Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

Selective Disruption of Drp1-Independent Mitophagy and Mitolysosome Trafficking by an Alzheimer’s Disease Relevant Tau Modification in a Novel C. elegans Model

Background: Accumulation of inappropriately phosphorylated tau into neurofibrillary tangles (NFT) is a defining feature of Alzheimer’s Disease (AD), with specific epitopes such as Tau pT231 emerging early in the development of tau pathology. Previously, we demonstrated that a phosphomimetic mutant (T231E) of human tau drove the loss of neuronal function and structural integrity with age in a novel C. elegans single-copy gene insertion AD model. A critical finding was that T231E, unlike wild type tau, suppressed oxidative stress-induced mitochondrial autophagy, or mitophagy. Regulation of mitochondrial morphology by fission is important for mitophagy, which has been reported to be dysregulated by AD-relevant tau species. Dynamin Related Protein 1 (Drp1) is a GTPase that plays a central role in mediating mitochondrial fission, and its altered function may contribute to AD pathogenesis. Methods: Genetically-encoded fluorescent biosensors and dynamic imaging approaches were combined with a genomic drp-1(-) loss-of-function and transgenic tau mutants to derive a comprehensive in vivo analysis of age-associated changes in mitochondria and mitolysosome (ML) morphology, abundance, neurite trafficking, and stress-induced mitophagy. Results: Strain expressing disease-associated PTM mimetic Tau T231E demonstrated a surprisingly selective effect on ML development and trafficking, with no effect on lysosomes or autolysosomes, and a subtle effect on mitochondria that was apparent mainly in older animals. Unexpectedly, we found that drp-1(-) mutants mount a robust mitophagy response to oxidative stress, consistent with recent observations that adaptive mitophagy may occur independent of the canonical DRP1 pathway. Moreover, T231E continued to suppress oxidative stress-induced mitophagy in the drp-1(-) background. Conclusions: Our C. elegans single-copy gene insertion model unveils multiple levels of selectivity – phenotypic selectivity for mutations that mimic pathologic tauopathy-associated PTM and physiologic selectivity for organelles that contain damaged mitochondria. In addition, our novel findings provide compelling support for DRP1-independent mechanisms playing a pivotal role in regulating mitochondrial dynamics and function in the context of AD-relevant tau species and age-associated stress.

Structural Diversity, Fitness Cost, and Stability of a BlaNDM-1-Bearing Cointegrate Plasmid in Klebsiella pneumoniae and Escherichia coli

Cointegrate/hybrid plasmids combine the genetic elements of two or more plasmids and generally carry abundant antimicrobial resistance determinants. Hence, the spread of cointegrate plasmids will accelerate the transmission of AMR genes. To evaluate the transmission risk caused by cointegrate plasmids, we investigated the structural diversity, fitness cost, and stability of a cointegrate plasmid in Klebsiella pneumoniae YZ6 and Escherichia coli EC600. The cointegrate plasmid pSL131_IncA/C_IncX3 was from a clinical Salmonella Lomita strain. After transferring the plasmid into E. coli EC600 by conjugation, we observed plasmids with different structures, including a full-length original plasmid and two truncated versions. By contrast, DNA fragment deletion and blaCTX-M-14 gene insertion in the plasmid were detected in a transconjugant derived from K. pneumoniae YZ6. These results suggest that the structure of the plasmid was unstable during conjugation. Furthermore, both the full-length plasmid in EC600 and the structurally reorganized plasmid in YZ6 imposed a fitness cost on the bacterial host and enhanced biofilm formation ability. Serial passaging in antibiotic-free medium resulted in a rapid decline of the plasmid in YZ6. However, the stability of the structurally reorganized plasmid in YZ6 was improved via serial passaging in antibiotic-containing medium. SNP calling revealed that mutations of the outer membrane porin may play an essential role in this process. These findings indicate that structural versatility could contribute to the dissemination of cointegrate plasmids. Although the plasmid incurred a fitness cost in other Enterobacteriaceae species, positive selection could alleviate the adverse effects.

