Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 145 6 Challenges and Limitations of Multiplex CRISPR in Rice 6.1 Off-target effects and editing specificity When it comes to CRISPR systems, whether it is Cas9 or Cas12a, their accuracy is generally good. But once things get complicated, such as when you want to edit dozens of genes at the same time, trouble may come. It's not to say that there will be problems every time, but whole-genome sequencing of rice shows that once the number of editing sites exceeds 50, some rare but serious conditions may sometimes occur, such as deletion or duplication of certain regions of the chromosome (Zhang et al., 2022). But then again, if you only move a few points, such as less than ten, it is safe most of the time. But to be safe, you still have to check what needs to be checked. In order to improve the accuracy of the test, researchers used technologies such as "multiple ligation-dependent probe amplification". It can not only confirm whether the target site has been actually changed, but also find out whether there is "accidental injury" to other places that should not be moved (Biswas et al., 2020). As for how to reduce these off-target risks, the commonly used method at present is to optimize the design of sgRNA. Some people directly replace it with a high-fidelity Cas variant, which has stricter target recognition and is safer to use (Mishra et al., 2018; Ren et al., 2019). But even so, the more editing is done, the more risks you have to keep an eye on. 6.2 Trait trade-offs and genetic interactions When multiple genes are modified simultaneously in the same rice plant, some complex trait changes may occur. Some genes may reinforce each other, while others may conflict, making it more difficult to predict the final performance (Zhou et al., 2018). For example, if the genes that control grain size, number, and plant structure are edited at the same time, many different phenotypes may be obtained, and sometimes unexpected situations may occur. Therefore, detailed observation and screening in the field are required to confirm whether these traits are ideal (Shen et al., 2017). In this case, completely knocking out a gene is not necessarily the best option. Sometimes some "fine-tuning" of gene expression is more conducive to maintaining plant yield and adaptability (Zhou et al., 2023). 6.3 Technical and regulatory barriers Technical challenges include complex vector construction, inconsistent editing efficiency at different gene loci, and the delivery system of editing tools is not ideal enough (Hu et al., 2020). Although there have been significant improvements in tRNA processing systems and simpler vector systems, which have indeed improved the ability of multi-gene editing, there is still a long way to go before it can be widely used in actual breeding (Xie et al., 2015; Xiong et al., 2023). In addition, different countries and regions have different policies on gene-edited crops. In particular, there are still many controversies about whether non-GMO and unmarked plants should be classified as GMOs and whether they need approval (Pan and Qi, 2023). These policy uncertainties are one of the key issues for the successful commercialization of multi-edited rice. 7 Future Perspectives and Research Directions 7.1 Towards next-generation CRISPR technologies Technology updates are never done in one go, especially in complex operations such as multi-gene editing. CRISPR-Act3.0 is a typical example. It can activate multiple genes at one time, which sounds ideal and is especially suitable for dealing with complex problems involving multiple traits or metabolic pathways (Pan et al., 2021). However, whether this type of "all-round" technology can really be used depends on whether the specific system is compatible. There is also a bottleneck - the limitation of PAM sequence. In the past, the editing range was very limited, but now Cas9 variants like SpRY, which are almost not picky about PAM, are much more flexible. There is also Cpf1 (also called Cas12a), which is also used as an alternative enzyme to Cas9, and more and more people are using it (Li et al., 2018). The emergence of these tools has gradually shifted multi-gene editing from "can it be done" to "how to do it more smoothly". A more impressive point is that some people can now use a vector to target dozens of gene sites for editing at the same time (Wu et al., 2024). What does this mean? Simply put, breeding does not need to be repeated over and over again, and genetic diversity can be increased immediately, saving time and effort. The real challenge is how to select the best batch.
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