Figure 2.

AsCas12a guides can exhibit variable behavior across strains that can be countered by blocking the homologous recombination pathway or opening the predicted secondary structure. (A) Transformation-based targeting assay in different Klebsiella pneumoniae strains with the same four guides used for K. pneumoniae ATCC 10031. (B) Targeting assay in two Klebsiella strains with the recA gene deleted. (C) Folding predictions and targeting assay with gRNAs with processed repeats against the rdgC and ftsZ genes. (D) Folding predictions and targeting assay with gRNA guides with open secondary structure and against the rdgC and ftsZ genes. (E) Targeting assay with two Klebsiella strains expressing the dominant negative inhibitor of the RecA protein (RecA56). In all the experiments, the dashed lines represent the limit of detection of colony numbers. *: The sample yielded a lawn that was uncountable. ftsZ and rpoE: essential genes. rdgC and lacZ: non-essential genes. Each dot represents a biological replicate obtained by preparing electrocompetent cells from a separate colony. The bars represent average values and are absent for samples in which at least one replicate had no colonies.