Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

First report of prevalence and assemblage analysis of Giardia duodenalis in pigs from Guangxi Zhuang Autonomous Region, southern China.

Giardia duodenalis is a common intestinal protozoan that can cause diarrhea and intestinal disease in animals and in humans. However, the prevalence and assemblages of G. duodenalis in pigs from Guangxi Zhuang Autonomous Region have not been reported. In this study, a total of 724 fecal samples (201 from nursery pigs, 183 from piglets, 175 from breeding pigs, and 165 from fattening pigs) were obtained in four areas of the region (Nanning, Yulin, Hezhou, and Guigang). The gene of the small subunit ribosomal RNA (SSU rRNA) of G. duodenalis was amplified by nested PCR. The results show that the prevalence of G. duodenalis in pigs was 3.59% (26/724), of which 14 samples belonged to assemblage A (53.85%) and 12 samples belonged to assemblage E (46.15%). The infection rates of G. duodenalis in Hezhou, Yulin, Nanning, and Guigang were 0%, 0.7%, 10.8% and 1.1%, respectively (χ2 = 45.616, p < 0.01); whereas 5.1% of breeding pigs, 6.0% of piglets, 2.4% of fattening pigs, and 1.0% of nursery pigs were infected with G. duodenalis (χ2 = 8.874, p < 0.05). The SSU rRNA-positive samples were amplified by PCR based on the ß-giardin (bg), glutamate dehydrogenase (gdh), and triphosphate isomerase (tpi) genes. Ten, eight and seven positive samples were detected, respectively. Based on phylogenetic analysis of the three genetic loci sequences, a multilocus genotyping A1 was found. The findings of this study provide basic data for the development of prevention and control of G. duodenalis infections in pigs and humans in the Guangxi Zhuang Autonomous Region.

Title: Premier rapport sur la prévalence et l'analyse des assemblages de Giardia duodenalis chez les porcs de la région autonome Zhuang du Guangxi, dans le sud de la Chine. Abstract: Giardia duodenalis est un protozoaire intestinal commun qui peut provoquer des diarrhées et des maladies intestinales chez les animaux et les humains. Cependant, la prévalence et les assemblages de G. duodenalis chez les porcs de la région autonome Zhuang du Guangxi n'ont pas été rapportés. Dans cette étude, un total de 724 échantillons fécaux (201 provenant de jeunes porcelets, 183 de porcelets, 175 de porcs reproducteurs et 165 de porcs à l'engrais) ont été obtenus dans quatre zones de la région (Nanning, Yulin, Hezhou et Guigang). Le gène de la petite sous-unité de l'ARN ribosomal (ARNr SSU) de G. duodenalis a été amplifié par PCR nichée. Les résultats ont montré que la prévalence de G. duodenalis chez les porcs était de 3,59 % (26/724), dont 14 échantillons appartenaient à l'assemblages A (53,85 %) et 12 échantillons à l'assemblage E (46,15 %). Les taux d'infection par G. duodenalis à Hezhou, Yulin, Nanning et Guigang étaient respectivement de 0, 0,7 %, 10,8 % et 1,1 % (χ2 = 45,616, p < 0,01), alors que 5,1 % des porcs reproducteurs, 6,0 % des porcelets, 2,4 % de porcs à l'engrais et 1,0 % des jeunes porcelets étaient infectés par G. duodenalis (χ2 = 8,874, p < 0,05). Les échantillons positifs pour l'ARNr SSU ont été amplifiés par PCR basée sur les gènes de la ß-giardine (bg), de la glutamate déshydrogénase (gdh) et de la triphosphate isomérase (tpi), et dix, huit et sept échantillons positifs ont été détectés, respectivement. Sur la base de l'analyse phylogénétique des trois séquences de loci génétiques, un génotypage multilocus A1 a été trouvé. Les résultats de cette étude fournissent des données de base pour le développement de la prévention et du contrôle des infections à G. duodenalis chez les porcs et les humains dans la région autonome Zhuang du Guangxi.

First Molecular Detection and Genotype Identification of Toxoplasma gondii in Chickens from Farmers' Markets in Fujian Province, Southeastern China.

