Development and Application of CRISPR/Cas in Microbial Biotechnology.

Wentao Ding,Shuobo Shi,Yang Zhang

doi:10.3389/fbioe.2020.00711

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.

Highlights

Microbial cell factories producing fuels, chemicals, and pharmaceutics are perspective production mode to replace petrol relied methods because microbial methods are usually clean and renewable
This review focuses on the establishment and development of clustered regularly interspaced short palindromic repeats (CRISPR) toolbox for genome editing and gene regulation, and applications of these techniques in metabolic engineering and synthetic biology in microorganisms
The CRISPR based tools are generally with higher efficiency, more convenience, more efficient multiplex targets editing/regulation and timesaving compared with traditional ones

Summary

INTRODUCTION

Microbial cell factories producing fuels, chemicals, and pharmaceutics are perspective production mode to replace petrol relied methods because microbial methods are usually clean and renewable. The CRISPR/Cas systems have been categorized into two classes and six major types based on the constitution of effector protein and signature genes, protein sequence conservation, and organization of the respective genomic loci (Koonin et al, 2017; Tang and Fu, 2018) Among these CRISPR systems, the Cas (Type II), Cas12a (previously known as Cpf, type V) and their mutant variants are most investigated effectors, and have shown broad applicational potentials in genome editing, gene regulation, DNA detection, DNA imaging, etc. Considering the guide RNAs are easy to design and expressed, Cas protein can be programmed to introduce DSBs at one or more DNA targets, making CRISPR/Cas an convenient and precise platform for genome editing (Doetschman and Georgieva, 2017). The Cas9-sgRNA (or Cas9-crRNA-tracrRNA) complex binds to DNA target arising from Watson-Crick base pairing of spacer sequence, and triggers double strand break (DSB) when next to a short protospacer adjacent motif (PAM, ‘NGG’ for Cas from S. pyogenes). Shmakov et al (2015) further classified three class 2 CRISPR systems, including C2c1, C2c3, and C2c2, which further expands CRISPR toolbox for genome editing

Design and Expression of Guide RNA

Findings

CONCLUSION AND FUTURE PERSPECTIVES