We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxinconverting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted Al fragment of the SLT-1 A subunit. SI nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene op plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Shigella dysenteriae 1 produces a toxin which is cytotoxic to eucaryotic cell lines (8). Binding to the glycolipid globotriaosylceramide (Gb3) membrane receptor is mediated by a pentamer of 7-kilodalton (kDa) B subunits, while the 31-kDa A subunit, after proteolytic nicking and reduction, inhibits protein synthesis by catalytic inactivation of the 60S ribosomal subunit (8,20,34,35). O'Brien and LaVeck (30,31) have shown that some Escherichia coli strains produce large amounts of a cytotoxin which appeared very similar to Shiga toxin with respect to its subunit structure and mechanism of action. It was completely neutralized by antiserum raised against Shiga toxin and was named Shiga-like toxin 1 (SLT-1) (28, 42). Recently, Strockbine et al. (42) have characterized a second cytotoxin, also produced by E. coli, which is related to SLT-1 at the DNA sequence level but is not neutralized by antiserum raised against Shiga toxin or SLT-

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.