CRISPR-Cas9, TALENs and ZFNs - the battle in gene editing

Technological advances are crucial for innovative biological research. ZFNs, TALENs, and CRISPR/CRISPR-associated Cas have revolutionized genome editing.

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Summary

• Recognition: ZFNs and TALENs use protein-DNA binding; CRISPR uses a simpler, more programmable RNA-DNA guide system.
• Design: CRISPR is the most user-friendly and fastest to engineer, while ZFNs are the most complex due to unpredictable protein interactions.
• Precision: All three utilize specific enzymes (FokI or Cas9) to cut DNA, but CRISPR "nickases" offer higher specificity with fewer off-target errors.
• Multiplexing: CRISPR is uniquely capable of editing several different genes simultaneously, a significant advantage over earlier tools.
• Versatility: Beyond cutting, "deactivated" CRISPR (dCas9) can be used to regulate gene expression or visualize genomic locations.

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Advances in DNA genome editing

Genome editing is the process of making permanent modifications to DNA sequences at specific locations. Genomic editing was initially performed by introducing breaks to double-stranded DNA via radiation or using cleavage proteins called endonucleases. DNA was repaired either by non-homologous end joining (NHEJ), where the broken ends are directly joined back together, or by homologous repair (HR) where a homologous (similar) DNA sequence of is used as a guide for repair. Repair templates may also contain selection markers, such as those for antibiotic resistance or fluorescent tags; these selection tools can be used to screen mixed populations of cells for those containing the desired DNA modifications.

The genome editing process until recently, was time-consuming, requiring mutagenesis, cleavage, and the synthesis and delivery of long homologous repair DNA templates for specific changes. Genome editing in mammalian cells was often inefficient and required multiple steps. Fortunately, the discovery of novel nucleases allowed improvements and advances in the field of genomic editing. In this article, we will briefly describe the four groups of genome-editing nucleases: meganucleases (MNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-associated nucleases (CRISPR-Cas9).

The era of nucleases in genome editing

Meganucleases (MNs)

Meganucleases are Double-stranded DNAses (enzymes that cleave DNA) with characteristically long recognition sites, for example, upward of 40 nucleotides ¹ (Table 1).

Meganucleases are often referred to as “DNA scissors”, due to their ability to recognize and cleave large pieces of DNA. Meganucleases are encoded by mobile introns, and their targets are found in homologous genes that lack the mobile intron they carry. First, the meganucleases pair with a recognition sequence and cleave DNA, then the mobile genetic element is inserted into the cut site ². The benefits of using meganucleases include their low toxicity in mammalian cells, as they’re naturally occurring. Another benefit is their high specificity due to their long recognition sequences, which are often not duplicated in the genome due to their large size.

Zinc finger nucleases (ZFNs)

Zinc finger nucleases are based on zinc finger proteins, a family of naturally occurring transcription factors, fused on an endonuclease Fok ³.

Zinc finger nucleases were one of the first enzymes utilized in targeted genome engineering and are created when a zinc finger is fused to a restriction endonuclease, usually FokI ³. Zinc finger domains recognize a three base pair sequences on DNA; a series of linked zinc finger domains can therefore recognize longer DNA regions, providing desired on-target specificity. The FokI endonuclease works as a dimer, which means that the double-strand DNA cleavage occurs only at sites of binding of two ZFNs onto the opposite DNA strands (Figure 1A). Two ZFNs are engineered to recognize different closely located nucleotide sequences within the target site, which requires the simultaneous recognition and binding of both ZFNs, and naturally limits off-target effects. However, multiple zinc finger motifs aligned in an array also influence the specificity of neighboring zinc fingers, making the design and selection of modified zinc finger arrays more challenging. Predicting specificity of the final product is can often be difficult.

Transcription activator-like effector nucleases (TALENs)

Transcription activator-like effector nucleases are fusion proteins of a bacterial TALE protein and the FokI endonuclease ⁴.

