Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

11 Pages · 2017 · 1.81 MB · English

Mihris I.S. Naduthodi1, Alex Gussak1, Rudolf B.L. Brinkman2, Richard van Kranenburg 1,2 & John van der Oost1. CRISPR-Cas9-based genome engineering tools have selected for culturing under microaerobic lactate-producing conditions for 24h44. The growth of the pThermoCas9i_ldhL cultures 

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ARTICLE Characterizing a thermostable Cas9 for bacterial genome editing and silencing Ioannis Mougiakos 1, Prarthana Mohanraju 1, Elleke F Bosma 1,3, Valentijn Vrouwe 1, Max Finger Bou 1, Mihris IS Naduthodi 1, Alex Gussak 1, Rudolf BL Brinkman 2, Richard van Kranenburg 1,2 & John van der Oost 1 CRISPRCas9based 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 bacteriumGeobacillus thermodenitrificansT12 We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAMpreference at lower temperatures, tolerates fewer spacerprotospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNAstructure We develop ThermoCas9based engineering tools for gene deletion and transcriptional silencing at 55 °C inBacillus smithiiand for gene deletion at 37 °C inPseudomonas putida Altogether, our findings provide fundamental insights into a thermophilic CRISPRCas family member and establish a Cas9based bacterial genome editing and silencing tool with a broad temperature range DOI: 101038/s41467017015914 OPEN 1Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands 2Corbion, Arkelsedijk 46, 4206 AC Gorinchem, The Netherlands 3Present address: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet B220, 2800 Kgs Lyngby, Denmark Ioannis Mougiakos, Prarthana Mohanraju and Elleke F Bosma contributed equally to this work Correspondence and requests for materials should be addressed to JvdO (email:[email protected]) NATURE COMMUNICATIONS |8: 1647 |DOI: 101038/s41467017015914 |wwwnaturecom/naturecommunications 1 C lustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPRassociated (Cas) proteins provide adaptive and heritable immunity in prokaryotes against invading genetic elements 1–4 CRISPRCas systems are subdivided into two classes (1 and 2) and six types (IVI), depending on their complexity and signature proteins 5 Class 2 systems, including typeII CRISPRCas9 and type V CRISPR Cas12a (previously called CRISPR Cpf1) have recently been exploited as genome engineering tools for both eukaryotes 6–10 and prokaryotes 11–13 These systems are among the simplest CRISPRCas systems known as they introduce targeted double stranded DNA breaks (DSBs) based on a ribonucleoprotein (RNP) complex formed by a single Cas endonuclease and an RNA guide The guide of Cas9 consists of a crRNA (CRISPR RNA): tracrRNA (transactivatingCRISPRRNA) duplex For engi neering purposes, the crRNA:tracrRNA duplex has been simpli fied by generating a chimeric, single guide RNA (sgRNA) to guide Cas9 upon coexpression 14 In addition, cleavage of the target DNA requires a protospacer adjacent motif (PAM): a 3–8 nucleotide (nt) long sequence located next to the targeted pro tospacer that is highly variable between different Cas9 proteins 15–17 Cas9 endonucleases contain two catalytic domains, denoted as RuvC and HNH Substituting catalytic residues in one of these domains results in Cas9 nickase variants, and in both domains in an inactive variant 18–20 The inactive or dead Cas9 (dCas9) has been instrumental as an efficient gene silencing system and for modulating the expression of essential genes 11,21,22 To date,Streptococcus pyogenesCas9 (SpCas9) is the best characterized and most widely employed Cas9 for genome engi neering Although a few other typeII systems have been exploited for bacterial genome engineering purposes, none of them is derived from a thermophilic organism 23 Characterization of suchCRISPRCas systems would be interesting to gain fundamental insights as well as to develop novel applications Although basic genetic tools are available for a number of thermophiles 24–27 , the efficiency of these tools is still too low to enable full exploration and exploitation of this interesting group of organisms Based on ourfinding that SpCas9 is not active in vivo at or above 42 °C, we have previously developed a SpCas9 based engineering tool for facultative thermophiles, combining homologous recombination at elevated temperatures and SpCas9 based counterselection at moderate temperatures 28 However, a Cas9based editing and silencing tool for obligate thermophiles is not yet available as SpCas9 is not active at elevated tempera tures 28,29 , and to date no thermophilic Cas9 has been adapted for such purpose Here we describe the characterization of Ther moCas9: an RNAguided DNAendonuclease from the CRISPR Cas typeIIC system of the thermophilic bacteriumGeobacillus thermodenitrificansT12 30 We show that ThermoCas9 is active in vitro between 20 and 70 °C

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