Genes Associated with Disturbed Cerebral Neurogenesis in the Embryonic Brain of Mouse Models of Down Syndrome

Down syndrome (DS), also known as trisomy 21, is the most frequent genetic cause of intellectual disability. Although the mechanism remains unknown, delayed brain development is assumed to be involved in DS intellectual disability. Analyses with human with DS and mouse models have shown that defects in embryonic cortical neurogenesis may lead to delayed brain development. Cre-loxP-mediated chromosomal engineering has allowed the generation of a variety of mouse models carrying various partial Mmu16 segments. These mouse models are useful for determining genotype–phenotype correlations and identifying dosage-sensitive genes involved in the impaired neurogenesis. In this review, we summarize several candidate genes and pathways that have been linked to defective cortical neurogenesis in DS.

Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers. Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation. Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.

Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target

The widespread application of high-throughput sequencing methods is resulting in the identification of a rapidly growing number of novel gene fusions caused by tumour-specific chromosomal rearrangements, whose oncogenic potential remains unknown. Here we describe a strategy that builds upon recent advances in genome editing and combines ex vivo and in vivo chromosomal engineering to rapidly and effectively interrogate the oncogenic potential of genomic rearrangements identified in human brain cancers. We show that one such rearrangement, an microdeletion resulting in a fusion between Brevican (BCAN) and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), is a potent oncogenic driver of high-grade gliomas and confers sensitivity to the experimental TRK inhibitor entrectinib. This work demonstrates that BCAN-NTRK1 is a bona fide human glioma driver and describes a general strategy to define the oncogenic potential of novel glioma-associated genomic rearrangements and to generate accurate preclinical models of this lethal human cancer.

Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion

SummaryDNA fragment editing (DFE) or chromosomal engineering including inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate structural genome variations and developmental gene regulation, but little is known about the underlying mechanisms. Here we report that debilitating CtIP, which is thought to function in NHEJ, enhances precise DNA fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA fragment inversions, deletions, and duplications, we find that Cas9 cleaves the noncomplementary strand with a flexible profile upstream of the PAM site and rationally-designed Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions of DFE are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DFEs could be achieved by perturbing DNA repair genes and using appropriate PAM configurations. These findings have important applications regarding 3D chromatin folding and enhancer insulation during gene regulation.

Generation of Targeted Deletions in the Genome of Rhodothermus marinus

ABSTRACTThe aim of this work was to develop an approach for chromosomal engineering of the thermophileRhodothermus marinus. A selection strategy forR. marinushad previously been developed; this strategy was based on complementing a restriction-negativetrpBstrain with theR. marinustrpBgene. The current work identified an additional selective marker,purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp+selection was used during the deletion ofpurAfrom theR. marinuschromosome. The alternative Ade+selection was in turn used while deleting the endogenoustrpBgene. Since both deletions are unmarked, thepurAandtrpBmarkers may be reused. Through the double deletant SB-62 (ΔtrpBΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency inR. marinusstrain SB-62 (ΔtrpBΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes,crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5′ and 3′ homologous sequences, respectively. The resulting Trp+transformants were colorless rather than orange-red. The correct replacement of an internalcrtBIfragment with thetrpBmarker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in theR. marinuschromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.