How Did We Edit Genes Before CRISPR?

Since the discovery of the DNA double-helix structure in the 1950s, scientists have gradually recognized that alterations in gene sequences can influence cellular functions, paving the way for the exploration of gene-editing technologies. Broadly speaking, any intentional modification to an endogenous gene sequence—such as knockout, insertion, replacement, or disruption of regulatory regions—can be considered gene editing.

With advances in sequencing technologies and molecular genetics, it became increasingly clear that many important biological phenotypes and even diseases arise from subtle changes in genetic sequences. Single-nucleotide substitutions, or point mutations, are among the most common forms of genetic variation, involving the alteration of a single nucleotide or nucleotide pair within DNA. These mutations may occur spontaneously or be induced by environmental factors, and can influence protein function or gene expression, resulting in synonymous, missense, or nonsense mutations.

Among the tens of thousands of identified disease-associated variants, single-nucleotide substitutions account for the largest proportion. This has driven the development of more precise gene-editing tools that allow researchers to model, correct, or investigate these mutations with greater accuracy-advancing basic research, disease-mechanism studies, and drug-target validation.

Figure 1. Overview of common gene-editing approaches

Early gene-editing technologies evolved from relying on natural cellular mechanisms to employing engineered nucleases. In this section, we highlight three pivotal approaches: gene targeting, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). These methods laid the groundwork for modern gene editing and significantly advanced research in genetics, developmental biology, and biomedical science.

Gene Targeting: The First Milestone in Mammalian Gene Editing

Gene targeting is an early gene-engineering strategy that enables precise modification of genomic DNA. By designing a DNA fragment homologous to the target gene—often carrying specific edits or selectable markers—researchers can leverage the cell’s own homologous recombination machinery to insert the exogenous DNA at the intended locus.

▌ Technical Principle

The core mechanism behind gene targeting is homologous recombination—a natural DNA repair process in which cells use a matching DNA template to repair double-strand breaks. Researchers design a targeting vector containing two “homology arms” identical to the sequences flanking the desired genomic locus, with the intended insertion or replacement—such as selectable markers or reporter genes—placed in between.

The gene-targeting workflow generally involves two major steps:
1. Gene editing in mouse embryonic stem (ES) cells
A targeting vector is constructed and introduced into ES cells using electroporation. Through homologous recombination, precise modifications can be introduced into the ES-cell genome.

2. Generation of gene-modified mice
ES cells carrying the desired mutation are selected and microinjected into mouse blastocysts. After chimeric mice are born, germline transmission is confirmed through breeding, completing the gene-targeting process.

Gene targeting opened the era of mammalian gene editing and laid the technological and conceptual foundation for many gene-editing tools that followed. Several fundamental laboratory techniques developed alongside gene targeting—such as stem-cell isolation, culture, and characterization, electroporation-based transfection, and microinjection—remain widely used in research today.

Zinc Finger Nuclease Technology (ZFN):
The First Generation of Engineered Gene Editing

As the first generation of engineered gene-editing technology, zinc finger nucleases (ZFNs) were discovered and developed in the late 1990s by Aaron Klug’s laboratory.

This technology fuses the DNA-binding domains of zinc finger proteins with the cleavage domain of the FokI endonuclease, creating an artificial chimeric nuclease.

ZFNs induce site-specific double-strand breaks in cellular DNA, significantly enhancing the efficiency of homologous recombination-mediated repair. This effectively overcomes the low efficiency and limited applicability of traditional gene-targeting approaches.

▌ Technical Principle

The core mechanism of ZFNs is “recognition and cleavage”—specific DNA sequence recognition followed by targeted double-strand break (DSB) formation.

Figure 2. Schematic representation of Zinc Finger structure

1. DNA Recognition Domain: Zinc Finger Arrays
Zinc finger proteins are common transcription factor domains stabilized by a zinc ion coordinated with multiple amino acids, such as cysteine and histidine, forming “finger-like” structures. Each “finger” can specifically recognize a triplet of nucleotides. By linking 3-6 zinc fingers in tandem, a zinc finger array capable of recognizing a 9-18 base pair sequence is created, enabling precise targeting of a specific genomic locus.

