Zhe Zeng

Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.

Cloning of large genomic sequences is an enabling technology in synthetic biology. To obtain large gene fragments, traditional cloning methods are faced with various defects, for instance, random library cloning relies always on high-throughput screening. It is difficult to get gene fragments more than 10 kb by PCR amplification. Assembly of small fragments is labor intensive with high mutation rates. It is difficult to find suitable cleavage sites on the fragment ends by restriction endonuclease. Recently genome-wide editing creates a new high-performance large fragments cloning methods. For example, CRISPR/cas9 system can identify and cut 20 bp nucleic acid sequences recognition sites used to obtain any desired gene fragments; if combined with Gibson or transformation associated recombination (TAR) assembly technology, these methods can efficiently clone large fragments. This article introduces large fragments cloning technology by classification, then proposes the choice criteria of methods for cloning gene fragments of different sizes.

ABSTRACT Rice processing quality, a key driver of consumer acceptance and global market competitiveness, is primarily determined by two critical parameters: brown rice rate (BRR) and milled rice rate (MRR). Despite their significant agricultural importance, the genetic mechanisms underpinning these traits remain poorly understood. This study analysed 447 genetically diverse rice accessions from the 3K Rice Genomes Project across multiple environments. Significant variation in processing traits was observed, with BRR from 73.1% to 79.3% and MRR from 58.3% to 74.3%. Seven quantitative trait loci (QTL) were identified via genome‐wide association studies (GWAS), including four novel loci ( qBRR10 , qBRR10‐1 , qMRR5 and qMRR11‐1 ) and three loci co‐localized with functionally validated genes ( OsGME2 , du3 and OsPK5 ). These results confirm the reliability of the analytical framework. Haplotype‐based stratification revealed statistically significant variations in traits ( p < 0.05) across subpopulations, with distinct geographic clustering patterns identified through cluster analysis. This study enhances the molecular understanding of mechanisms regulating processing quality and delivers specific genetic targets for marker‐assisted breeding, establishing a foundation for developing superior rice cultivars with enhanced processing traits.