Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium.

Michael E Pyne,Duane A Chung,C Perry Chou,Mark R Bruder,Murray Moo-Young

doi:10.1038/srep25666

Abstract

Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium.

Highlights

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-based immunity encompasses three distinct processes, termed adaptation, expression, and interference[9,10]
We report development of broadly applicable strategies of markerless genome editing based on exploitation of both heterologous (Type II) and endogenous (Type I) bacterial CRISPR-Cas systems in C. pasteurianum, an organism possessing substantial biotechnological potential for conversion of waste glycerol to butanol as a prospective biofuel[48]
To determine if the S. pyogenes machinery could function for genome editing in C. pasteurianum, we constructed a Type II CRISPR-Cas[9] vector by placing cas[9] under constitutive control of the C. pasteurianum thiolase gene promoter and designing a synthetic guide RNA (gRNA) expressed from the C. beijerinckii sCbei_5830 small RNA promoter[46]

Summary

Introduction

CRISPR-based immunity encompasses three distinct processes, termed adaptation, expression, and interference[9,10]. CrRNAs enlist and form complexes with specific Cas proteins, including the endonucleases responsible for attack of invading nucleic acids during the interference stage of CRISPR immunity. Owing to the simplicity of CRISPR-Cas[9] interference in Type II systems, the S. pyogenes CRISPR-Cas[9] machinery has recently been implemented for extensive genome editing in a wide range of organisms, such as E. coli[27,28,29], yeast[30,31], mice[32], zebrafish[33], plants[34], and human cells[35,36]. CRISPR-based methodologies provide a powerful means of selecting rare recombination events, even in strains suffering from poor homologous recombination Such strategies have been shown to be highly robust, frequently generating editing efficiencies up to 100%27,29,45. Our strategy is broadly applicable to any bacterium or archaeon that encodes a functional CRISPR-Cas locus and appears to yield more edited cells compared to the commonly employed heterologous Type II CRISPR-Cas[9] system

Methods

Results

Conclusion