Table 4. Duplex and triplex gRNA constructs developed and their impact on disease phenotype.

Sl No.	Multiplex level	gRNA combination	Final clone ID after joining with Cas9	Target region in ChiLCV genome	Disease phenotype grade*
1	Duplex	gRNA1+ gRNA2	gRNA1+2	IR	Leaf curl– 1, Stunting—2
2	Duplex	gRNA1+ gRNA4	gRNA1+4	IR and V2-V1 overlap	Leaf curl– 1, Stunting—2
3	Duplex	gRNA1+ gRNA6	gRNA1+6	IR and C1-C4 overlap	Leaf curl– 2, Stunting—2
4	Duplex	gRNA3+ gRNA2	gRNA3+2	IR and V2-V1 overlap	Leaf curl– 2, Stunting—2
5	Duplex	gRNA3+gRNA4	gRNA3+4	V2-V1 overlap	Leaf curl– 2, Stunting—2
6	Duplex	gRNA3+gRNA6	gRNA3+6	V2-V1 overlap and C1-C4 overlap	Leaf curl– 2, Stunting—2
7	Duplex	gRNA5+gRNA2	gRNA5+2	IR and C1-C4 overlap	Leaf Curl– 0, Stunting—1
8	Duplex	gRNA5+gRNA4	gRNA5+4	V2-V1 overlap and	Leaf curl– 0, Stunting– 0
9	Duplex	gRNA5+gRNA6	gRNA5+6	C1-C4 overlap	Leaf curl– 1, Stunting– 2
1	Triplex	gRNA1+gRNA4+gRNA5-1	gRNA1+4+5	IR, V2-V1 overlap and C1-C4 overlap	Leaf curl– 2, Stunting—2
1	Triplex	gRNA1+gRNA4+gRNA5-1	gRNA3+2+5	IR, V2-V1 overlap and C1-C4 overlap	Leaf curl– 2, Stunting—2

*In mock vector infiltrated ChiLCV inoculated plant (positive control) both the leaf curl and stunting grades were 3, while only mock vector inoculated plant (negative control) both these grades were 0.