Variants of the gene scissors CRISPR/Cpf1, also known as CRISPR/Cas12a, from different types of bacteria were used in this study to simultaneously change several different genes in rice. This process is known as multiplexing. The scientists tested different strategies to alter as many genes as possible. A variant of the bacterium M. bovoculi, Mb2Cas12a, was particularly effective and made it possible to alter a total of 16 different gene regions in a genome-edited rice variety. The genes that were altered in the process regulate various agronomically relevant properties as well as bacterial resistance.
The complexity of multiplexing applications means that the risk of unwanted changes in the genome also increases. Each guide RNA, which is the recognition component of the gene scissors, can guide the gene scissors to unintended parts of the genome, which can cause unwanted changes, i.e. so-called off-target effects, at these locations. Thus, if several different guide RNAs are used in one experiment, the risk of unwanted changes at several other locations in the genome increases. In addition, unintended on-target effects can occur at the target sites. On-target effects are unintended changes that affect the area around the target site of an SDN application. This can lead, for example, to a restructuring of the area at the target site or the unintentional insertion of DNA fragments. Such unwanted “technical” errors can have various consequences, depending on where they occur in the genome. For example, genes can be switched off, gene sequences can be changed or gene activity can be influenced.
In the study, only a few regions in the genome of the rice plants were analysed for off-target effects and none were found. The plants should, however, be subjected to an unbiased examination using genomic methods to systematically investigate the genome for unintended changes in DNA. This would enable the detection of both off-target and on-target effects. It would also make sense to use other omics methods, such as transcriptomics or metabolomics, to investigate whether the simultaneous change of many different genes interferes with other metabolic pathways.
Zhang Y, Ren Q, Tang X, Liu S, Malzahn AA, Zhou J, Wang J, Yin D, Pan C, Yuan M, Huang L, Yang H, Zhao Y, Fang Q, Zheng X, Tian L, Cheng Y, Le Y, McCoy B, Franklin L, Selengut JD, Mount SM, Que Q, Zhang Y, Qi Y (2021) Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems. Nature Communications 12 (1):1944. doi:10.1038/s41467-021-22330-w