Field Crop 2025, Vol.8, No.1, 20-31 http://cropscipublisher.com/index.php/fc 28 Now, countries have clearly relaxed their attitudes towards CRISPR technology, especially those edited varieties that do not introduce foreign DNA-in other words, rice that only changes its own genes and does not touch foreign genes. This "clean" editing method is indeed easier to pass approval (Zegeze et al., 2022). Farmers and consumers are also much more accepting, after all, it sounds more reliable than genetic modification. But then again, although the policy is being relaxed, it still has to go through many hurdles to promote it on a large scale. But in any case, this brings the new high-yield and stress-resistant rice varieties one step closer to the field, which is definitely a good thing for solving food security problems. 7 Challenges and Solutions 7.1 Improving editing efficiency Currently, CRISPR technology has encountered a headache in rice breeding-the editing efficiency is sometimes high and sometimes low, and it is not very stable. Take OsPIN5b and GS3, the popular target genes, for example. The editing success rate can jump from 42% to 66% in different experiments, like a roller coaster ride (Zeng et al., 2020). If the efficiency cannot be improved, it will not only be time-consuming and labor-intensive, but also costly. Researchers have found that this matter is affected by many factors: it is important to choose the right target site, the gRNA design must also be particular, and even the method of delivering CRISPR components must be particular. To put it bluntly, if you want to improve the success rate, you have to adjust the parameters of each link to the best state like a tuner. Fortunately, with the accumulation of experience, everyone is slowly getting the hang of it, and the editing efficiency is also steadily improving. Researchers now have many tricks to deal with the problem of editing efficiency. The most direct one is "multi-pronged approach"-designing several different gRNAs for the same gene, there is always one that can hit the bull's eye. The development of bioinformatics has also helped a lot. Now it is much more accurate to use computers to predict target sites, unlike before when you have to rely on guessing (Zegeye et al., 2022). What is even more exciting is the new generation of editing tools, such as "upgraded scissors" such as Cas12 and base editors, which not only cut more accurately, but also more efficiently (Tabassum et al., 2021). For example, some teams have found that when using Cas12 to process certain genes, the success rate can be about 20% higher than that of Cas9. These technological advances combined have given rice breeders more and more confidence in producing stable and reliable improved varieties in the field. 7.2 Reducing off-target effects The most troublesome thing about CRISPR editing is the "accidental injury" problem-originally wanted to modify gene A, but accidentally moved gene B as well. For example, this situation occurred when editing the OsMYB30 gene before, and the entire breeding experiment was almost wasted (Zeng et al., 2020). This off-target effect is like injuring other organs during surgery. At the least, it will affect the experimental results, and at worst, it may cause various problems in the plants. Therefore, the gene editing teams are now very careful. Some use computer simulations to predict possible off-target locations, and some have developed new Cas proteins to reduce accidental injuries. After all, if you want to cultivate a truly reliable new rice variety, you cannot fail to meet the accuracy standards. Scientists now have several tricks to deal with off-target problems. The first is to upgrade the Cas9 protein to a "high-precision version", just like adding an anti-shake function to scissors, which can cut accurately and not easily slip (Zegeye et al., 2022). Designing gRNA is also more sophisticated. Now we use intelligent algorithms to screen the most specific targets, which is completely different from the past decision-making (Romero and Gatica-Arias, 2019). Another smart way is to use a "time-limited tool"-let the editing component disappear automatically after staying in the cell for a while, so that even if you want to cut randomly, you won't have the chance (Wang et al., 2022). These methods can be used together to basically minimize the risk of off-target. But to be honest, no matter how good the technology is, it must be repeatedly verified, so now everyone will do a whole genome scan after editing rice to ensure that there is no risk before they dare to plant it in the field.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==