Development and Application of CRISPR/Cas in Microbial Biotechnology

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.

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

“…The development in genome engineering has revolutionized many areas (Dai et al ., 2018; Ding et al ., 2020). Its movements into the environmental field have advanced the elucidation of environmental microbial functions and the engineering of sophisticated biosystems for environmental applications (Jaiswal et al ., 2019; Li et al ., 2020).…”

CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

“…On other hand, better understanding of repair pathways such as DNA double-strand break repair in parasites may help exploit alternative pathways for extensive genome functional studies [2,90,91,106]. Several other microbes have been targeted successfully by CRISPR methods (Table 2, [107]). However, the different challenges of application CRISPR technology to microbe transmission or control, resistance, off-target editing, and mutations still challenge its applicability in the field against emerging resistant pathogens, as discussed by Shabbir et al (2019) [108].…”

CRISPR/Cas9 Systems for the Development of Saccharomyces cerevisiae Cell Factories

“…The CRISPR/Cas9 system is suitable for simultaneous multigene editing in S. cerevisiae because of the high HDR rate ( Table 1 ). The execution of multigene editing requires the expression of multiple gRNAs ( Figure 1B ), which can be transcribed individually by RNA polymerase promoters ( Jakociunas et al, 2015a ; Ding et al, 2020 ) or transcribed in a single long transcript. Then, individual gRNAs can be released through different strategies.…”

“…Besides the precise manipulation of genomic DNA, the CRISPR/Cas9 system serves as a transcriptional regulation platform with the adoption of inactive Cas protein (e.g., dCas9, with H840A and D10A mutations, loses its endonuclease activity but retains its capability of sequence-specific binding). Further, dCas9 can be combined with effector domains as artificial scaffolds, thus influencing genomic structure and transcriptional regulation ( Lian et al, 2018a ; Ding et al, 2020 ).…”