Abstract
To reduce the operational friction and scale DNA engineering, we report here an in vivo DNA assembly technology platform called SCRIVENER (Sequential Conjugation and Recombination for In Vivo Elongation of Nucleotides with low ERrors). SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and in vivo homologous recombination to seamlessly stitch blocks of DNA together by mating E. coli in large arrays or pools. This workflow is simpler, cheaper, and higher throughput than current DNA assembly approaches that require DNA to be moved in and out of cells at different procedural steps. We perform over 5,000 assemblies with two to 13 DNA blocks that range from 240 bp to 8 kb and show that SCRIVENER is capable of assembling constructs as long as 23 kb at relatively high throughput and fidelity. Most SCRIVENER errors are deletions between long interspersed repeats. However, SCRIVENER can overcome these errors by enabling assembly and sequence verification at high replication at a nominal additional cost per replicate. We show that SCRIVENER can be used to build combinatorial libraries in arrays or pools, and that DNA blocks onboarded into the platform can be repurposed and reused with any other DNA block in high throughput without a PCR step. Because of these features, DNA engineering with SCRIVENER has the potential to accelerate design-build-test-learn cycles of DNA products.
Competing Interest Statement
Ethics declarations Geoffrey J. Taghon was supported in part by an appointment to the NRC Research Associateship Program at the National Institute of Standards and Technology, administered by the Fellowships Office of the National Academies of Sciences, Engineering, and Medicine. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification implies neither recommendation or endorsement by the National Institute of Standards and Technology nor that the materials or equipment identified are necessarily the best available for the purpose. The views expressed in this publication are those of the authors and do not necessarily represent the views of the U.S. Department of Commerce or the National Institute of Standards and Technology. <br> Competing interests X.L., T.M., W.L., D.M., and S.F.L filed patent applications based on the methods presented in this work (WO2022187697A1, EP4301854A4, GB202315184D0, KR20230152124A, JP2024509194A, CA3212642A1, DE112022001365T5, AU2022228362A1). S.F.L and G.S. co-founded BacStitch DNA, Inc. to commercialize methods presented in this work. T.M., P.H.H., F.L., J.C., N.W. G.S. and S.F.L have financial interest in BacStitch DNA, Inc..
