FIG 3 .

Targeted chromosomal translocations generated by targeting sites from two different chromosomes simultaneously. (A) The double-gRNA vector used to coexpress LdBPK_241510.1 targeting gRNA (241510), the LdMT targeting gRNAa (MT), and a schematic of chromosomes 13 and 24, with the Cas9 cleavage sites and the polycistronic transcription directions indicated. (B) Schematic of the 4 types of chromosomal translocations detected following transfection with the gRNA241510+MT coexpression vector. The chromosomal translocation junction sequences joined by MMEJ or a transfected oligonucleotide donor-directed repair are also included. Note that the polycistronic transcription directions and the numbers indicating chromosome size, which could have been altered in the newly generated fused chromosomes after translocation, are directly transferred from the parent chromosomes 13 and 24. (C) PCR detection of type II and type IV chromosomal translocations in cells expressing 241510- and MT-targeting gRNAs following transfection with the mixture of type II and type IV oligonucleotide donors (see the sequences in panel B). Primer 13L2, Ld131590L2; 24L1, Ld241510L1; 24R1, Ld241510R1. (D) Chromosomal translocation detected after L. donovani cells transfected with gRNA A2a+b+MT coexpression vector (Fig. 2B). For simplicity, only one A2 gene and one Cas9 cleavage site are represented for the A2-A2rel gene cluster loci in chromosome 22. See the supplemental material for all primer pairs used to detect these chromosomal translocations.