Cas12a (previously known as Cpf1), belonging to the Class II type V CRISPR system, has gained extensive utilization in plant genome editing and has distinctive characteristics compared to Cas9. Despite being recognized as one of the most robust Cas12a nucleases, LbCas12a generally exhibits lower genome editing efficiency than the widely used SpCas9 in animals and plants. This is partially due to reduced nuclease activity of LbCas12a at room temperatures. Therefore, developing novel LbCas12a variants provides an opportunity for enhanced genome editing in plants. In our recent study, we engineered a suite of LbCas12a variants that possess improved genome editing activity compared to wildtype LbCas12a. Among these new LbCas12a variants, LbCas12a-RRV (G146R/D156R/R182V) exhibited improved editing efficiency in rice and poplar, at both canonical TTTV PAM (protospacer adjacent motif) sites and noncanonical TTV PAM sites. Consequently, LbCas12a-RRV represents one of the most efficient Cas12a nucleases for genome editing in plants. In this chapter, we describe a comprehensive protocol for constructing CRISPR-LbCas12a-RRV vectors through Golden Gate and Gateway assembly for generating T-DNA vectors for multiplexed genome editing in plants. This highly efficient CRISPR-Cas12a system can tolerate low temperatures and is anticipated to broaden the scope of genome editing applications across diverse plant species.