Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Abstract

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a

“…As the 5 0 -NGG motif occurs quite often, the PAM is generally not limiting Cas9 applications, unless a very precise cleavage position is desired. It should be noted that PAM recognition by Cas9 can be either strict or very relaxed in different Cas9 orthologs [39,[43][44][45].…”

CRISPR-Cas9/Cas12a biotechnology and application in bacteria

“…After combination of sgRNA and optimization of temporal control system, the final strain could synthesize 103.1 g/L of GlcNAc in a 3-L fermenter via fed-batch culture. In a thermophilic B. smithii study, to overcome obstacles of the regular spCas9's working temperature (<42 °C), Mougiakos et al [ 225 , 226 ] developed a thermos-tolerant Cas9 (ThermoCas9) from a thermophilic bacterium G. thermodenitrificans T12. Finally, the CRISPR-mediated gene deletion can be achieved at 55 °C in vivo.…”

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…Proof of principle of CRISPR interference activity in L. lactis was demonstrated by silencing the upp gene [47]. For some species, Cas9 is toxic and cannot be used for genetic engineering [10]; therefore, alternative Cas proteins have been developed, such as the Cas9 variant ThermoCas9 [50], Cpf1 and C2c1/2/3 [51], and Cas12a, with this last combined with single-stranded DNA recombineering to improve precision [52][53][54]. Another improvement to the field is base editing, where a fusion protein that contains a catalytically impaired Cas9 variant coupled to cytidine deaminase allows C to T (or G to A) substitutions [10,55,56].…”