Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations.

Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74-77%), followed by HDR (21-24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.

Massively targeted evaluation of therapeutic CRISPR off-targets in cells.

Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context.

A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 "sliding" on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design.

Circle-Seq reveals genomic and disease-specific hallmarks in urinary cell-free extrachromosomal circular DNAs.

BACKGROUND: Extrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine. METHODS: Here, we report the discovery and analysis of urinary cell-free eccDNAs (ucf-eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle-Seq. RESULTS: Millions of unique ucf-eccDNAs were identified and comprehensively characterised. The ucf-eccDNAs are GC-rich. Most ucf-eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf-eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein-coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf-eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf-eccDNA. CONCLUSIONS: This work discovers and provides the first deep characterisation of ucf-eccDNAs and suggests ucf-eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.

Systematical analysis reveals a strong cancer relevance of CREB1-regulated genes.

The transcription factor cyclic-AMP response element-binding protein 1 (CREB1) responds to cAMP level and controls the expression of target genes, which regulates nutrition partitioning. The promoters of CREB1-targeted genes responsive to cAMP have been extensively investigated and characterized with the presence of both cAMP response element and TATA box. Compelling evidence demonstrates that CREB1 also plays an essential role in promoting tumor development. However, only very few genes required for cell survival, proliferation and migration are known to be constitutively regulated by CREB1 in tumors. Their promoters mostly do not harbor any cAMP response element. Thus, it is very likely that CREB1 regulates the expressions of distinct sets of target genes in normal tissues and tumors. The whole gene network constitutively regulated by CREB1 in tumors has remained unrevealed. Here, we employ a systematical and integrative approach to decipher this gene network in the context of both tissue cultured cancer cells and patient samples. We combine transcriptomic, Rank-Rank Hypergeometric Overlap, and Chipseq analysis, to define and characterize CREB1-regulated genes in a multidimensional fashion. A strong cancer relevance of those top-ranked targets, which meet the most stringent criteria, is eventually verified by overall survival analysis of cancer patients. These findings strongly suggest the importance of genes constitutively regulated by CREB1 for their implicative involvement in promoting tumorigenesis.

Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning.

The design of CRISPR gRNAs requires accurate on-target efficiency predictions, which demand high-quality gRNA activity data and efficient modeling. To advance, we here report on the generation of on-target gRNA activity data for 10,592 SpCas9 gRNAs. Integrating these with complementary published data, we train a deep learning model, CRISPRon, on 23,902 gRNAs. Compared to existing tools, CRISPRon exhibits significantly higher prediction performances on four test datasets not overlapping with training data used for the development of these tools. Furthermore, we present an interactive gRNA design webserver based on the CRISPRon standalone software, both available via https://rth.dk/resources/crispr/ . CRISPRon advances CRISPR applications by providing more accurate gRNA efficiency predictions than the existing tools.

Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs.

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.

LION: a simple and rapid method to achieve CRISPR gene editing.

The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.

Isolation and characterization of a bacteriophage and its potential to disrupt multi-drug resistant Pseudomonas aeruginosa biofilms.

A lytic Pseudomonas aeruginosa bacteriophage, vB_PaeM_LS1, was isolated and characterized herein. To examine the eligibility of bacteriophage vB_PaeM_LS1 as a therapeutic bacteriophage, we analysed its genome and compared it to similar bacteriophages. Genome of bacteriophage vB_PaeM_LS1 consisted of a linear, double-stranded DNA molecule 66,095 bp in length and with 55.7% G + C content. Neighbor-joining analysis of the large subunit terminase showed that bacteriophage vB_PaeM_LS1 had similarity to the Pbunavirus genus. The potential of the lytic bacteriophage to disrupt Pseudomonas aeruginosa biofilms was assessed by scanning electron microscopy and bacterial counts. This study revealed that the bacteriophage vB_PaeM_LS1 with its lytic effect showed a high potential impact on the inhibition of the growth of Pseudomonas aeruginosa biofilm formation.

Synbiotic dietary supplement affects growth, immune responses and intestinal microbiota of Apostichopus japonicus.

