Figure 2.
Effect on transformation and genome-editing (positive rates) efficiencies when the length and position of the RF are modified. Transformation efficiency and positive rate in relation to the length and position of the RF. Shown are the transformation efficiencies [cfu (2 μg DNA)−1] for the CRISPR/LbCas12a pMM002P-derived plasmids that were used to transform M. maripaludis. (a) CRISPR/LbCas12a plasmid p002-218, in which the lengths of the homology arms flanking the RF are 250, 500, and 1000 bp (p002-218-L250, p002-218-L500, and p002-218-L1000, respectively). The distance from the RF to DSB for all plasmids is ∼25 bp. (b) CRISPR/LbCas12a plasmid p002-218 with 1000 bp homologous arms, in which the distance between the RF and the DSB is ∼25, ∼500, and ∼1000 bp (p002-218-L1000, p002-218-D500, and p002-218-D1000, respectively). p002-218 without the RF is included as a control. Error bars represent the standard deviation of the values obtained for the transformation efficiency (n = 3). Positive rates representing the fraction of correctly edited colonies per colonies tested by PCR are shown for all plasmid transformations (numbers above bars).