2. DNA Cleavage Domain: FokI Endonuclease
FokI is a restriction endonuclease derived from Flavobacterium okeanokoites. In ZFNs, only its cleavage domain is retained, without any inherent DNA sequence recognition capability.

3. Dimerization and DNA Cleavage
ZFNs must be designed and applied as pairs, each targeting sequences upstream and downstream of the intended DNA site. When both ZFN molecules bind to their respective target sites, the two FokI cleavage domains are brought into proximity, dimerize, and become activated. They cleave the DNA in the intervening region (typically 5-7 base pairs apart), generating a double-strand break.

The cell then repairs the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR), inducing site-specific recombination that can result in gene knockout, knock-in, or correction.

▌ Clinical Breakthroughs
ZFN technology has also made significant strides in clinical applications. In November 2017, the first human clinical trial assessing the safety and tolerability of in vivo genome editing using ZFNs commenced, targeting patients with mucopolysaccharidosis I (MPS I), MPS II, and hemophilia B. The study demonstrated successful genome editing in vivo and, in some patients, transiently increased enzyme activity to approximately half of normal levels. Despite its limitations, ZFN paved the way for the development of subsequent gene-editing technologies.

Transcription Activator-Like Effector Nucleases (TALEN):
The Second-Generation Modular Gene-Editing Tool

Following ZFNs, TALENs (Transcription Activator-Like Effector Nucleases) represent the second generation of engineered gene-editing technologies. Their core principle involves fusing TALE proteins—derived from the plant pathogen Xanthomonas, capable of recognizing specific DNA sequences—with the FokI nuclease responsible for DNA cleavage. This fusion enables precise double-strand breaks at targeted genomic loci, subsequently inducing gene knockout, knock-in, or repair. Compared to ZFNs, TALENs offer greater flexibility in sequence design and improved targeting specificity, making them easier to use in practice.

Figure 3. Schematic representation of TALEN structure

▌ Technical Principle

The core of TALENs lies in the TALE protein, which consists of three main components: a nuclear localization signal, a transcriptional activation domain, and a DNA-binding domain. The DNA-binding domain is composed of multiple tandem repeat units, each recognizing a single nucleotide. The amino acids at positions 12 and 13 of each repeat—known as the repeat-variable di-residue (RVD)—determine the nucleotide recognition specificity.

In TALENs, the TALE DNA-binding domain is fused to the FokI nuclease cleavage domain, creating an artificial nuclease. Only when a pair of TALENs binds to adjacent target sequences does the FokI domain dimerize, become activated, and cleave the DNA to generate a double-strand break (DSB). The cell then repairs the break through non-homologous end joining (NHEJ) or homology-directed repair (HDR), resulting in gene knockout or precise knock-in.

Comparison of Advantages and Disadvantages

Technology	Advantages	Disadvantages
Gene Targeting	High precision; Does not rely on specific sequences	Low efficiency; Long experimental cycle; High technical requirements
ZFN	Suitable for inserting relatively large fragments; Relatively high editing efficiency	Complex design; Risk of off-target effects and cytotoxicity; Sequence constraints; High construction cost
TALEN	High specificity with low off-target effects; Flexible sequence design; Can target most gene sequences	Large protein size complicates vector delivery; Labor-intensive construction and assembly; High cost and operational complexity

TALEN technology, with its modular protein design, achieved a revolutionary decoupling of DNA recognition and cleavage functions, marking a major leap in gene-editing specificity, flexibility, and safety. It effectively overcame many limitations of ZFN technology by employing programmable DNA-binding domains, greatly expanding the targeting range while significantly reducing off-target effects and cytotoxicity, thereby enabling nearly any genomic sequence to be edited.

The successful application of TALENs not only demonstrated the broad feasibility of efficient and precise gene editing but also provided critical design principles and practical experience that informed the development and optimization of subsequent, more streamlined, and powerful tools such as CRISPR.

EDITGENE focuses on CRISPR technology, offering a comprehensive range of high-quality gene-editing services and in vitro diagnostic products. These include CRISPR library screening, cellular gene editing and CRISPR detection, among others. We are dedicated to providing the most efficient technical support for scientific research related to CRISPR, gene function studies, in vitro diagnostics, and therapeutic applications.