Abstract
Industrial biology plays a crucial role in the fields of medicine, health, food, energy, and so on. However, the lack of efficient genetic engineering tools has restricted the rapid development of industrial biology. Recently, the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system brought a breakthrough in genome editing technologies due to its high orthogonality, versatility, and efficiency. In this review, we summarized the barriers of CRISPR/Cas9 and corresponding solutions for efficient genetic engineering in industrial microorganisms. In addition, the advances of industrial biology employing the CRISPR/Cas9 system were compared in terms of its application in bacteria, yeast, and filamentous fungi. Furthermore, the cooperation between CRISPR/Cas9 and synthetic biology was discussed to help build complex and programmable gene circuits, which can be used in industrial biotechnology.
Highlights
Industrial biotechnology has advanced significantly in recent years due to the improvement of genomic engineering tools
Previous methods of manufacturing targeted double-strand breaks (DSBs) relied on protein–DNA recognition systems, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)
In this review, the potential of CRISPR/Cas9 in industrial biotechnology was demonstrated by introducing its applications in bacteria, yeasts, and filamentous fungi
Summary
Industrial biotechnology has advanced significantly in recent years due to the improvement of genomic engineering tools. During the long-time evolution of microorganisms, a unique adaptive immune system, named as clustered regularly interspaced short palindromic repeat sequences and CRISPR-associated protein 9 (CRISPR/Cas9), was employed by bacteria and archaea to defend against foreign-invading DNA (Figure 1; Horvath and Barrangou, 2010) This system is consisted of a Cas nuclease, a target-recognizing CRISPR RNA (crRNA), and auxiliary non-coding trans-activating crRNAs (tracrRNAs) (Jiang and Doudna, 2017). Jiang et al established a two-plasmid-based CRISPR/Cas system in Escherichia coli, in which Streptococcus pyogenes Cas and crRNA array were expressed in the lowcopy plasmid (pCas) and high-copy plasmid (pCRISPR) series (Jiang et al, 2013; Mali et al, 2013) This novel genetic engineering tool had a better performance than did the traditional one, it still needed further modifications to obtain higher efficiency. The disruption efficiency using the SNR52′-tRNAGly promoter was improved by 28% than the initial SNR52 promoter
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