DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.

Konstantin Severinov,Sergey Shmakov,Tatyana Zyubko,Anatolii Arseniev,Georgii Pobegalov,Olga Musharova,Marina Abramova,Aleksandra Vasileva,Iana Fedorova,Tatyana Artamonova,Mikhail Khodorkovskii,Daria Artamonova,Maksim Kazalov,Polina Selkova,Ignatiy Goryanin

doi:10.1093/nar/gkz1225

Abstract

Type II CRISPR–Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Highlights

CRISPR–Cas systems are bacterial and archaeal immune systems that protect their hosts from invaders such as plasmids or bacteriophages
Most of these genome editing approaches rely on the use of the SpCas9 protein, the most investigated to date effector nuclease from Streptococcus pyogenes Type II-A CRISPR–Cas system [5]
The C. cellulolyticum H10 type II-C CRISPR–Cas locus was bioinformatically found by Fonfara et al in 2014 but up to date there is no information about the activity of this system

Summary

Introduction

CRISPR–Cas systems are bacterial and archaeal immune systems that protect their hosts from invaders such as plasmids or bacteriophages. The crRNAs with investigator defined spacer sequences allow one to guide Cas nucleases to virtually any desirable target Because of their relative simplicity, single-subunit Cas nucleases of Type II CRISPR–Cas systems form the basis of multiple genome editing applications. Alongside with eukaryotic genome editing, there is a large demand for genome engineering of microorganisms useful in biotechnology and several efficient CRISPR-based methods of bacterial genome editing have been developed [2,3,4]. Most of these genome editing approaches rely on the use of the SpCas protein, the most investigated to date effector nuclease from Streptococcus pyogenes Type II-A CRISPR–Cas system [5]. Despite high DNA cleavage efficiency, SpCas has several limitations due to its large size, a strict requirement for an NGG PAM (protospacer adjacent motif essential for target DNA recognition) and imperfect specificity

Methods

Results

Conclusion