Fig. 2

Development of a proof-of-concept for genome editing in T. kivui with its endogenous type I-B CRISPR system. A Cloning design with hierarchical Golden Gate assembly to generate the CRISPR plasmids. B Genome interference assay design. A plasmid bearing a synthetic mini array with crRNAs targeting the pyrE gene is transformed in T. kivui. Functionality of the CRISPR system is determined by antibiotic challenge. C Genome interference assay shows strain transformation is impaired for plasmids with pyrE crRNAs (CCA or CCC PAM). Controls: empty vector, mini array without cRNA. Data represent three biological replicates (average ± standard deviation). Statistical significance was determined using a two-tailed t-test for independent samples (*p < 0.05). D Design of CRISPR-based pyrE deletion. The functionality of the CRISPR system is determined by antibiotic challenge and PCR. E Transformation efficiencies of type I-B CRISPR vectors and editing efficiency of pyrE deletion. Controls: empty vector, mini array without crRNA. Transformation efficiency was measured for four independent experiments (average ± standard deviation), and editing efficiency is shown as the fraction of edited colonies (ten colonies, average ± standard deviation). F Gel electrophoresis of PCR results from (E). Data are representative of ten colonies per sample. G Effect of homology arm length and plasmid nature (purified from E. coli or Golden Gate assembly mixture) on pyrE deletion. Control: mini array without crRNA plasmid. Transformation efficiency was measured for four independent experiments (average ± standard deviation), and editing efficiency is shown as the fraction of edited colonies (ten colonies, average ± standard deviation)