Effect of Angiotensin-I Converting Enzyme Gene Insertion/Deletion Polymorphism on genome instability in children living in Russian Arctic

Main risks of arterial hypertension manifest in childhood. Children living in the Far North are especially susceptible to this. There is a need for an inexpensive, non-invasive and simple diagnosis of the risk of childhood pathologies. It was previously found that the genotype DD of the in/del polymorphic marker of the ACE gene is found in people at risk of developing cardiovascular pathologies. Buccal micronucleus cytome assay and genetic analysis were used in the work. In total, 77 schoolchildren from the city of Apatity, aged 15-17 years old, were examined. We have shown that carriers of the D allele have a tendency to an increase in the frequency of cells with micronuclei. In the case of homozygous I/I variant, the frequency of occurrence of cells with karyopycnosis is significantly higher than in carriers of allele D. Polymorphic marker in/del of the ACE gene is associated with apoptotic changes in the cells of the studied children. The in/del polymorphic marker of the ACE gene can be used as a prognostic marker of the processes of genome destabilization at the early stages of development of the human body.

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9

Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

RNAi Suppression of LEAFY Gives Stable Floral Sterility, and Reduced Growth Rate and Leaf Size, in Field-Grown Poplars

The central floral development gene LEAFY (LFY), whose mutation leads to striking changes in flowering and often sterility, is commonly expressed in non-floral structures; however, its role in vegetative development is poorly understood. Sterility associated with suppression of LFY expression is an attractive means for mitigating gene flow by both seeds and pollen in vegetatively propagated forest trees, but the consequences of its suppression for tree form and wood production are unclear. To study the vegetative effects of RNAi suppression of LFY, we created a randomized, multiple-year field study with 30–40 trees (ramets) in each of two sterile gene insertion events, three transgenic control events, and a wild-type control population. We found that floral knock-down phenotypes were stable across years and propagation cycles, but that several leaf morphology and productivity traits were statistically and often substantially different in sterile vs. normal flowering RNAi-LFY trees. Though trees with suppressed LEAFY expression looked visibly normal, they appear to have reduced growth and altered leaf traits. LFY appears to have a significant role in vegetative meristem development, and evaluation of vegetative impacts from LFY suppression would be prudent prior to large-scale use for genetic containment.

Gene Flow Risks From Transgenic Herbicide-Tolerant Crops to Their Wild Relatives Can Be Mitigated by Utilizing Alien Chromosomes

Integration of a transgene into chromosomes of the C-genomes of oilseed rape (AACC, 2n = 38) may affect their gene flow to wild relatives, particularly Brassica juncea (AABB, 2n = 36). However, no empiric evidence exists in favor of the C-genome as a safer candidate for transformation. In the presence of herbicide selections, the first- to fourth-generation progenies of a B. juncea × glyphosate-tolerant oilseed rape cross [EPSPS gene insertion in the A-genome (Roundup Ready, event RT73)] showed more fitness than a B. juncea × glufosinate-tolerant oilseed rape cross [PAT gene insertion in the C-genome (Liberty Link, event HCN28)]. Karyotyping and fluorescence in situ hybridization–bacterial artificial chromosome (BAC-FISH) analyses showed that crossed progenies from the cultivars with transgenes located on either A- or C- chromosome were mixoploids, and their genomes converged over four generations to 2n = 36 (AABB) and 2n = 37 (AABB + C), respectively. Chromosome pairing of pollen mother cells was more irregular in the progenies from cultivar whose transgene located on C- than on A-chromosome, and the latter lost their C-genome-specific markers faster. Thus, transgene insertion into the different genomes of B. napus affects introgression under herbicide selection. This suggests that gene flow from transgenic crops to wild relatives could be mitigated by breeding transgenic allopolyploid crops, where the transgene is inserted into an alien chromosome.