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.
Highlights
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR Associated Systems (Cas) are native to bacteria and archaea, which provide adaptive immunity against invading nucleic acids such as those from viruses[1,2]
In order to edit the genome of Mycoplasma mycoides, we cloned it as a circular yeast artificial chromosome (YAC)[27] in the Saccharomyces cerevisiae str
We utilized the potential of CRISPR/Cas[9] and the innate homologous recombination capacity of yeast to engineer the small-subunit ribosomal RNA in the bacterial genome of M. mycoides
Summary
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR Associated Systems (Cas) are native to bacteria and archaea, which provide adaptive immunity against invading nucleic acids such as those from viruses[1,2]. Homologous recombination is efficiently accomplished in conjunction with the CRISPR/Cas9-targeted cleavage by native machineries present in the eukaryotic chromosomes[3,4,5,6,7], without the need for heterologous proteins Bacteria such as E. coli have traditionally been used as model organisms to study and understand the fundamental processes in biology such as replication, transcription and translation, due to the simplicity in their genome architecture and tractable genetics. We describe a unique genome-editing platform by combining the precise editing capability of the CRISPR/Cas[9] system and the yeast homologous recombination machinery, and demonstrate robust and extensive chromosomal engineering of an essential bacterial gene that is conserved across all kingdoms, the rrs gene encoding the small-ribosomal subunit (16S) RNA. We eliminated the commonly-used two-plasmid CRISPR/Cas[9] expression system[7,28] by using in vitro transcribed guide-RNAs and chromosomally-encoded Cas[9], thereby simplifying this tool significantly without compromising its genome-editing efficiency
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