Fig. 1

Establishment of multiple U6 promoter tandem sgRNA expression cassettes The strategy of U6 promoter tandem sgRNA expression cassette targeting multiple sites at the same time is described. The strategy is composed of tandem U6-sgRNA units. Each sgRNA contains a target-specific spacer (represented by different color diamonds) and a conserved sgRNA skeleton (represented by yellow rectangles). The U6 promoter is represented by a blue rectangle. Each U6 promoter is transcribed to release mature sgRNA, which guides Cas9 to target multiple sites"

Fig. 2

Schematic diagram of tRNA-mediated polycistronic sgRNA processing The PTG/Cas9 strategy diagram targeting multiple sites at the same time shows that the PTG straegy consists of a series of tRNA-gRNA units, each gRNA contains a target-specific spacer (represented by different color diamonds) and a conserved gRNA skeleton (represented by a rectangle), tRNA is represented by a rounded rectangle containing A and B elements, and the primary transcript of PTG is cleaved by endogenous RNase P and RNase Z (labeled as scissors) to release mature gRNAs and tRNA (clover leaf structure brown line). The cleavage and release of mature gRNA will guide Cas9 to target multiple sites"

Fig. 3

The schematic diagram of polycistronic sgRNA processing mediated by nuclease Csy4 was established The polycistronic sgRNA processing maturation strategy mediated by nuclease Csy4 is illustrated. This strategy consists of tandemly arranged Csy4-gRNA units. Each gRNA contains a target-specific spacer (represented by different color diamonds) and a conserved gRNA skeleton (represented by a yellow rectangle). Csy4 is represented by a red ellipse. Csy4 cleaves and releases mature gRNA, guiding Cas9 to target multiple sites at the same time"

Fig. 4

Establishment of a schematic diagram of polycistronic sgRNA processing mediated by self-cleaving ribozyme The self-cleaving ribozyme-mediated polycistronic sgRNA processing maturation strategy is illustrated. The strategy consists of a tandem arrangement of HH-gRNA-HDV units. Each gRNA contains a target-specific spacer (represented by different color diamonds) and a conserved gRNA skeleton (represented by a yellow rectangle). HH is represented by a white rectangle, and HDV is represented by a gray rectangle. Under the combined action of HH ribozyme installed at the 5′ end and HDV ribozyme installed at the 3′ end, mature gRNA is cleaved and released, guiding Cas9 to target multiple sites at the same time"