Science https://doi.org/10.1126/science.aax9181 (2019)
Some CRISPR–Cas variants with deficient nuclease activity are reported to associate with Tn7-like transposons. However, whether these variants are functional in DNA transposition is unclear. Strecker at al. characterized two CRISPR-associated transposase (CAST) loci from the cyanobacteria Scytonema hofmanni and Anabaena cylindrica and found that four proteins (TnsC, TnsB, TniQ, and Cas12k) encoded in both CASTs are essential to mediate DNA transposition in vitro and in Escherichia coli with the assistance of tracrRNA and crRNA targeting a synthetic protospacer. Sequencing analysis revealed that both CASTs have a preference for NGTN protospacer adjacent motifs (PAMs), and DNA insertions localize within a small window downstream of the PAMs. Further studies showed that the S. hofmanni CAST can mediate transposition of DNA segments as long as 10 kb and insertion of endogenous DNA in the E. coli genome with a frequency of up to 80%. This study enriches the diversity of the CRISPR toolbox and paves the way for developing gene knock-in tools with high efficiency.