title: A semisynthetic organism engineered for the stable expansion of the genetic alphabet.
abstract: All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: Yorke
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Lamb
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Feldman
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: Anne Xiaozhou
authors.initials: AX
authors.lastname: Zhou
authors.affiliation: Département de Chimie Moléculaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5250, Université Grenoble Alpes, F-38000 Grenoble, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lavergne
authors.affiliation: School of Chemistry and Chemical Engineering, Henan Normal University, Henan 453007, People's Republic of China.
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; floyd@scripps.edu.
authors.forename: Floyd E
authors.identifier: 0000-0001-6317-1315
authors.initials: FE
authors.lastname: Romesberg
mesh_terms: D064113:CRISPR-Cas Systems; D005815:Genetic Code; D009841:Oligonucleotides; D010957:Plasmids; D058615:Synthetic Biology
chemical_list: D009841:Oligonucleotides
keywords: CRISPR; Cas9; DNA replication; nucleotide transporter; unnatural base pair