Fig 1.
Experimental scheme of chromosome splitting.
(A) pbcas9 is the transgenic parasite expressing the Cas9 nuclease in the nucleus. (B) Chromosome 1 is cleaved by the Cas9, which generates a centric fragment and an acentric fragment. The white circle is the original centromere. (C) The cleaved end of the centric chromosome fragment is repaired by homologous recombination using the donor DNA with a telomere sequence (blue triangle). While, that of the acentric chromosome fragment is repaired using the donor template with both a centromere (red circle) and telomere (blue triangle). The split chromosomes will be maintained in the parasite due to the centromere and telomere. (D and E) In contrast, if the cleaved end of the acentric chromosome fragment is repaired using the donor template with only a telomere, the parasite will lose the split chromosome due to the failure of its segregation. As a result, the parasite will die due to this loss of a chromosome.
Fig 2.
CRISPR/Cas9-based chromosome split.
(A) The PBANKA_011600 was cleaved by the Cas9-sgRNA complex, followed by HDR with the donor template DNA including only telomere, and telomere and centromere. (B) The genotyping PCR was performed using the sets of primers indicated at the bottom. (C) Southern hybridization analyses of the transgenic parasites, which are split-Ch1-1116 and -1053, detected the split chromosomes. The probe DNA used in each analysis is shown at the bottom. The information about the probe are described in S2 and S3 Figs. (D) Southern hybridization shows the telomere extension in the transgenic parasite.
Fig 3.
Asexual development of the split-Ch1-1116 parasites.
(A) The growth of the split-Ch1-1116 parasites, which is indicated by the blue line, was comparable to that of the parental pbcas9 parasites, which is shown by the black line. The points and error bars represent the mean and standard error of the mean of triplicate values. Distributions for each day were compared using the unpaired t-test (not significant). (B) The morphologies of the parasites during asexual development were similar between pbcas9 (upper) and split-Ch1-1116 (lower). The bar indicates 5 μm. (C) The number of merozoites per schizonts of the split-Ch1-1116 parasite was comparable to that of the parental pbcas9 strain. The middle line, top, and bottom of the box, top and bottom whiskers are the median, 75th and 25th percentiles, and the maximum and minimum values respectively. Distributions were compared using the unpaired t-test (not significant).
Fig 4.
Sexual development of the split-Ch1-1116 parasites.
(A) The number of exflagellation centers of split-Ch1-1116 parasites were comparable to that of pbcas9 parasites. The column and error bars indicate the mean and standard error from biological triplicates. (B and C) Ookinete shape and conversion rate were normal in the split-Ch1-1116 parasites. The columns and error bars indicate the mean and standard error of the mean from biological triplicates. Distributions in A and C were compared using the unpaired t-test (not significant). The bar indicates 5 μm.