Genome editing techniques and its principles

In the last few decades, progresses in breeding approaches, especially forward genetic approaches, have played vital roles in elucidating the molecular mechanism that control agriculturally important traits in tomato. The advantage of conventional plant breeding consists of increasing the availability of genetic resources for crop improvement through introgression of the desired traits [7,10,11]. However, some plants are at risk of becoming susceptible to environmental stress and losing genetic diversity. Thus, traditional cultivation methods are not sufficient to resolve global food security issues [12].

The newly developed technologies in genome-editing have overcome the limitations of traditional breeding methods in elaborating functional genomics and crop improvement in tomato. These genetic innovations provide more accurate, timesaving, efficient targeted genomic modifications, including whole-gene insertion or deletion, stacking, or pyramiding of genes, in a transgene-free manner [13-15].

Gene editing is a molecular biology technique that intentionally targets user-defined DNA sites within the genome for the purpose of elucidating functions of unknown genes. Since modified genetic information in the parental lines is passed to next generations, gene editing can be employed to purposely alter traits of agricultural importance to develop new cultivars or breeding lines [1,16].  Various gene editing techniques have been established including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and cluster regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) [8]. All these tools rely on the specificity of the endonucleases that recognize and cleave DNA at desired sites to facilitate mutations induced by cellular repair mechanism. In this review, we aim to provide the latest updates of CRISPR/Cas9 application on swine breeding (what do you mean?), although TALEN and ZFN can obtain the same outcomes [3,17].

ZFN and TALEN are two early gene editing techniques that employs similar conceptual nuclease structure to introduce genetic mutation. Both systems depend on the specificity of the DNA-binding domain of zinc finger protein (ZFP) in the ZFN system and transcription activator-like effector (TALE) in the TALEN system. Since each zinc finger in the ZFP recognizes every triplet on single-strand DNA, designing 3-6 zinc finger components in combination will therefore attach to 9-18 base pairs on aimed regions to achieve specificity [18]. On the other hand, the improved targeting property of TALE relies on the programmable tandem repeat modules, of which each module specifically binds to a single base pair. The order of the tandem repeat modules can be rearranged to obtain better directing at chosen DNA sequence. After the binding to DNA region, both ZFP and TALE will orchestrate the dimerized endonuclease Fok1 to break the double strand DNA at predetermined regions [7,19].

The introduction of DSB generated by ZFN and TALEN will trigger the DNA repair mechanisms including non-homologous end-joining (NHEJ) or homologous recombination (HR) [20]. In the error prone NHEJ pathway, the two ends of the cleaved DNA are joined and ligated, resulting in the generation of insertion or deletion at the site of DSB, thus producing knock-out mutation [17].  In the HE pathway, a site-directed nuclease and an exogenous DNA template harboring homologous sequence to the DSB regions are required to facilitate the insertion of single or multiple transgenes, thereby gaining knock-in mutation. Accumulation of reports has demonstrated the successful application of ZFN [4,21].

The latest CRISPR/Cas9 is extensively employed in genome editing research thanks to its reliability, efficiency, and simplicity [22]. Basically, CRISPR/Cas9 is a RNA- mediated adaptive immune system that can be found in bacteria, and archaea [23]. This immune protection provides resistance against genetic attacks and later stores infection histories in a form of spacer sequences for future safety. These spacers function in concert with Cas9 endonuclease proteins to monitor, recognize and degrade exogenous DNA.

This process can be divided in three stages: spacer acquisition, biogenesis, and immunity. In the spacer acquisition stage, the foreign DNA is identified, captured, and embedded into the CRISPR locus in a form of spacer. Subsequently, the expression of the CRISPR/Cas9 system will be initiated in the biogenesis stage, in which the primary CRISPR-RNAs (crRNAs) is synthesized from the CRISPR locus and subsequently undergone many processes to become crRNAs. Finally, in the immunity stage, the crRNAs, together with the trans-activating RNAs (tracrRNAs), will associate with Cas9 endonuclease, forming a ribonucleotide complex. This complex will initiate interference and consequent degradation of the targeted foreign DNA by base pairing recognition mechanism and endonucleases, respectively.

It was not until the work of Jinek et al., the significant contribution of CRISPR/Cas9 technology to genome editing begins to emerge by the establishment of the programmable version of CRISPR/Cas9 [24]. This modified version of CRISPR/Cas9 is made up of the customizable single strand RNA (sgRNA), which is the fusion product of crRNA and tracrRNA, the recombinant Cas9 protein and. This combination will result in Cas9/sgRNA complex that targets and initiates DSB at specific DNA sequences. Once DSBs are introduced, NHEJ or HDR strategy is activated to repair the DNA damages, leading to gene knockout, or gene knock-in, respectively [12,25]. CRISPR/Cas9 system has been widely employed in various research model research, including Prokaryotes (Escherichia coli) [26] and Eukaryotes (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, etc.) [27-30].