Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Comparing genomes of Fructus Amomi-producing species reveals genetic basis of volatile terpenoid divergence.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are 2 primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoid divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contributes to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.

[Genetic diagnosis for a Chinese Han family with hereditary multiple osteochondromas].

OBJECTIVE: To identify the genetic cause for a Chinese Han family affected with hereditary multiple osteochondromas. METHODS: Two patients, five unaffected relatives of the family and 100 unrelated healthy controls were collected. The coding sequences and intron/exon boundaries of EXT1 gene were amplified with polymerase chain reaction (PCR) and sequenced. RESULTS: A heterozygous c.600G>A (p.Trp200X) mutation in exon 1 of the EXT1 gene was detected in the patients. The same mutation was not found in unaffected family members and 100 healthy controls. CONCLUSION: The hereditary multiple osteochondromas in the family is caused by a nonsense mutation (p.Trp200X) in the EXT1 gene.

A de novo 1.38 Mb duplication of 1q31.1 in a boy with hemifacial microsomia, anophthalmia, anotia, macrostomia, and cleft lip and palate.

We reported a 2-year-old boy with developmental delay, mild mental retardation, and severe craniofacial malformation, including facial asymmetry with hypoplasia of the left zygoma, maxilla, and mandible, and left anophthalmia and anotia. A genome-wide screen revealed a 1.38 Mb duplication on chromosome 1q31.1, which was absent in his parents and 27 healthy controls. The duplication region contains two Refseq genes, PLA2G4A and C1orf99, which have not been reported to be implicated in craniofacial malformation. Functional studies of these genes and additional clinical analysis are necessary to elucidate the pathogenesis of craniofacial malformation.

[Clinical investigation of a Chinese family with hypotrichosis simplex of the scalp and mutational analysis of CDSN gene].

OBJECTIVE: To analyze clinical symptoms and disease-causing mutations of corneodesmosin (CDSN) gene in a Chinese family affected with hypotrichosis simplex of the scalp and to establish a method for prenatal diagnosis. METHODS: Family survey and clinical examinations were carried out to determine the inheritance pattern. Three patients and 7 unaffected relatives from the family, in addition with 100 unrelated healthy controls were recruited. Genomic DNA from peripheral blood leukocytes was extracted. Five pairs of primers were designed based on the CDSN gene sequence. Exons and flanking regions of the CDSN gene were amplified using polymerase chain reaction (PCR). Potential mutations were analyzed through direct sequencing and comparison by BLAST. RESULTS: The type of alopecia of the family was diagnosed as hypotrichosis simplex of the scalp with an autosomal dominant inheritance pattern. A nonsense mutation (C717G) in cDNA sequence of the CDSN gene was identified in all three patients of the family, which resulted in a premature stop codon (Y239X). The same mutation was not found among healthy members of the family and 100 healthy controls. CONCLUSION: A Chinese family was diagnosed with hypotrichosis simplex of the scalp, which was caused by a novel nonsense mutation (Y239X) in the CDSN gene.

[Mutation analysis of FGFR3 gene in a family featuring hereditary dwarfism].

OBJECTIVE: To investigate the clinical symptoms and potential mutation in FGFR3 gene for a family featuring hereditary dwarfism in order to attain diagnosis and provide prenatal diagnosis. METHODS: Five patients and two unaffected relatives from the family, in addition with 100 healthy controls, were recruited. Genome DNA was extracted. Exons 10 and 13 of the FGFR3 gene were amplified using polymerase chain reaction (PCR). PCR products were sequenced in both directions. RESULTS: All patients had similar features including short stature, short limbs, lumbar hyperlordosis but normal craniofacial features. A heterozygous mutation G1620T (N540K) was identified in the cDNA from all patients but not in the unaffected relatives and 100 control subjects. A heterozygous G380R mutation was excluded. CONCLUSION: The hereditary dwarfism featured by this family has been caused by hypochondroplasia (HCH) due to a N540K mutation in the FGFR3 gene.

Genome-wide scanning reveals complex etiology of oculo-auriculo-vertebral spectrum.

Oculo-auriculo-vertebral spectrum (OAVS) is a common developmental disorder involving first and second pharyngeal arches. Although some family cases and such patients showing chromosomal aberrations suggest that OAVS have a genetic basis, no consistent genetic defects have been recorded at present time. Thus, we conducted genetic studies of a three-generation family with five OAVS patients to identify a causative variant for OAVS. Cytogenetic studies revealed those family members had a normal karyotype and no causative mutations were founded in SALL1 and TCOF1, which known to be responsible for two other syndromes that have clinical overlapping with OAVS. Genotyping with commercially available BeadChips was performed on 13 individuals in the same family, showing no significant difference between the affected and normal members in terms of copy number variations (CNVs) in either number or size and no definitive causative CNV. A total of 8,224 informative autosomal SNPs that are evenly distributed throughout the genome were selected for both parametric and non-parametric linkage analysis. Significant negative LOD scores were obtained for the reported OAVS locus, providing further evidence for genetic heterogeneity of this complex disorder. The highest LOD score of 1.60 was noted on chromosome 15q26.2-q26.3 showing a potential linkage to this locus. The variable phenotypes of the affected members and the failure to identify a causative variant indicate that a complex etiology may be present even in a consanguineous family, which makes it more challenging to ascertain the cause of OAVS in further analysis.

Two neighboring microdeletions of 5q13.2 in a child with oculo-auriculo-vertebral spectrum.

We describe a patient with multiple congenital anomalies, including hemifacial microsomia, asymmetric macrostomia, dysplastic mandible, multiple preauricular tags, atresia of the external auricular canal, and vertebral anomalies, which coincide with oculo-auriculo-vertebral spectrum. G-banding ( approximately 850 band level) showed a normal 46, XY karyotype. A genome-wide screen for copy number variations (CNVs) using single nucleotide polymorphism (SNP) arrays revealed a 1Mb and a 167 kb deletion both on chromosome 5q13.2, which were absent in the parents and in 27 controls. Sixteen genes were located in the deleted region, including BIR1C and OCLN, which are involved in apoptosis. Haploinsufficiency of these genes may be contributing to the phenotype in this patient. To our knowledge, there are no previous reports of this 5q13.2 deletion in a patient with oculo-auriculo-vertebral spectrum.