FIGURE 5.

Results of simultaneous recombination at multiple sites. (A) Efficiency of simultaneous integration at two and three sites. The helper plasmid p46Cpf1-OP2 was used. Different combination of two cRNAs against the three selected target sites (galK, lacZ, and pyrF) were used in the recombination experiment. The donor plasmids pTc-GL was used for recombination at the galK and lacZ sites; pTc-GP, galK and pyrF sites; pTc-PL, lacZ, and pyrF sites. And the donor plasmid pTc-GLP was used for simultaneous recombination at all the three sites (galK, lacZ, and pyrF). (B) Effect of different ways of supplying the crRNAs. The donor plasmid pTc-PL expresses two crRNAs separately, while the donor plasmids pTc-arrayLP and pTc-arrayPL express crRNAs arranged in a CRISPR array. The helper plasmid p46Cpf1-OP2 was used. The data represent the averages of three independent experiments.