Recent Advances in CRISPR-Cas Technologies for Synthetic Biology

Abstract

With developments in synthetic biology, “engineering biology” has emerged through standardization and platformization based on hierarchical, orthogonal, and modularized biological systems. Genome engineering is necessary to manufacture and design synthetic cells with desired functions by using bioparts obtained from sequence databases. Among various tools, the CRISPR-Cas system is modularly composed of guide RNA and Cas nuclease; therefore, it is convenient for editing the genome freely. Recently, various strategies have been developed to accurately edit the genome at a single nucleotide level. Furthermore, CRISPR-Cas technology has been extended to molecular diagnostics for nucleic acids and detection of pathogens, including disease-causing viruses. Moreover, CRISPR technology, which can precisely control the expression of specific genes in cells, is evolving to find the target of metabolic biotechnology. In this review, we summarize the status of various CRISPR technologies that can be applied to synthetic biology and discuss the development of synthetic biology combined with CRISPR technology in microbiology.

AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach

“…Incorporating alternative PAM patterns for the same Cas into the AlPaCas database acknowledges the inherent Cas promiscuity documented in the literature, as well as the variability in some Cas-PAM interactions ( 68 , 69 ), thus enhancing the tool's versatility. However, this expansion comes with a caveat of potentially ‘over-representing’ targetable PAMs, particularly considering that certain patterns can significantly vary in their affinity for a given Cas.…”

Biotechnological production of omega-3 fatty acids: current status and future perspectives

“…This gene editing technology can not only precisely knock out and insert genes into target species, but it can also utilize its accurate gene targeting function to perform gene editing tasks such as gene activation (CRISPRa) (Bikard et al, 2013), interference (CRISPRi) (Qi et al, 2013), and point mutation (CRISPR-nCas9) . This precise gene editing of the target species allows for subtle adjustments of carbon and energy flow (Jeong et al, 2023), balancing the carbonenergy balance within the organism, thereby enhancing the high yield and high-productivity production of omega-3 fatty acids.…”

Efficient CRISPR-Cas12f1-Mediated Multiplex Bacterial Genome Editing via Low-Temperature Recovery

“…The function of the CRISPR-Cas system is divided between the guide RNA (gRNA) for target nucleic acid recognition and the Cas nuclease for cleavage [ 1 ]. This modular system allows for the cleavage of desired nucleotide sequences by modifying the target recognition sequence (TRS) on the gRNA [ 2 ], thereby enabling genome editing across various organisms, including microbes [ 3 , 4 ].…”