An engineered genetic circuit for lactose intolerance alleviation.

BACKGROUND: Lactose malabsorption occurs in around 68% of the world's population, causing lactose intolerance (LI) symptoms, such as abdominal pain, bloating, and diarrhea. To alleviate LI, previous studies have mainly focused on strengthening intestinal ß-galactosidase activity while neglecting the inconspicuous drop in the colon pH caused by the fermentation of non-hydrolyzed lactose by the gut microbes. A drop in colon pH will reduce the intestinal ß-galactosidase activity and influence intestinal homeostasis. RESULTS: Here, we synthesized a tri-stable-switch circuit equipped with high ß-galactosidase activity and pH rescue ability. This circuit can switch in functionality between the expression of ß-galactosidase and expression of L-lactate dehydrogenase in response to an intestinal lactose signal and intestinal pH signal, respectively. We confirmed that the circuit functionality was efficient in bacterial cultures at a range of pH levels, and in preventing a drop in pH and ß-galactosidase activity after lactose administration to mice. An impact of the circuit on gut microbiota composition was also indicated. CONCLUSIONS: Due to its ability to flexibly adapt to environmental variation, in particular to stabilize colon pH and maintain ß-galactosidase activity after lactose influx, the tri-stable-switch circuit can serve as a promising prototype for the relief of lactose intolerance.

Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse.

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.

[Advance in genetic research on multiple morphological abnormalities of sperm flagellum].

Multiple morphological abnormalities of sperm flagella (MMAF) is a type of teratospermia caused by genetic defects. The sperm motility is low due to absence of flagella, shortness, curling, bending or irregularity of sperms, and combination of various abnormalities. Ultrastructure may show flagellum assembly abnormalities, which are mainly manifested by the absence of microtubules in the axoneme and defects of various structures such as fibrous sheath, outer dense fiber, mitochondrial sheath and dynein arms. MMAF males are unable to reproduce naturally and require assisted reproductive technology to obtain offsprings. For the heterogeneity of molecular etiology of MMAF, the outcome of assisted reproduction may be different. Here the candidate genes of MMAF and their functional mechanisms are summarized, which may provide a reference for clinical diagnosis, treatment and research for this disorder.

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

Patients with severe asthenoteratospermia carrying SPAG6 or RSPH3 mutations have a positive pregnancy outcome following intracytoplasmic sperm injection.

PURPOSE: To investigate the relation between mutations in ciliopathy-related SPAG6 and RSPH3 and male infertility with severe asthenoteratospermia characterized by multiple flagellar malformations and reveal the intracytoplasmic sperm injection (ICSI) outcomes of those primary ciliary dyskinesia (PCD) patients. METHODS: Whole-exome sequencing was applied to identify the pathogenic genes for the five PCD patients. The ICSI outcomes of those patients were compared with eight DNAH1-mutated patients and 215 oligo-asthenospermia (OAT) patients. RESULTS: We identified, for the first time, the compound heterozygous SPAG6 mutations (c.143_145del: p.48_49del, c.585delA: p.Lys196Serfs*6) in a sporadic PCD patient. Further, a novel homozygous nonsynonymous RSPH3 mutation (c.C799T: p.Arg267Cys) was identified in another PCD patient with consanguineous parents. The pathogenicity of these mutations in the assembly of sperm flagella was confirmed by flagellar ultrastructure analysis, immunofluorescence, and quantitative real-time PCR. All five patients underwent six ICSI cycles. The fertilization rate, blastocyst development rate, and clinical pregnancy rate were 69.3%, 50.0%, and 66.7%, respectively. Four of the five couples, including the subjects carrying mutations in SPAG6 or RSPH3, got healthy children born after ICSI. Additionally, the ICSI outcomes of the five PCD couples were statistically comparable with those of the eight DNAH1-mutated couples and the 215 OAT couples. CONCLUSIONS: Mutations in ciliopathy-related SPAG6 and RSPH3 cause severe asthenoteratospermia characterized by multiple flagellar malformations, resulting in sterility. ICSI is an optimal management with a positive pregnancy outcome.

Homozygous mutations in DZIP1 can induce asthenoteratospermia with severe MMAF.

BACKGROUND: Asthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes. METHODS AND RESULTS: Two asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF. CONCLUSION: Our study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.

Homozygous mutations in SPEF2 induce multiple morphological abnormalities of the sperm flagella and male infertility.

BACKGROUND: Male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated. METHODS AND RESULTS: We conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 (sperm flagellar 2) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2-affected subjects. CONCLUSIONS: We identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins.

Biallelic mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella in humans and mice.

BACKGROUND: Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases. METHODS AND RESULT: We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF. CONCLUSIONS: Our experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.

Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice.

