Journal: Communications Biology

Article Title: Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium berghei with linear donor template

doi: 10.1038/s42003-020-01138-2

Figure Lengend Snippet: a The pbcas9 parasites were transfected with a linear donor template (green box) and a plasmid containing the sgRNA, resulting in a mutation of the imc1i gene. Top: HDR possibly occurred between the linear donor template with the mutation (asterisk) and the cleaved imc1i locus during the first round of the cell cycle after transfection. In addition, there were no unexpected single crossover recombinations in the resultant imc_mut_L. Middle: when the double-strand break at the target genomic locus was not repaired by HDR during the first round of the cell cycle after transfection, the parasite died due to the instability of chromosome. Bottom: when the target genomic locus was not cleaved during the first round of the cell cycle after transfection, the linear donor template was lost from the parasite and the daughter parasites would have thus only the plasmid containing sgRNA. These parasites eventually died during further cell cycles. b The genotyping PCR of imc_mut_L was performed using the sets of primers indicated at the bottom. c Southern hybridization analysis of imc_mut_L was carried out using the donor template as the DNA probe. The genomic DNA used for this analysis was purified from the transfected parasite population before cloning by limiting dilution. d A 41-bp sequence of the imc1i gene was deleted by transfecting pbcas9 with a linear donor template (green). e The imc_Δ41 parasites formed abnormal ookinetes. f The mNG gene was incorporated at the C-terminus of MTIP. g The mNG signal was detected in the pellicule of mtip::mNG ookinetes. h When pbcas9 were transfected with only the plasmid containing the sgRNA for MTIP, they died due to instability of the cleaved genome (black lines). In contrast, transgenic parasites were readily obtained using the linear donor template and the plasmid containing the sgRNA (blue lines). The uncropped images of gel or blot are shown in Supplementary Fig. .

Article Snippet: The obtained genomic DNA was further purified using the Nucleospin gDNA Clean-up kit (Macherey-Nagel).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Hybridization, Purification, Cloning, Sequencing, Transgenic Assay