Figure 2.

Identification of novel I-CreI derivatives with locally altered specificity. (a) Yeast screening assay principle. A strain expressing the meganuclease (MEGA) to be assayed is mated with a strain harboring a reporter plasmid containing the chosen target. The target is flanked by overlapping truncated LacZ genes (LAC and ACZ). Upon target cleavage, tandem repeat recombination restores a functional LacZ gene, which can be monitored by standard methods. (b) Examples of profiling. Each novel endonuclease is profiled in yeast on a series of 64 palindromic targets, differing from the sequence shown in Figure 1c at positions ±8, ±9 and ±10. These targets are arrayed as in Figure 2c. As described previously (11), blue staining indicates cleavage. (c) Numbers of mutants cleaving each target, and average intensity of cleavage. Each sequence is named after the −10, −9, −8 triplet (10NNN). The number of proteins cleaving each target is shown below, and the level of gray coloration is proportional to the average signal intensity obtained with these cutters in yeast.