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      CRISPR-Mediated Base Editing: From Precise Point Mutation to Genome-Wide Engineering in Nonmodel Microbes

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      Biology
      MDPI AG

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          Abstract

          Nonmodel microbes with unique and diverse metabolisms have become rising stars in synthetic biology; however, the lack of efficient gene engineering techniques still hinders their development. Recently, the use of base editors has emerged as a versatile method for gene engineering in a wide range of organisms including nonmodel microbes. This method is a fusion of impaired CRISPR/Cas9 nuclease and base deaminase, enabling the precise point mutation at the target without inducing homologous recombination. This review updates the latest advancement of base editors in microbes, including the conclusion of all microbes that have been researched by base editors, the introduction of newly developed base editors, and their applications. We provide a list that comprehensively concludes specific applications of BEs in nonmodel microbes, which play important roles in industrial, agricultural, and clinical fields. We also present some microbes in which BEs have not been fully established, in the hope that they are explored further and so that other microbial species can achieve arbitrary base conversions. The current obstacles facing BEs and solutions are put forward. Lastly, the highly efficient BEs and other developed versions for genome-wide reprogramming of cells are discussed, showing great potential for future engineering of nonmodel microbes.

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          Development and applications of CRISPR-Cas9 for genome engineering.

          Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine. Copyright © 2014 Elsevier Inc. All rights reserved.
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            Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

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              CRISPR–Cas9 Structures and Mechanisms

              Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
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                Author and article information

                Contributors
                Journal
                BBSIBX
                Biology
                Biology
                MDPI AG
                2079-7737
                April 2022
                April 09 2022
                : 11
                : 4
                : 571
                Article
                10.3390/biology11040571
                35453770
                79018b43-46ba-4819-9870-60ee26632363
                © 2022

                https://creativecommons.org/licenses/by/4.0/

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