A feeding experiment was conducted to investigate the effects of dietary administration of synbiotic with Bacillus lincheniformis WS-2 (CGMCC No. 12813) and alginate oligosaccharides (AOS) on the growth, innate immune response, and intestinal microbiota of the sea cucumber Apostichopus japonicus and its resistance to Vibrio infection. Sea cucumbers were given a control diet (non-supplemented), pro diet (basal diet plus 1 × 109 cfu (g diet)-1B. lincheniformis WS-2), syn diet (basal diet plus 1 × 109 cfu (g diet)-1B. lincheniformis WS-2 and 10 g (kg diet) -1 AOS) or pre diet (basal diet plus 10 g (kg diet) -1 AOS) over a period of 60 days, and the growth performance and various innate immune parameters of the animals were evaluated after 30 and 60 days of feeding. No significant difference in growth performance was observed between the group fed with the syn and the group fed with the pro diet, but both these groups exhibited significant (P < 0.05) enhancement in growth performance compared to the control group. At the same time, both syn and pro diets also resulted in the animals having significantly higher levels of amylase, protease and alginate lyase activities compared to the con diet. Individuals fed with the syn or pro diet showed enhanced levels of various immune enzyme activities, compared to those fed with the con diet. At the end of the growth period, the sea cucumbers were challenged with Vibrio splendidus via intraperitoneal injection. The survival rates of sea cucumbers fed with the syn, pro or pre diet were significantly improved compared to that of sea cucumbers fed with the con diet, with sea cucumbers fed with synbiotic having the highest survival. In addition, increased proportions of Bacillus and Lactococcus were found in the intestinal tract of sea cucumbers fed with the syn diet (9.5% and 7.3%) compared to those of sea cucumbers fed with the pro diet (6.1% and 4.6%), con diet (4.0% and 3.4%), or pre diet (5.2% and 6.8%) after 60 days of feeding. Furthermore, the proportion of Vibrio in the intestinal tracts of sea cucumbers fed with the pro diet (2%) or syn diet (3.1%) was lower than that of sea cucumbers fed with the con diet (5.5%) or pre diet (3.8%), although no significant difference was detected between the pro diet and syn diet groups (P > 0.05). Overall, the results suggested that dietary synbiotic consisting of Bacillus lincheniformis and alginate oligosaccharides (AOS) could have positive benefit for sea cucumber aquaculture.

Use of Bacteriophages to Control Escherichia coli O157:H7 in Domestic Ruminants, Meat Products, and Fruits and Vegetables.

Escherichia coli O157:H7 is an important foodborne pathogen that causes severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Ruminant manure is a primary source of E. coli O157:H7 contaminating the environment and food sources. Therefore, effective interventions targeted at reducing the prevalence of fecal excretion of E. coli O157:H7 by cattle and sheep and the elimination of E. coli O157:H7 contamination of meat products as well as fruits and vegetables are required. Bacteriophages offer the prospect of sustainable alternative approaches against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes. This article reviews the use of phages administered orally or rectally to ruminants and by spraying or immersion of fruits and vegetables as an antimicrobial strategy for controlling E. coli O157:H7. The few reports available demonstrate the potential of phage therapy to reduce E. coli O157:H7 carriage in cattle and sheep, and preparation of commercial phage products was recently launched into commercial markets. However, a better ecological understanding of the phage E. coli O157:H7 will improve antimicrobial effectiveness of phages for elimination of E. coli O157:H7 in vivo.

Traditional Herbal Medicine-Derived Sulforaphene LFS-01 Reverses Colitis in Mice by Selectively Altering the Gut Microbiota and Promoting Intestinal Gamma-Delta T Cells.

Sulforaphene (LFS-01) is a natural compound derived from traditional herbal medicine. Here, we show that oral administration of LFS-01 is able to dramatically alter the skewed gut microbiota and reverse colitis in model mice associated with an increase of intestinal γδT cells. Through 16S rDNA sequencing, we showed that LFS-01 can selectively suppress enteric pathogens such as Escherichia-Shigella and Helicobacter whereas the protective strains including Lactobacillus and Lachnospiraceae were significantly expanded after LFS-01 treatment. Interestingly, we demonstrated that LFS-01 administration can significantly promote the IL-17+γδT cells in model mice in response to the expanded Lactobacillus. We verified that the intracellular components of Lactobacillus can stimulate the growth of IL-17+γδT cells upon preincubation. The increased IL-17A after LFS-01 treatment in turn recovers the disrupted occludin subcellular location and protects the epithelial barrier in the colon of model mice. Remarkably, LFS-01 does not show apparent toxicity to animals and we demonstrated that LFS-01 also exerts strong protective effects in TNBS-induced colitis rats. Therefore, LFS-01 holds great promise for the treatment of inflammatory bowel disease (IBD) and warrants translation for use in clinical trials. Our work provided a new avenue for the treatment of IBD based on the strategy of harnessing intestinal symbiosis.