lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7–independent positive regulation of hbl-1 through its 3′UTR to control L2 stage-specific cell fates; and second, a let-7–dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.

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Recombinant DNA technology mediated by restriction enzymes and ligases allows in vitro manipulation of a DNA segment isolated from the genome. Short overhangs generated by restriction enzymes facilitate efficient pasting together a DNA sequence and a vector. We adopted this recombinant DNA strategy to develop an in vivo recombinant-genome genome editing approach. Using the programmable endonuclease Cas9 or Cas12a as a restriction enzyme, we devised an in situ cut-and-paste (iCAP) genome editing method that was tested in both mouse germline and human cell line platforms. Mouse gene loci Slc35f2 and Slc35f6 were each edited with in-frame insertion of a large APEX2-Cre cassette and concurrent FRT3 insertion at a second location providing proof of principle for the iCAP method. Further, a de nova single nucleotide mutation associated with MED13L syndrome was efficiently corrected in patient cells. Altogether, the iCAP method provides a single genome editing platform with flexibility and multiutility enabling versatile and precise sequence alterations, such as insertion, substitution, and deletion, at single or multiple locations within a genomic segment in mammalian genomes.

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