Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression in vitro and in vivo.

Chronic hepatitis B virus (HBV) infection remains a global health problem due to the lack of treatments that prevent viral rebound from HBV covalently closed circular (ccc)DNA. In addition, HBV DNA integrates in the human genome, serving as a source of hepatitis B surface antigen (HBsAg) expression, which impairs anti-HBV immune responses. Cytosine base editors (CBEs) enable precise conversion of a cytosine into a thymine within DNA. In this study, CBEs were used to introduce stop codons in HBV genes, HBs and Precore. Transfection with mRNA encoding a CBE and a combination of two guide RNAs led to robust cccDNA editing and sustained reduction of the viral markers in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Furthermore, base editing efficiently reduced HBsAg expression from HBV sequences integrated within the genome of the PLC/PRF/5 and HepG2.2.15 cell lines. Finally, in the HBV minicircle mouse model, using lipid nanoparticulate delivery, we demonstrated antiviral efficacy of the base editing approach with a >3log10 reduction in serum HBV DNA and >2log10 reduction in HBsAg, and 4/5 mice showing HBsAg loss. Combined, these data indicate that base editing can introduce mutations in both cccDNA and integrated HBV DNA, abrogating HBV replication and silencing viral protein expression.

Identification of Bioactive Peptides from a Laminaria digitata Protein Hydrolysate Using In Silico and In Vitro Methods to Identify Angiotensin-1-Converting Enzyme (ACE-1) Inhibitory Peptides.

Bioactive peptides range in size from 2-30 amino acids and may be derived from any protein-containing biomass using hydrolysis, fermentation or high-pressure processing. Pro-peptides or cryptides result in shorter peptide sequences following digestion and may have enhanced bioactivity. Previously, we identified a protein hydrolysate generated from Laminaria digitata that inhibited ACE-1 in vitro and had an ACE-1 IC50 value of 590 µg/mL compared to an ACE-1 IC50 value of 500 µg/mL (~2.3 µM) observed for the anti-hypertensive drug Captopril©. A number of peptide sequences (130 in total) were identified using mass spectrometry from a 3 kDa permeate of this hydrolysate. Predicted bioactivities for these peptides were determined using an in silico strategy previously published by this group utilizing available databases including Expasy peptide cutter, BIOPEP and Peptide Ranker. Peptide sequences YIGNNPAKGGLF and IGNNPAKGGLF had Peptide Ranker scores of 0.81 and 0.80, respectively, and were chemically synthesized. Synthesized peptides were evaluated for ACE-1 inhibitory activity in vitro and were found to inhibit ACE-1 by 80 ± 8% and 91 ± 16%, respectively. The observed ACE-1 IC50 values for IGNNPAKGGLF and YIGNNPAKGGLF were determined as 174.4 µg/mL and 133.1 µg/mL. Both peptides produced sequences following simulated digestion with the potential to inhibit Dipeptidyl peptidase IV (DPP-IV).

Angiotensin-I-Converting Enzyme Inhibitory Activity of Protein Hydrolysates Generated from the Macroalga Laminaria digitata (Hudson) JV Lamouroux 1813.

