**3.2. Gene insertion**

Gene insertion or addition is another famous technique where more than one nucleotide base pairs are added into a DNA sequence. The newly inserted sequence can be designed in such a way where it can specifically encode proteins that bring crucial benefits. However, designing the inserted-to-be DNA sequence is not an easy task as imperfection could pose a risk to the health of the host cell or organism, or it can be simply nulled as it fails to function as predicted [55]. In the terms of gene addition for crop improvement, the desired goal is usually the addition of beneficial traits such as pest resistance, high yield or quality.

In fact, adding multiple genes that confer different trait improvement in a single plant is a common practice to produce elite cultivar. For instance, the TC1507 maize that contains both the *Bacillus thuringiensis* (*Bt*) gene that confers insect resistance and *acetyltransferase* gene that is herbicide tolerance [56]. To date, the most common and efficient method used is to collocate all the desired genes into a single molecular stack, whereby all of the genes will now behave as a single locus or better known as gene stacking [52]. The colocation is no easy task as it has two major limitations. First, each of the genes may potentially affect each other if they are placed adjacently too close [57]. Second, once the transgenes are collocated, they can no longer be moved as they are placed too close to each other to the point where it acts as a single locus.

There is another unconventional substitute to molecular stacks where it can only be generated through the CRISPR/Cas9 system. That substitute is known as complex trait loci (CTL) or quantitative trait loci (QTL) and where transgenes can also be genetically collocated [58, 59]. An example of CTLs is constructed through the CRISPR/Cas9 system by specifically inserting the transgenes into the desired region in the genome through HDR. To start, the transgenes in the CTL can be separated by a larger distance (50 kb to more than 1 Mb) compared to the molecular stacks (few hundred or thousand bp) while retaining their genetic linkage [60]. The changes of distance solves both the limitations of the molecular stacks as adjacent transgene will no longer affect each of their function and they can now be individually moved and swapped.

Similarly, with the help of the CRISPR/Cas9 system, the DsRED2 gene, which encodes a protein that expresses red fluorescence, was also successfully inserted into the genome of an immature sorghum embryo [61]. As a result, the plant now displays red fluorescence.
