CRISPR/Cas is a genome-editing technology derived from a natural defense mechanism found in bacteria and archaea. The CRISPR system, which stands for "Clustered Regularly Interspaced Short Palindromic Repeats," works in conjunction with Cas (CRISPR-associated) proteins to recognize and destroy invading genetic material, such as that from viruses. Scientists have adapted this system to enable precise and efficient editing of DNA in a wide range of organisms. The technique involves two main components: the Cas9 protein, which acts as molecular scissors, and a guide RNA (gRNA), which directs Cas9 to the specific DNA sequence to be edited. The gRNA is engineered to match the desired target sequence, ensuring high specificity. Once bound, Cas9 creates a double-strand break at the target site. The cell then repairs this break using one of two mechanisms. Non-homologous end joining (NHEJ) typically introduces small insertions or deletions, often disrupting the gene, while homology-directed repair (HDR) allows precise modifications if a repair template is provided. CRISPR/Cas has transformed genetic engineering due to its simplicity, efficiency, and versatility.

TALENs (Transcription Activator-Like Effector Nucleases) are advanced tools used for precise genetic editing by targeting specific DNA sequences. They consist of two main components: a DNA-binding domain derived from Transcription Activator-Like Effectors (TALEs) and a nuclease domain. When two TALEN molecules bind to opposite DNA strands near each other, the nuclease domains come into proximity, causing a double-strand break in the DNA. These breaks trigger natural cellular repair mechanisms. Non-homologous end joining (NHEJ) often introduces small insertions or deletions (indels), which can disable genes, while homology-directed repair (HDR) enables precise DNA changes if a template is provided. TALENs have found diverse applications in research, medicine, and agriculture. They are used to study gene functions, develop gene therapies for genetic disorders, and engineer crops with enhanced traits such as disease resistance. TALENs offer high specificity and flexibility, capable of editing nearly any genome. However, challenges include the complexity of constructing large TALENs and the potential for off-target effects. While newer techniques like CRISPR-Cas systems often dominate the field, TALENs remain an important tool due to their precision and adaptability in certain applications.

Zinc Finger Nucleases (ZFNs) are powerful tools for precise genome editing that combine the DNA-binding specificity of zinc finger proteins with the DNA-cleaving activity of a nuclease. Zinc finger proteins are naturally occurring DNA-binding domains that recognize specific DNA sequences through modular structures, each capable of binding to a triplet of DNA bases. By engineering arrays of these zinc fingers, scientists can target virtually any DNA sequence. In ZFNs, the zinc finger DNA-binding domain is fused to the nuclease. For effective DNA cleavage, two ZFN molecules must bind to opposite DNA strands near each other, bringing the nucleases into proximity. This dimerization creates a double-strand break at the target site. The cell repairs this break via one of two pathways: non-homologous end joining (NHEJ), which can introduce insertions or deletions that disrupt the gene, or homology-directed repair (HDR), which allows precise sequence modifications using a repair template. While ZFNs offer high specificity and flexibility, the design and optimization of zinc finger arrays are technically challenging, requiring significant expertise. Additionally, off-target effects can occur if the zinc fingers bind unintended DNA sequences. Despite these challenges, ZFNs paved the way for modern genome editing techniques like TALENs and CRISPR, and they remain a valuable tool for applications requiring precise genetic modifications.

When an SDN is used without a DNA repair template, it creates a targeted but random genetic deletion mutation. In this case, the DSB is precisely located, but the host cell’s DNA repair mechanisms act unpredictably, resulting in small nucleotide deletions, insertions, or substitutions. Alternatively, if two DSBs are introduced flanking a specific DNA sequence, SDN-1 can remove larger DNA segments, such as a promoter or an entire gene. In both cases, screening and selecting for the desired change ensures the identification of the intended genomic mutation.

Applications:

• Gene knockouts or loss-of-function mutations.
• Generating disease resistance or altering traits in plants.

SDN-2 creates targeted DNA breaks, coupled with a repair template to guide precise modifications during the cell’s repair process. The repair template, provided as an external DNA molecule, allows the introduction of small, predefined changes, such as point mutations or short sequence insertions. Unlike SDN-3, SDN-2 does not result in the integration of large foreign DNA, making it useful for subtle and precise genome edits.

Applications:

• Correcting point mutations causing genetic diseases.
• Introducing precise genetic modifications for trait enhancement in agriculture.

SDN-3 introduces double-strand DNA breaks and integrates larger pieces of foreign DNA into the genome through homology-directed repair (HDR). Unlike SDN-1 and SDN-2, this approach allows for the insertion of entire genes or regulatory elements, enabling more substantial genetic modifications.

Applications:

• Generating transgenic organisms by integrating new genes.
• Developing organisms with novel traits, such as pest resistance or enhanced nutritional profiles in crops.