PCR genotype analysis to determine RNP-mediated knockout efficiency in C. lusitaniae. PCR genotype analysis to determine RNP-mediated knockout efficiency.

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PCR genotype analysis to determine RNP-mediated knockout efficiency in C. lusitaniae. PCR genotype analysis to determine RNP-mediated knockout efficiency in C. lusitaniae. (A) Schematic indicating the locations of primers used to detect transformants with the C. lusitaniae CLUG_04072Δ::NAT1 genotype. Amplification of the left and right flanking regions (LF and RF, respectively) was performed using sets of primers that included one annealing within the NAT1 locus and another annealing to the genome immediately outside the deletion construct flank (depicted in blue or green). The NAT1 gene is shown in orange. The predicted sizes for the LF and RF amplicons are shown. (B) Amplicons from reactions using primers 1 and 9 (LF) and primers 4 and 10 (RF) are shown. Genomic DNA isolated from the parental strain (P) and 10 randomly selected NATr colonies (1 to 10) from transformation reactions that included either the gene deletion construct and CRISPR RNPs (+RNPs) or the gene deletion construct alone (−RNP) was used as the template. Transformants for which there is a band present in both the LF and RF reactions were considered positive transformants with the NAT1 gene properly integrated at the CLUG_04072 locus. Black arrows indicate the location of the correct band size for each amplicon. Nora Grahl et al. mSphere 2017; doi:10.1128/mSphere.00218-17