Fig 3. Single-step assembly of a dual sgRNA expression vector and efficient genomic deletion.

(A) Schematic of the dual sgRNA expression vector designed for single-step assembly. The yellow highlights indicate the BpiI sites, and the green and orange letters show the target sequences of the sgRNAs and the overhangs for the Golden Gate cloning, respectively. The BpiI sites in both the first and second sgRNA sites generate different overhangs after digestion. act15, act15 promoter; act8, act8 terminator; tRNA, isoleucine tRNA; act6, act6 promoter. (B) Correct insertion of the B1-2 and T1 sgRNA sequences into pTM1544 via one-step cloning. Correct insertion was confirmed via colony PCR. The B1-2 sequence was inserted as the first target (F), and T1 was inserted as the second target (S). (C) Genomic deletions generated via transient introduction of pTM1544. PCR products produced using primers flanking the target sites are shown. The arrowhead indicates product size found in the control (the AX2 genome).