Unusual Removal of T-DNA in T1 Progenies of Rice after Agrobacterium-mediated CRISPR/Cas9 Editing

In the present study, we attempted to knock out the bar gene selection marker in the fixed Bt- and herbicide-resistant transgenic line BT-T07 (T8 generation) to generate a marker-free Bt-resistant rice line. A binary vector containing a CRISPR/Cas9 system targeting the 108–130 bp region of bar was transformed into rice embryonic calli, and plantlets were regenerated under non-selective conditions. Three T0 plants were observed to have non-target point mutations and deletions in the targeted gene and were putatively heterozygous and chimeras. One T0 plant, 130-4, was confirmed to have a 76-nt deletion, from 140 bp to 225 bp, and it showed the segregation of bar in its T1 progenies, with 16 bar-knockout lines and seven normal bar-expressing lines. However, the CRISPR/Cas9 editing vector sequences were not detected in any of the T1 plants. In addition, unusual removal of pre-existing T-DNA was observed in all bar-knockout T1 plants. Illumina sequencing of a bar-knockout line, 130-4-36, revealed a small fraction of read residues of pre-existing T-DNA from the bar sequence to the right border at the original junction site. We speculate that this rare phenomenon might be directed by the homology between pre-existing T-DNA and CRISPR/Cas9 editing vector sequences during meiotic recombination. We report imprecise modifications and unpredictable outcomes of gene-editing techniques, providing valuable perspectives on gene-editing applications.

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

Inactivation of mrpigH Gene in Monascus ruber M7 Results in Increased Monascus Pigments and Decreased Citrinin with mrpyrG Selection Marker

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are currently used around the world via Asian catering. The MPs biosynthetic pathway has been well-illustrated; however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In the current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.

Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

Targeted gene knock-in reduces variation between transformants in the mushroom-forming fungus Schizophyllum commune

Gene integration in mushroom-forming fungi currently occurs by the ectopic integration of a plasmid. The locus of integration is unpredictable and, problematically, this generally results in a high variability in gene expression and phenotypes between the transformants. Here, we developed an approach for targeted gene integration (knock-in) in the basidiomycete Schizophyllum commune by replacing a 75-bp non-coding region of the genome with a selection marker and an arbitrary gene of interest using CRISPR-Cas9 ribonucleoproteins. To assess the suitability of our method, we compared targeted integration and ectopic integration of the gene encoding the red fluorescent protein dTomato. Targeted integration resulted in a higher average fluorescence intensity and less variability between the transformants. This method may be applied to any gene construct and may therefore greatly increase the efficiency of functional gene analysis in S. commune.

Inactivation of MrPigH in Monascus ruber M7 Results in its Monascus Pigments Increase and Citrinin Decrease with mrpyrG Selection Marker

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and nowadays in the whole world via Asian catering. The MPs biosynthetic pathway has been well-illustrated, however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.

Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation

Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

Exploiting Type I-B CRISPR Genome Editing System in Thermoanaerobacterium Aotearoense SCUT27 And Engineering The Strain For High-Level Ethanol Production

Background: Thermophilic microbes for biofuels and chemicals have attracted great attention due to their tolerance of high temperature and wide range of substrate utilization. Thermoanaerobacterium aotearoense SCUT27 has the ability of glucose and xylose co-utilization in lignocellulosic biomass. Polygene manipulation was a bottleneck since it was hindered by available markers for selection. In this study, the endogenous Type I-B CRISPR/Cas system was developed for multiplex genome editing in SCUT27. Results: The protospacer-adjacent motif (PAM) was identified by in silico and orotidine-5’-phosphate decarboxylase (pyrF) and then lactate dehydrogenase (ldh) were chosen as the editing target to assess the toxicity of this immune system and gene editing efficiency. The mutants could be repeatedly obtained with an editing efficiency of 58.3-100%. Higher transformation efficiency was observed after optimization of some editing strategies. Furthermore, a new method was performed for screening mutants of plasmid curing (recycling of the editing plasmid) for multiplex genome editing based on the negative selection marker tdk, and then ldh and arginine repressor (argR) were knocked out successively. The mutant SCUT27/Δldh/ΔargR had the prominent advantages over SCUT27 for ethanol production with enhanced ability to metabolize xylose. When cultured under various lignocellulosic hydrolysates, the mutant showed a satisfactory performance with the ethanol titer and yield improved by 147.42–739.40% and 112.67–267.89%, respectively, compared with SCUT27, as well as the enhanced tolerance to inhibitors.Conclusion: The multi-gene editing by native CRISPR/Cas system is a promising strategy to engineer SCUT27 for higher ethanol production with lignocellulosic hydrolysates.