As a type of severe asthenoteratospermia, multiple morphological abnormalities of the flagella (MMAF) are characterized by the presence of immotile spermatozoa with severe flagellar malformations. MMAF is a genetically heterogeneous disorder, and the known MMAF-associated genes can only account for approximately 60% of human MMAF cases. Here we conducted whole-exome sequencing and identified bi-allelic truncating mutations of the TTC29 (tetratricopeptide repeat domain 29) gene in three (3.8%) unrelated cases from a cohort of 80 MMAF-affected Han Chinese men. TTC29 is preferentially expressed in the testis, and TTC29 protein contains the tetratricopeptide repeat domains that play an important role in cilia- and flagella-associated functions. All of the men harboring TTC29 mutations presented a typical MMAF phenotype and dramatic disorganization in axonemal and/or other peri-axonemal structures. Immunofluorescence assays of spermatozoa from men harboring TTC29 mutations showed deficiency of TTC29 and remarkably reduced staining of intraflagellar-transport-complex-B-associated proteins (TTC30A and IFT52). We also generated a Ttc29-mutated mouse model through the use of CRISPR-Cas9 technology. Remarkably, Ttc29-mutated male mice also presented reduced sperm motility, abnormal flagellar ultrastructure, and male subfertility. Furthermore, intracytoplasmic sperm injections performed for Ttc29-mutated mice and men harboring TTC29 mutations consistently acquired satisfactory outcomes. Collectively, our experimental observations in humans and mice suggest that bi-allelic mutations in TTC29, as an important genetic pathogeny, can induce MMAF-related asthenoteratospermia. Our study also provided effective guidance for clinical diagnosis and assisted reproduction treatments.

Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice.

Male infertility is a major concern affecting human reproductive health. Asthenoteratospermia can cause male infertility through reduced motility and abnormal morphology of spermatozoa. Several genes, including DNAH1 and some CFAP family members, are involved in multiple morphological abnormalities of the sperm flagella (MMAF). However, these known genes only account for approximately 60% of human MMAF cases. Here, we conducted further genetic analyses by using whole-exome sequencing in a cohort of 65 Han Chinese men with MMAF. Intriguingly, bi-allelic mutations of TTC21A (tetratricopeptide repeat domain 21A) were identified in three (5%) unrelated, MMAF-affected men, including two with homozygous stop-gain mutations and one with compound heterozygous mutations of TTC21A. Notably, these men consistently presented with MMAF and additional abnormalities of sperm head-tail conjunction. Furthermore, a homozygous TTC21A splicing mutation was identified in two Tunisian cases from an independent MMAF cohort. TTC21A is preferentially expressed in the testis and encodes an intraflagellar transport (IFT)-associated protein that possesses several tetratricopeptide repeat domains that perform functions crucial for ciliary function. To further investigate the potential roles of TTC21A in spermatogenesis, we generated Ttc21a mutant mice by using CRISPR-Cas9 technology and revealed sperm structural defects of the flagella and the connecting piece. Our consistent observations across human populations and in the mouse model strongly support the notion that bi-allelic mutations in TTC21A can induce asthenoteratospermia with defects of the sperm flagella and head-tail conjunction.

NovelCFAP43 andCFAP44 mutations cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF).

RESEARCH QUESTION: Multiple morphological abnormalities of the sperm flagella (MMAF) comprise a rare congenital disease that can cause primary male infertility. Several pathogenic genes (e.g. AKAP4, DNAH1, CFAP43 and CFAP44) are associated with MMAF but the pathogenic mechanisms have not been elucidated. DESIGN: Whole-exome sequencing (WES) was applied to identify the pathogenic genes in 13 Chinese patients with MMAF; the patients were unrelated but all had consanguineous parents (usually first cousins). Real-time polymerase chain reaction and immunofluorescence staining were employed to assess the pathogenicity of these mutations. RESULTS: Four novel homozygous CFAP43 mutations in four (30.8%) MMAF patients and one novel homozygous CFAP44 mutation in one (7.7%) other case were identified. The four novel homozygous CFAP43 mutations included one frameshift mutation (c.1140_1143del: p.Asn380Lysfs*3), one nonsense mutation (c.739A>T: p.Lys247*) and two missense mutations (c.1474G>C: p.Gln492Arg; c.4600C>G: p.Leu1534Val). The novel mutation in CFAP44 was a homozygous nonsense mutation (c.4963C>T: p.Arg1655*). Co-segregation of the mutations was verified by Sanger sequencing of the families. The relative mRNA expression levels of CFAP43 in patients 1 and 9 and the levels of CFAP44 in patient 5 were significantly lower than those in control sperm samples. Immunofluorescence analysis of CFAP43 showed the protein was absent in the sperm flagella of patients 1 and 9. Furthermore, two previously reported mutations of DNAH1 were also identified in another four (30.8%) patients. CONCLUSIONS: This study demonstrated that CFAP43 and CFAP44 mutations are important causes of MMAF in the Chinese population. These novel mutations broaden the spectrum of CFAP43 and CFAP44 mutations.

Novel homozygous CFAP69 mutations in humans and mice cause severe asthenoteratospermia with multiple morphological abnormalities of the sperm flagella.

