Cell engineering is commonly limited to the serial manipulation of a single gene or locus. The recently discovered CRISPR-associated transposases (CASTs) could manipulate multiple sets of genes to achieve predetermined cell diversity, with orthogonal CASTs being able to manipulate them in parallel. Here, a novel CAST from Pseudoalteromonas translucida KMM520 (PtrCAST) was characterized without a protospacer adjacent motif (PAM) preference which can achieve a high insertion efficiency for larger cargo and multiplexed transposition and tolerate mismatches out of 4-nucleotide seed sequence. More importantly, PtrCAST operates orthogonally with CAST from Vibrio cholerae Tn6677 (VchCAST), though both belonging to type I-F3. The two CASTs were exclusively active on their respective mini-Tn substrate with their respective crRNAs that target the corresponding 5 and 2 loci in one Escherichia coli cell. The multiplexed orthogonal MUCICAT (MUlticopy Chromosomal Integration using CRISPR-Associated Transposases) is a powerful tool for cell programming and appears promising with applications in synthetic biology.

/pdf/orthogonal-crispr-associated-transposases-for-parallel-and-54oy6a738p.pdf

Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration.

Recent advances in phage defense systems and potential overcoming strategies.

Abstract Background Natronobacterium gregoryi Argonaute (NgAgo) was found to reduce mRNA without generating detectable DNA double-strand breaks in a couple of endogenous genes in zebrafish, suggesting its potential as a tool for gene knockdown. However, little is known about how it interacts with nucleic acid molecules to interfere with gene expression. Results In this study, we first confirmed that coinjection of NgAgo and gDNA downregulated target genes, generated gene-specific phenotypes and verified some factors (including 5’ phosphorylation, GC ratio, and target positions) of gDNAs affecting gene downregulation. Therein, the sense and antisense gDNAs were equally effective, suggesting that NgAgo possibly binds to DNA. NgAgo-VP64 with gDNAs targeting promoters upregulated the target genes, further providing evidence that NgAgo interacts with genomic DNA and controls gene transcription. Finally, we explain the downregulation of NgAgo/gDNA target genes by interference with the process of gene transcription, which differs from that of morpholino oligonucleotides. Conclusions The present study provides conclusions that NgAgo may target genomic DNA and that target positions and the gDNA GC ratio influence its regulation efficiency. 

/pdf/efficient-manipulation-of-gene-expression-using-2c0oxeoz.pdf

Efficient manipulation of gene expression using Natronobacterium gregoryi Argonaute in zebrafish

Traditional Chinese food therapies often motivate the development of modern medicines, and learning from them will bring bright prospects. Monascus, a conventional Chinese fungus with centuries of use in the food industry, produces various metabolites, including natural pigments, lipid-lowering substances, and other bioactive ingredients. Recent Monascus studies focused on the metabolite biosynthesis mechanisms, strain modifications, and fermentation process optimizations, significantly advancing Monascus development on a lab scale. However, the advanced manufacture for Monascus is lacking, restricting its scale production. Here, the synthetic biology techniques and their challenges for engineering filamentous fungi were summarized, especially for Monascus. With further in-depth discussions of automatic solid-state fermentation manufacturing and prospects for combining synthetic biology and process intensification, the industrial scale production of Monascus will succeed with the help of Monascus improvement and intelligent fermentation control, promoting Monascus applications in food, cosmetic, agriculture, medicine, and environmental protection industries. 

Synthetic biology for Monascus: From strain breeding to industrial production

Argonaute proteins: Opening new horizons for the detection of non-nucleic acid targets in food

Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5′ phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3′ of the target RNA which is recognized by 5′ sequence of the gDNA. These results confirmed that NgAgo–gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3′ of the target RNA, which is a new finding that has not been reported in the past.

Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets

The invention relates to a method for rapidly preparing sgRNA by using a plurality of overlapping primers PCR method, which belongs to the field of molecular biology. The invention designs a pluralityof overlapping primers, amplifies and synthesizes a complete DNA fragment by PCR, and the complete DNA fragment is transcribed into sgRNA in vitro, and is combined with Cas9 enzyme to perform nucleicacid cleavage function. The invention uses multiple overlapping primers to synthesize DNA template of sgRNA in one step for the first time, and the experiment proves that the DNA template has high cleavage efficiency. The method established by the invention has value in the aspects of fast synthesis of sgRNA and reduction of experiment consumption.

Method for rapidly preparing sgRNA by PCR with multiple overlap primers

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Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets

Method for rapidly preparing sgRNA by PCR with multiple overlap primers