Similar to ZFNs, target specificity comes from the protein-DNA association. In the case of TALENs, a single TALE motif recognizes one nucleotide, and an array of TALEs can associate with a longer sequence (Figure 1B). The activity of each TALE domain is restricted only to one nucleotide and does not affect the binding specificity of neighboring TALEs, making the engineering of TALENs much easier than ZFNs. Like ZFNs, TALE motifs are linked with FokI endonuclease, which requires dimerization for the cleavage to occur. This means that the binding of two different TALENs at opposite strands in close vicinity to the target DNA is needed.

The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats)

The system is based on the bacterial immune system.

It is composed of Cas9 nuclease and two types of RNAs: trans-activating crRNA (tracrRNA) and a single guide RNA (sgRNA) that recognizes the target sequence by standard Watson-Crick base pairing. It has to be followed by a DNA motif called a protospacer adjacent motif (PAM). Each Cas9 protein has a specific PAM sequence, ^5,6 e.g., for standard Cas9 it is 5’-NGG-3’. DNA cleavage is performed by Cas9 nuclease and can lead to a double-strand break in the case of a wild-type enzyme, or to a single-strand break when using mutant Cas9 variants called nickases (Figure 1C). Recognition of the DNA site in the CRISPR-Cas9 system is controlled by RNA–DNA interactions. This offers many advantages over ZFNs and TALENs, including easy design for any genomic targets, easy prediction regarding off-target sites, and the possibility of modifying several genomic sites simultaneously (multiplexing). Furthermore, the recognition sequence of the sgRNA is small (20bp), which is useful for cloning, compared to traditional homologous repair design.

Mutations to the Cas9 protein are also used for different applications of experimental design. One example is dCas9, which that renders Cas9 catalytically dead (no activity in both catalytic domains). Many Cas9 systems, however, often achieved low off-target effects at the cost of target specificity. One means to resolve these issues, Cas9 ‘nickases’ were developed. Cas9 d10A and H840A are two mutations that render one of the domains inactive, in Cas9, such that the enzyme is only capable of single-strand DNA cleavage, compared to the double-strand cleavage of WT Cas9. The Cas9 nickases are often used in pairs, creating a double-strand break only where each nickase cleaves at each single-strand, enhancing both specificity and reducing off-target effects ⁷.

Feature	MN's	ZFNs	TALENs	CRISPR-Cas9
Length of recognized DNA target	~14-40bp	9–18 bp	30–40 bp	22 bp + PAM sequence
Mechanism of target DNA recognition	Protein-DNA	DNA–protein interaction	DNA–protein interaction	DNA–RNA interaction via Watson-Crick base pairing
Mechanism of DNA cleavage and repair	Double-strand break	Double-strand break induced by FokI	Double-strand break induced by FokI	Single- or double-strand break induced by Cas9
Design	Difficult due to large sizes of recognition sites.	Challenging. Motifs can affect neighboring specificity.	Easy. TALE motifs are well defined.	Easy. Based on sgRNA complementarity.
Cloning	Requires I-CreI or I-SceI scaffold	Requires engineering linkages.	No linkages required. Golden Gate assembly.	Cas9 vectors available. SgRNA can be DNA or RNA.

Table 1. Target specificity, mechanism of action, and experimental design for commonly used editing nucleases.

Figure 1. Editing nucleases. A. ZFNs – two discrete ZFNs recognize and bind to specific sites; assembled FokI dimer cleaves DNA. B. TALENs – two discrete TALENs bind opposite strands; FokI dimer cleaves DNA. C. CRISPR-Cas9 – site recognition via sgRNA complementarity.

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Reducing off-target effects

The design of editing nucleases in a way that reduces off-target effects was a major issue not only for the basic science approaches, but also for their putative clinical and industrial use.