BACKGROUND: Male infertility is a major issue of human reproduction health. Asthenoteratospermia can impair sperm motility and cause male infertility. Asthenoteratospermia with multiple morphological abnormalities of the flagella (MMAF) presents abnormal spermatozoa with absent, bent, coiled, short and/or irregular-calibre flagella. Previous studies on MMAF reported that genetic defects in cilia-related genes (eg, AKAP4, DNAH1, CFAP43, CFAP44 and CFAP69) are the major cause of MMAF. However, the known MMAF-associated genes are only responsible for approximately 30% to 50% of human cases. We further investigated the cases with MMAF in search of additional genes mutated in this condition. METHODS AND RESULTS: We conducted whole exome sequencing in a male individual with MMAF from a consanguineous Han Chinese family. Sanger sequencing was also conducted in additional individuals with MMAF. Intriguingly, a homozygous frameshift mutation (p.Leu357Hisfs*11) was identified in the gene encoding CFAP69 (cilia and flagella-associated protein 69), which is highly expressed in testis. The subsequent Sanger sequencing of the CFAP69 coding regions among 34 additional individuals with MMAF revealed a case with homozygous nonsense mutation (p.Trp216*) of CFAP69. Both of these CFAP69 loss-of-function mutations were not present in the human population genome data archived in the 1000 Genomes Project and ExAC databases, nor in 875 individuals of two Han Chinese control populations. Furthermore, we generated the knockout model in mouse orthologue Cfap69 using the CRISPR-Cas9 technology. Remarkably, male Cfap69-knockout mice manifested with MMAF phenotypes. CONCLUSION: Our experimental findings elucidate that homozygous loss-of-function mutations in CFAP69 can lead to asthenoteratospermia with MMAF in humans and mice.

Biallelic mutations of CFAP251 cause sperm flagellar defects and human male infertility.

Multiple morphological abnormalities of flagella (MMAF) are human reproduction disorders due to the dysplastic development of sperm flagella. The spermatozoa of men with MMAF manifest absent, short, coiled, bent, and/or irregular-caliber flagella. Previous studies revealed genetic contributions to human MMAF, but known MMAF-associated genes only explained approximately 50% MMAF cases. In this study, we employed human whole-exome sequencing for genetic analysis and identified biallelic mutations of CFAP251 (cilia- and flagella-associated protein 251, also known as WDR66) in three (5%) of 65 Han Chinese men with MMAF. All these CFAP251 mutations are loss-of-function. The population genome data suggested that these CFAP251 mutations are extremely rare (only heterozygous) or absent from human populations. Our functional assays of gene expression and immunofluorescence staining in a CFAP251-deficient man, together with previous experimental evidence from model organisms, suggested that CFAP251 is involved in flagellar functions. Our observations suggested that CFAP251 is associated with sperm flagellar development and human male infertility.

Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella.

Sperm motility is vital to human reproduction. Malformations of sperm flagella can cause male infertility. Men with multiple morphological abnormalities of the flagella (MMAF) have abnormal spermatozoa with absent, short, coiled, bent, and/or irregular-caliber flagella, which impair sperm motility. The known human MMAF-associated genes, such as DNAH1, only account for fewer than 45% of affected individuals. Pathogenic mechanisms in the genetically unexplained MMAF remain to be elucidated. Here, we conducted genetic analyses by using whole-exome sequencing and genome-wide comparative genomic hybridization microarrays in a multi-center cohort of 30 Han Chinese men affected by MMAF. Among them, 12 subjects could not be genetically explained by any known MMAF-associated genes. Intriguingly, we identified compound-heterozygous mutations in CFAP43 in three subjects and a homozygous frameshift mutation in CFAP44 in one subject. All of these recessive mutations were parentally inherited from heterozygous carriers but were absent in 984 individuals from three Han Chinese control populations. CFAP43 and CFAP44, encoding two cilia- and flagella-associated proteins (CFAPs), are specifically or preferentially expressed in the testis. Using CRISPR/Cas9 technology, we generated two knockout models each deficient in mouse ortholog Cfap43 or Cfap44. Notably, both Cfap43- and Cfap44-deficient male mice presented with MMAF phenotypes, whereas the corresponding female mice were fertile. Our experimental observations on human subjects and animal models strongly suggest that biallelic mutations in either CFAP43 or CFAP44 can cause sperm flagellar abnormalities and impair sperm motility. Further investigations on other CFAP-encoding genes in more genetically unexplained MMAF-affected individuals could uncover novel mechanisms underlying sperm flagellar formation.

A Living Eukaryotic Autocementation Kit from Surface Display of Silica Binding Peptides on Yarrowia lipolytica.

With the development of civil engineering, the demand for suitable cementation materials is increasing rapidly. However, traditional cementation methods are not eco-friendly enough and more sustainable approach such as biobased cementation is required. To meet such demand, Euk.cement, a living eukaryotic cell-based biological autocementation kit, was created in this work. Through the surface display of different silica binding peptides on the fungus Yarrowia lipolytica, Euk.cement cells can immobilize onto any particles with a silica containing surface with variable binding intensity. Meanwhile, recombinant MCFP3 released from the cells will slowly consolidate this binding of cells to particles. The metabolism of immobilized living cells will finally complete the carbonate sedimentation and tightly stick the particles together. The system is designed to be initiated by blue light, making it controllable. This autocementation kit can be utilized for industrial and environmental applications that fit our concerns on making the cementation process eco-friendly.