title: One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.
abstract: Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life.
authors.affiliation: Synthetic Genomics, Inc., La Jolla, CA 92037, USA.
authors.forename: Krishna
authors.initials: K
authors.lastname: Kannan
authors.affiliation: Synthetic Genomics, Inc., La Jolla, CA 92037, USA.
authors.forename: Billyana
authors.initials: B
authors.lastname: Tsvetanova
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Ray-Yuan
authors.initials: RY
authors.lastname: Chuang
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Noskov
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Nacyra
authors.initials: N
authors.lastname: Assad-Garcia
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Hutchison Iii
authors.affiliation: Synthetic Genomics, Inc., La Jolla, CA 92037, USA.|J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Chuck
authors.initials: C
authors.lastname: Merryman
authors.affiliation: Synthetic Genomics, Inc., La Jolla, CA 92037, USA.|J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: J Craig
authors.initials: JC
authors.lastname: Venter
authors.affiliation: Synthetic Genomics, Inc., La Jolla, CA 92037, USA.|J. Craig Venter Institute, La Jolla, CA 92037, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gibson
mesh_terms: D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D009176:Mycoplasma mycoides / Q000235:genetics*; D010802:Phylogeny; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S