Seaweeds have a long history of use as both food and medicine, especially in Asian cultures. Moreover, there is growing interest in the use of seaweed ingredients and bioactive compounds in pharmaceutical and nutraceutical products. One ailment that seaweed bioactive compounds may impact is hypertension caused by the enzyme Angiotensin Converting Enzyme 1 (ACE-1; EC 3.4.15.1), found within the Renin-Angiotensin Aldosterone System (RAAS), which causes vasoconstriction of blood vessels, including veins and arteries. The aim of this paper is to generate bioactive peptide containing protein hydrolysates from the brown seaweed Laminaria digitata (Hudson) JV Lamouroux 1813. Proteins were extracted from this seaweed by disrupting the seaweed cell wall using a combination of carbohydrases and proteolytic enzymes. Bioactive peptide containing permeates were generated from L. digitata protein hydrolysates, and both hydrolysates and permeates were screened for their ability to inhibit the enzyme ACE-1. The protein content of the permeate fractions was found to be 23.87% compared to the untreated seaweed, which contained 15.08% protein using LECO analysis. Hydrolysis and filtration resulted in a "white" protein powder, and the protein content of this powder increased by 9% compared to the whole seaweed. The total amino acid (TAA) content of the L. digitata protein permeate was 53.65 g/100 g of the sample, and contains over 32% essential amino acids (EAA). Furthermore, the L. digitata permeate was found to inhibit the ACE-1 enzyme by 75% when compared to the commercial drug Captopril© when assayed at a concentration of 1 mg/mL. The inhibition of ACE-1 (the IC50 value) of 590 µg/mL for the L. digitata permeate compares well with Captopril©, which had 100% inhibition of ACE-1, with an IC50 value of 500 µg/mL. This study indicates that there is potential to develop protein powders with ACE-1 inhibitory bioactivities from the brown seaweed L. digitata using enzymatic hydrolysis as a cell disruption and protein extraction/hydrolysate generation procedure.

Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a rare autosomal codominant disease caused by mutations within the SERPINA1 gene. The most prevalent variant in patients is PiZ SERPINA1, containing a single G > A transition mutation. PiZ alpha-1 antitrypsin (AAT) is prone to misfolding, leading to the accumulation of toxic aggregates within hepatocytes. In addition, the abnormally low level of AAT secreted into circulation provides insufficient inhibition of neutrophil elastase within the lungs, eventually causing emphysema. Cytosine and adenine base editors enable the programmable conversion of C⋅G to T⋅A and A⋅T to G⋅C base pairs, respectively. In this study, two different base editing approaches were developed: use of a cytosine base editor to install a compensatory mutation (p.Met374Ile) and use of an adenine base editor to mediate the correction of the pathogenic PiZ mutation. After treatment with lipid nanoparticles formulated with base editing reagents, PiZ-transgenic mice exhibited durable editing of SERPINA1 in the liver, increased serum AAT, and improved liver histology. These results indicate that base editing has the potential to address both lung and liver disease in AATD.

Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes.

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.

An Enlarged and Infected Prostatic Utricle as a Rare Cause of Lower Urinary Tract Symptoms in Adolescent Males.

Dysuria with lower abdominal pain is a common presentation for a urinary tract infection (UTI), and diagnosis is based on symptoms together with a urinalysis and urine culture suggestive of infection. UTI is uncommon in circumcised males who are not sexually active. When urine culture is negative, alternate diagnoses including, but not limited to, gastroenteritis, severe constipation, appendicitis, or epididymitis need to be considered. In patients with a known urologic history of proximal hypospadias and/or disorders of sexual development, rarer diagnoses also need to be considered. This paper reports the case of a 13-year-old male with a remote history of proximal hypospadias repair, who presented with nonspecific lower urinary tract symptoms. Initially he was treated for UTI. However, urine cultures remained negative despite persistent urinary tract symptoms. On further workup, he was found to have an enlarged and infected prostatic utricle. This report illustrates the importance of considering an enlarged prostatic utricle in the differential diagnoses of patients with chronic lower urinary tract symptoms and a history of hypospadias. Additionally, this case highlights the utility of magnetic resonance imaging (MRI) in clarifying lower urinary tract anatomy in cases where ultrasound is inconclusive.

Transvesical robotic excision of a Müllerian duct remnant.

Müllerian duct remnants are rare and found in patients with disorders of sexual development. Presenting symptoms vary and many parents opt for surgical management. Literature on robotic repair is limited to small series, single case reports and all were approached extravesically. We present our case of a unique transvesical approach. Perioperative parameters were favorable with no complications, suggesting robotic repair is a safe and effective treatment strategy for these unique patients.

Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation.

Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.

Aquaculture Production of the Brown Seaweeds Laminaria digitata and Macrocystis pyrifera: Applications in Food and Pharmaceuticals.

