CRISPRi-dCas12a: A dCas12a-Mediated CRISPR Interference for Repression of Multiple Genes and Metabolic Engineering in Cyanobacteria

2020 ◽  
Vol 9 (9) ◽  
pp. 2351-2361 ◽  
Author(s):  
Sun Young Choi ◽  
Han Min Woo
Keyword(s):  
Metabolic Engineering ◽  
Crispr Interference ◽  
Multiple Genes

Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway

2010 ◽  
Vol 4 (4) ◽  
pp. 269-280 ◽  
Author(s):  
Pejman Azadi ◽  
Ntui Valentaine Otang ◽  
Dong Poh Chin ◽  
Ikuo Nakamura ◽  
Masaki Fujisawa ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Carotenoid Biosynthesis ◽  
Biosynthesis Pathway ◽  
Multiple Genes ◽  
Carotenoid Biosynthesis Pathway

scholarly journals CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

2016 ◽  
Vol 38 ◽  
pp. 170-179 ◽  
Author(s):  
Gina C. Gordon ◽  
Travis C. Korosh ◽  
Jeffrey C. Cameron ◽  
Andrew L. Markley ◽  
Matthew B. Begemann ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Crispr Interference ◽  
Synechococcus Sp

scholarly journals An Alternative, Cas12a-based CRISPR Interference System for Mycobacteria

2021 ◽  
Author(s):  
Neil Fleck ◽  
Christoph Grundner
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Higher Order ◽  
Genetic Interactions ◽  
Gene Repression ◽  
Crispr Interference ◽  
Interference System ◽  
Multiple Genes ◽  
Technical Challenges

ABSTRACTThe introduction of CRISPR interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but technical challenges of the prototypical Cas9-based platform, for example in multigene regulation, remain. Here, we introduce an alternative CRSPRi platform that uses the minimal Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, and can regulate multiple genes simultaneously, providing a new tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb).

scholarly journals Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Dingyu Liu ◽  
Can Huang ◽  
Jiaxin Guo ◽  
Peiji Zhang ◽  
Tao Chen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
Genome Editing ◽  
Gene Editing ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Gene Deletions ◽  
Multiple Genes ◽  
Genetic Modifications ◽  
Riboflavin Operon

Abstract Background Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in multiplex genome editing technologies. However, simultaneously modulating multiple genes on the chromosome remains challenging in Bacillus subtilis. Thus, developing an efficient and convenient method for B. subtilis multiplex genome editing is imperative. Results Here, we developed a CRISPR/Cas9n-based multiplex genome editing system for iterative genome editing in B. subtilis. This system enabled us to introduce various types of genomic modifications with more satisfying efficiency than using CRISPR/Cas9, especially in multiplex gene editing. Our system achieved at least 80% efficiency for 1–8 kb gene deletions, at least 90% efficiency for 1–2 kb gene insertions, near 100% efficiency for site-directed mutagenesis, 23.6% efficiency for large DNA fragment deletion and near 50% efficiency for three simultaneous point mutations. The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by ligD gene, which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome. To demonstrate its potential, we applied our system to simultaneously fine-tune three genes in the riboflavin operon and significantly improved the production of riboflavin in a single cycle. Conclusions We present not only the iterative CRISPR/Cas9n system for B. subtilis but also the highest efficiency for simultaneous modulation of multiple genes on the chromosome in B. subtilis reported to date. We anticipate this CRISPR/Cas9n mediated system to greatly enhance the optimization of diverse biological systems via metabolic engineering and synthetic biology.

scholarly journals Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120

2017 ◽  
Vol 59 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Akiyoshi Higo ◽  
Atsuko Isu ◽  
Yuki Fukaya ◽  
Shigeki Ehira ◽  
Toru Hisabori
Keyword(s):  
Metabolic Engineering ◽  
Crispr Interference ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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