The delivery of ZFNs and TALENs, either in vivo or in vitro, can lead to toxicity or lethality due to binding at off-target sites and the induction of undesired DNA cleavage. Mutations in FokI in the case of ZFNs and TALENs were eventually introduced to promote FokI endonuclease activity only during heterodimerization events at sites bound by two discrete nucleases ^9,10. Off-target cleavage of CRISPR-Cas9 systems mostly comes from the recognition of fully or partially complementary genomic sites by sgRNAs ¹¹. Various approaches have been suggested to limit off-target cleavage, including limiting the amount and time of active Cas9 protein in cells by selective delivery methods or by altering Cas9 half-time ¹². A number of Cas9 variants with lower off-target specificity have been developed, including HF-Cas9, eCas9, and HypaCas9 ^13–15. New variants of Cas9 and Cas9 homologs, such as CRISPR-Cas12 (Cpf1) and CRISPR-Cas13a (C2c2), can recognize different PAMs, not only significantly increasing options for precision genome editing, but also potentially having higher on-target specificity ¹⁶. An interesting alternative is a Cas9 fusion with FokI nuclease, which combines the advantages of ZFNs/TALENs and the CRISPR-Cas9 system ^16,17. When fused to the FokI endonuclease, dCas9 has greater specificity compared to Cas9 alone ⁷. This specificity comes from the dCas9-FolkI cleavage activity, which depends strictly on the sgRNAs. The dCas9 protein proves to be a viable means to control gene expression by interfering with transcription ¹⁸.

Cas9 chimeras as novel research tools – not only for DNA cleavage

Various Cas9 chimeras, including loss-of-function and gain-of-function studies, have also been recently developed ¹⁹. Cas9 allows for genomic targeting, while the fused proteins can modulate Cas9 activity.

CRISPR interference (CRISPRi), occurs when a Cas9 protein is fused to a transcriptional effector such as the repressive KRAB domain, allowing downregulation of gene expression by transcriptional repression ²⁰. This approach provides an advantage over standard RNA interference (RNAi) techniques and allows for investigation of the function of non-protein coding genes. Additionally, the opposite approach (CRISPR activation, CRISPRa) is possible, meaning Cas9 can be used to activate gene expression. For example, Cas9 fusion with transcriptional activation domains (e.g., VP64) leads to increased expression of the desired genomic loci. Additional activation of transcription can be achieved with synergistic activation mediator (SAM), where the Cas9 fusion contains several activation domains to maximize activation efficiency ²¹. Some Cas9 chimeras do not possess nuclease activity. For example, Cas9 fusions with chromatin-modifying enzymes are used in epigenome studies to visualize protein-DNA binding sites, to examine chromatin structure, or for histones post-translational modification research ^22,23.

CRISPR-Cas9 and cell line gene-editing innovations

CRISPR-Cas9 can be used to reverse harmful genetic mutation in the human genome. This approach requires Cas9 protein, tracrRNA, and site-specific sgRNA together with the repair DNA template containing wild-type sequence. Cas9 creates a site-specific DNA break that is subsequently repaired by host DNA repair machinery, restoring wild-type allele (Figure 2A).

CRISPR-Cas9 can also serve as a reliable tool in cell line engineering. Cas9 creates a site-specific DNA break that is repaired by host DNA repair machinery. In the absence of a repair template, it can lead to truncations resulting in null mutants. A more targeted approach is available by introducing repair templates containing the desired mutation (Figure 2B).

Figure 2. Use of the CRISPR-Cas9 system in A) gene therapy, and B) cell line engineering.

Future avenues

Editing nucleases have revolutionized genomic engineering, allowing easy editing of the mammalian genome. They are commonly used not only in basic research but also in pre-clinical and clinical studies as a potential mechanism to reverse DNA-based disease-causing mutations in inherited genetic diseases. Non-commercial and commercial innovations have allowed continuous development of technologies that started only 20 years ago, and they will be even more widely used in the future in advancing science and biomedicine.

Posted by Dr. Karolina Szczesna, Senior Product Manager and Technical Support at Proteintech Ltd.
Last edited in 2023 by Nicole Lynn, PhD Candidate at University of California Los Angeles

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