with a 20-nucleotide portion of the sgRNA, then the endonuclease activity of Cas9 is activated and a DSB is introduced at the target loci.

Due to the RNA-based nature of the system, CRISPR is the most flexible, scalable, and user-friendly of the gene-editing platforms. Rather than relying on costly and challenging protein engineering to recognize a new target site, reprogramming requires just a change to the 20-nucleotide portion sgRNA. All components are delivered to the cell using standard molecular biology techniques so gene editing can be performed within the technical capabilities of most laboratories—and as CRISPR transfections have a higher efficiency, this results in robust levels of editing, and avoids mosaicism that can sometimes occur with TALENs. There is a greater tolerance for mismatch of the sgRNA to the target site compared to TALENs, which can result in off-target activity.⁴ But this can be reduced with careful selection of the sgRNA—there are several computational models that have been developed to help select the most specific and efficient sgRNA for your experiment.

Other strategies to improve precision have also been developed, such as a truncated sgRNA with a recognition sequence of 17 bp, using paired “nickase” Cas9 variants that introduce a single-strand break, or using a similar approach to ZFNs and TALENs, tethering the Fok1 domain to a catalytically inactive version of Cas9 and using offset sgRNA pairs that require dimerization to introduce the DSB.

A revolution in gene editing

When selecting a gene-editing platform to use, there are three main areas to consider:

1. Specificity or the frequency of off-target effects

2. The efficiency with which the target site will be edited

3. How easy is the system to implement and use.

The most important advantage CRISPR has over the other gene-editing platforms that have come before is arguably its simplicity and efficiency largely due to its RNA-based nature. CRISPR is much less labor intensive and cheaper than ZFN and TALENs, with an additional advantage that it can be used to target multiple loci simultaneously—or used to perform high-throughput, genome-wide functional screens.

Researchers can also utilize the expanded CRISPR toolkit to mediate other changes to the genome, such as modulation of gene expression with CRISPRi and CRISPRa, the introduction of single nucleotide variants with base editing, or even prime editing.⁵ There may be some cases where the use of TALENs is preferred—a recent Nature paper suggests that TALENs may be more efficient than Cas9 when targeting heterochromatin.⁶ But for most applications, the CRISPR system provides the best combination of accessibility, efficiency, and precision for gene editing, which will only increase as the platform is developed further.

Key takeaways

• Gene editing relies on the introduction of a double-strand break at the target site and activation of endogenous cellular DNA repair pathways
• Two other gene-editing technologies are ZFNs and TALENs, which require protein engineering to fuse a customizable, sequence specific DNA binding domain to a non-specific Fok1 DNA cleavage molecule
• More recently, the RNA-based CRISPR gene-editing technology has been developed, which involves using a Cas9 endonuclease targeted to the desired site by a short piece of RNA (sgRNA)
• The protein engineering required to reprogram ZFNs and TALENs can be time consuming, challenging, and costly, whereas CRISPR relies on just a change to the 20-nucleotide portion of the sgRNA to target the Cas9 endonuclease
• The CRISPR system has proven so simple to implement and use, and so effective for gene editing that it has seen widespread adoption and a publication count that already dwarfs that of ZFNs and TALENs combined

References

1. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, (2014)

2. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

3. Gaj, T., Gersbach, C. A. & Barbas, C. F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31, 397–405 (2013)

4. Tsai, S., Joung, J. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat Rev Genet 17, 300–312 (2016). 

5. Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). 

6. Jain, S., Shukla, S., Yang, C. et al. TALEN outperforms Cas9 in editing heterochromatin target sites. Nat Commun 12, 606 (2021).