Seaweeds have a long history of use as food, as flavouring agents, and find use in traditional folk medicine. Seaweed products range from food, feed, and dietary supplements to pharmaceuticals, and from bioenergy intermediates to materials. At present, 98% of the seaweed required by the seaweed industry is provided by five genera and only ten species. The two brown kelp seaweeds Laminaria digitata, a native Irish species, and Macrocystis pyrifera, a native New Zealand species, are not included in these eleven species, although they have been used as dietary supplements and as animal and fish feed. The properties associated with the polysaccharides and proteins from these two species have resulted in increased interest in them, enabling their use as functional foods. Improvements and optimisations in aquaculture methods and bioproduct extractions are essential to realise the commercial potential of these seaweeds. Recent advances in optimising these processes are outlined in this review, as well as potential future applications of L. digitata and, to a greater extent, M. pyrifera which, to date, has been predominately only wild-harvested. These include bio-refinery processing to produce ingredients for nutricosmetics, functional foods, cosmeceuticals, and bioplastics. Areas that currently limit the commercial potential of these two species are highlighted.

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

Directed evolution of adenine base editors with increased activity and therapeutic application.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

A critical control point approach to the removal of chemicals of concern from water for reuse.

The reuse of water in a range of potable and non-potable applications is an important factor in the augmentation of water supply and in improving water security and productivity worldwide. A key hindrance to the reuse of water is the cost of compliance testing and process validation associated with ensuring that pathogen and chemicals in the feedwater are removed to a level that ensures no acute or chronic health and/or environmental effects. The critical control point (CCP) approach is well established and widely adopted by water utilities to provide an operational and risk management framework for the removal of pathogens in the treatment system. The application of a CCP approach to barriers in a treatment system for the removal of chemicals is presented. The application exemplar is to a small community wastewater treatment system that aims to produce potable quality water from a secondary treated wastewater effluent, however, the concepts presented are generic. The example used seven treatment barriers, five of which were designed and operated as CCP barriers for pathogens. The work demonstrates a method and risk management framework by which three of the seven barriers could also include a CCP approach for the removal of chemicals. Analogous to a CCP approach for pathogens, the potential is to reduce the use of chemical analysis as a routine determinant of performance criteria. The operational deployment of a CCP approach for chemicals was augmented with the development of a decision tree encompassing the classification of chemicals and the total removal credits across the treatment train in terms of the mechanistic removal of chemicals for each barrier. Validation of the approach is shown for an activated sludge, ozone and reverse osmosis barrier.

Publisher Correction: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.

In this Article, owing to an error during the production process, in Fig. 1a, the dark blue and light blue wedges were incorrectly labelled as 'G•C → T•A' and 'G•C → A•T', instead of 'C•G → T•A' and 'C•G → A•T', respectively. Fig. 1 has been corrected online.

Greenshell™ Mussels: A Review of Veterinary Trials and Future Research Directions.

The therapeutic benefits of Greenshell™ mussel (GSM; Perna canaliculus) preparations have been studied using in vitro test systems, animal models, and human clinical trials focusing mainly on anti-inflammatory and anti-arthritic effects. Activity is thought to be linked to key active ingredients that include omega-3 polyunsaturated fatty acids, a variety of carotenoids and other bioactive compounds. In this paper, we review the studies that have been undertaken in dogs, cats, and horses, and outline new research directions in shellfish breeding and high-value nutrition research programmes targeted at enhancing the efficacy of mussel and algal extracts. The addition of GSM to animal diets has alleviated feline degenerative joint disease and arthritis symptoms, and chronic orthopaedic pain in dogs. In horses, GSM extracts decreased the severity of lameness and joint pain and provided improved joint flexion in limbs with lameness attributed to osteoarthritis. Future research in this area should focus on elucidating the key active ingredients in order to link concentrations of these active ingredients with their pharmacokinetics and therapeutic effects. This would enable consistent and improved efficacy from GSM-based products for the purpose of improved animal health.

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

Phage-assisted continuous evolution of proteases with altered substrate specificity.

Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications.Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities.

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

The biosynthesis of nitrous oxide in the green alga Chlamydomonas reinhardtii.

Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO.

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.

Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1-2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.