Toxoplasma gondii is an opportunistic pathogenic protozoan that can infect all nucleated cells in almost all warm-blooded animals, including humans. T. gondii infection has been reported in many food animals worldwide. However, the prevalence and genotypes of T. gondii in chickens from farmers' markets in Fujian province in southeastern China remain unreported. In the present study, four tissue samples from each of the 577 chickens (namely, the heart, liver, lungs, and muscles) were collected from farmers' markets in five regions of Fujian province (Zhangzhou, Sanming, Quanzhou, Fuzhou, and Longyan). We first analyzed the prevalence and genotypes of T. gondii using PCR targeting of the B1 gene of T. gondii. Of the 577 chickens, thirty-two (5.5%) tested positive for the B1 gene. Among the five regions, Sanming had the highest infection rate (16.8%, 16/95), followed by Quanzhou (8.0%, 8/100), Longyan (5.0%, 5/100), Zhangzhou (1.1%, 2/182), and Fuzhou (1.0%, 1/100). Among these thirty-two T. gondii-positive chickens, the infection rates of the lungs, heart, liver, and muscles were 68.8% (22/32), 34.4% (11/32), 28.1% (9/32), and 9.4% (3/32), respectively. Significant differences in prevalence were found among the different regions (χ2 = 35.164, p < 0.05) and tissues (χ2 = 25.874, p < 0.05). A total of 128 tissue and organ samples of the thirty-two T. gondii-positive chickens from the different regions were analyzed using PCR-restriction fragment length polymorphism (PCR-RFLP) on the basis of 10 genetic markers. Seven tissue samples (lung samples from five chickens, heart samples from one chicken, and liver samples from one chicken) underwent successful amplification at all the genetic markers, and all the T. gondii genotypes were identified as genotype I (ToxoDB #10). These findings serve as a foundation for evaluating the risk of T. gondii contamination in chicken products intended for human consumption and offer insight into preventing the transmission of the parasite from chickens to humans.

Detection of Rickettsia spp. and Anaplasma ovis in Melophagus ovinus from southern Xinjiang, China.

Melophagus ovinus is a hematophagous insect that is distributed worldwide and plays a crucial role in transmitting disease-causing pathogens. From June 2021 to March 2022, a total of 370 M. ovinus were collected from 11 sampling points in southern Xinjiang, China. The specimens were identified using morphological and molecular analyses. Rickettsia spp. and Anaplasma ovis were detected from all the samples using seven Rickettsia-specific genetic markers and the msp-4 gene of A. ovis. Approximately 11% of the M. ovinus specimens were positive for Rickettsia spp., and Candidatus Rickettsia barbariae was the most predominant species (35/41; 85.4%), while R. massiliae was least prevalent (6/41; 14.6%). Approximately 10.5% (39/370) of the M. ovinus specimens were positive for A. ovis of genotype III, which was co-detected with Candidatus R. barbariae in M. ovinus (3/370; 0.8%). To the best of our knowledge, this is the first report of the detection of R. massiliae and Candidatus R. barbariae in M. ovinus globally. The detection and control of insect-borne diseases originating from M. ovinus should be strengthened in southern Xinjiang, an area important to animal husbandry and production.

GREPore-seq: A robust workflow to detect changes after gene editing through long-range PCR and nanopore sequencing.

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Modulation of Immune Reaction in Hydrodynamic Gene Therapy for Hemophilia A.

Hemophilia A (HA) is a monogenic disease characterized by plasma clotting factor 8 (F8) deficiency due to F8 mutation. We have been attempting to cure HA permanently using a CRISPR-Cas9 gene-editing strategy. In this study, we induced targeted integration of BDDF8 (B-domain-deleted F8) gene into the albumin locus of HA mice by hydrodynamic tail vein injection of editing plasmid vectors. One week after treatment, a high F8 activity ranging from 70% to 280% of normal serum levels was observed in all treated HA mice but dropped to background levels 3-5 weeks later. We found that the humoral immune reaction targeting F8 is the predominant cause of the decreased F8 activity. We hypothesized that hydrodynamic injection-induced liver damage triggered the release of large quantities of inflammatory cytokines. However, coinjection of plasmids expressing a dozen immunomodulatory factors failed to curtail the immune reaction and stabilize F8 activity effectively. The spCas9 plasmid carrying a miR-142-3p target sequence alleviated the cellular immune response but could not deliver therapeutic efficacy. Strikingly, immunosuppressant cyclophosphamide virtually abolished the immune response, leading to a year-long stable F8 level. Our findings should have important implications in developing therapies in mouse models using the hydrodynamic gene delivery approach, highlighting the necessity of modulating the innate immune response triggered by liver damage.

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.