Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.

Lb2Cas12a and its engineered variants mediate genome editing in human cells.

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.

Gut microbial metabolism of dietary fibre protects against high energy feeding induced ovarian follicular atresia in a pig model.

To investigate the effects of dietary fibre on follicular atresia in pigs fed a high-fat diet, we fed thirty-two prepubescent gilts a basal diet (CON) or a CON diet supplemented with 300 g/d dietary fibre (fibre), 240 g/d soya oil (SO) or both (fibre + SO). At the 19th day of the 4th oestrus cycle, gilts fed the SO diet showed 112 % more atretic follicles and greater expression of the apoptotic markers, Bax and caspase-3, and these effects were reversed by the fibre diet. The abundance of SCFA-producing microbes was decreased by the SO diet, but this effect was reversed by fibre treatment. Concentrations of serotonin and melatonin in the serum and follicular fluid were increased by the fibre diet. Overall, dietary fibre protected against high fat feeding-induced follicular atresia at least partly via gut microbiota-related serotonin-melatonin synthesis. These results provide insight into preventing negative effects on fertility in humans consuming a high-energy diet.

Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.

Microbial and metabolomic mechanisms mediating the effects of dietary inulin and cellulose supplementation on porcine oocyte and uterine development.

BACKGROUND: Dietary fiber (DF) is often eschewed in swine diet due to its anti-nutritional effects, but DF is attracting growing attention for its reproductive benefits. The objective of this study was to investigate the effects of DF intake level on oocyte maturation and uterine development, to determine the optimal DF intake for gilts, and gain microbial and metabolomic insight into the underlying mechanisms involved. METHODS: Seventy-six Landrace × Yorkshire (LY) crossbred replacement gilts of similar age (92.6 ± 0.6 d; mean ± standard deviation [SD]) and body weight (BW, 33.8 ± 3.9 kg; mean ± SD) were randomly allocated to 4 dietary treatment groups (n = 19); a basal diet without extra DF intake (DF 1.0), and 3 dietary groups ingesting an extra 50% (DF 1.5), 75% (DF 1.75), and 100% (DF 2.0) dietary fiber mixture consisting of inulin and cellulose (1:4). Oocyte maturation and uterine development were assessed on 19 d of the 2nd oestrous cycle. Microbial diversity of faecal samples was analysed by high-throughput pyrosequencing (16S rRNA) and blood samples were subjected to untargeted metabolomics. RESULTS: The rates of oocytes showing first polar bodies after in vitro maturation for 44 h and uterine development increased linearly with increasing DF intake; DF 1.75 gilts had a 19.8% faster oocyte maturation rate and a 48.9 cm longer uterus than DF 1.0 gilts (P <  0.05). Among the top 10 microbiota components at the phylum level, 8 increased linearly with increasing DF level, and the relative abundance of 30 of 53 microbiota components at the genus level (> 0.1%) increased linearly or quadratically with increasing DF intake. Untargeted metabolic analysis revealed significant changes in serum metabolites that were closely associated with microbiota, including serotonin, a gut-derived signal that stimulates oocyte maturation. CONCLUSIONS: The findings provide evidence of the benefits of increased DF intake by supplementing inulin and cellulose on oocyte maturation and uterine development in gilts, and new microbial and metabolomic insight into the mechanisms mediating the effects of DF on reproductive performance of replacement gilts.

Effects of Energy and Dietary Fiber on the Breast Development in Gilt.

To study the effects of energy and dietary fiber on breast development in gilts and its possible mechanisms, 32 gilts (Landrace × Yorkshire) were randomly allocated into a 2 × 2 factorial design to receive a diet with low or high energy [LE: 33.37 MJ/d digestible energy (DE); HE: 41.87 MJ/d DE] and low or high fiber (LF: 0.3 kg/d dietary fiber, HF: 0.6 kg/d dietary fiber). The weight of breast tissue was recorded. The mammary glands were collected for further analyses. The high energy intake increased the relative weight of breast tissue (p < 0.05) and the content of breast fat (p < 0.05). At the same time, the oil red staining of breast slices also showed an increase in breast fat content in high-energy treatment. High energy intake increased the DNA concentration in breast tissues (p < 0.05). In addition, high energy intake increased the concentration of triglycerides, free fatty acids, and total cholesterol in the blood of gilts (p < 0.05), and the supplementation of high fiber tended to reduce free fatty acids, total cholesterol, and estradiol (p < 0.1). Proteomic analysis suggested that there were notable differences in the cytoskeleton, intracellular non-membrane-bounded organelle, apoptosis, receptor activity, and endopeptidase inhibitor activity in molecular function between the energy and fiber effects (p < 0.05). High fiber intake also decreased the mRNA expression of 5-HT7, Bax, and caspase-3 in the breast tissue of gilts (p < 0.05), which further confirmed the importance of fiber in regulating breast development in gilt. Our results indicate that increasing gilt energy intake improved breast weight and fat deposition and increased breast cell apoptosis. Increased fiber intake reduced breast fat deposition and breast cell apoptosis at high energy intake in gilts. These results provide a potential strategy for dietary intervention against high energy intake in gilts and even in humans.

Optimal Dietary Fiber Intake to Retain a Greater Ovarian Follicle Reserve for Gilts.

: Ovarian follicle activation and survival were recently found to be controlled by nutrient sensors AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) and apoptosis related markers Caspase-3, Bax, and Bcl-2, yet their expression as regulated by dietary fiber remained uncertain for gilts. To investigate the effects of dietary fiber levels on ovarian follicle development, and the cellular molecular components related to follicle activation and survival of gilts, 76 gilts with similar bodyweight and age were fed four diets, including a corn-soybean meal based control diet, or other three diets to consume 50%, 75%, and 100% more dietary fiber than the control gilts at different experimental phases. Inulin and cellulose (1:4) were added to the corn-soybean meal basal diet to increase dietary fiber content. The growth traits, and the age, bodyweight, and backfat thickness at puberty were not affected by diets. The number of primordial follicles and total follicles per cubic centimeter of ovarian tissue linearly increased with dietary fiber level at day 30 of the experiment and at the 19th day of the 3rd estrous cycle, without negatively affecting the formation of antral follicle with diameter between 1-3 mm or larger than 3 mm. These changes were associated with altered phosphorylation of mTOR, S6, Extracellular regulated protein kinases 1/2 (ERK1/2) and AMPK, and mRNA expression of Caspase-3, Bax, and Bcl-2 in ovarian tissues. Collectively, this study demonstrated a beneficial effect of dietary fiber on the ovarian follicle reserve in gilts, which provides a basis for enhancing reproduction in the short- or long-term.

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways.