Potential Off-target Sites Research Articles

Complementary Strategies for Installation of Thioimidates into Peptide Backbones.

Thioimidates are a precursor and synthetic branch point to access either thioamide or amidine isosteres of the native amide (peptide bond). Previous syntheses of thioimidate-containing peptides were prone to side reactivity and required slow, cumbersome steps that were difficult to monitor. We describe a more efficient approach to directly couple thioimidates onto the growing peptide chain. This work also outlines optimal conditions for thioimidate formation on solid support and identifies potential off-target sites for alkylation that impact the choice of protecting group.

Impact of predictive selection of LbCas12a CRISPR RNAs upon on- and off-target editing rates in soybean.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized creating targeted genetic variation in crops. Although CRISPR enzymes have been reported to have high sequence-specificity, careful design of the editing reagents can also reduce unintended edits at highly homologous sites. This work details the first large-scale study of the heritability of on-target edits and the rate of edits at off-target sites in soybean (Glycine max), assaying ~700 T1 plants each resulting from transformation with LbCas12a constructs containing CRISPR RNAs (crRNAs) predicted to be either "unique" with no off-target sites or "promiscuous" with >10 potential off-targets in the soybean genome. Around 80% of the on-target edits observed in T0 plants were inherited in the T1 generation, and ~49% of the total observed on-target edits in T1 were not observed at T0, indicating continued activity of LbCas12a throughout the life cycle of the plant. In planta editing at off-target sites was observed for the Promiscuous but not the Unique crRNA. Examination of the edited off-target sites revealed that LbCas12a was highly tolerant to mismatches between the crRNA and target site in bases 21-23 relative to the start of the protospacer, but even a single mismatch in the first 20 nt drastically reduced the editing rate. In addition, edits at off-target sites have lower inheritance rates than on-target edits, suggesting that they occur later in the plant's lifecycle. Plants with a desired on-target edit and no off-target edits could be identified in the T1 generation for 100% of the T0 plants edited with the Unique crRNA compared with the 65% of T0 plants edited with the Promiscuous crRNA. This confirms that proper crRNA selection can reduce or eliminate off-target editing. Even when potential off-target sites are predicted, plants containing only the intended edits can still be identified and propagated.

A systematic method for solving data imbalance in CRISPR off-target prediction tasks

Accurately identifying potential off-target sites in the CRISPR/Cas9 system is crucial for improving the efficiency and safety of editing. However, the imbalance of available off-target datasets has posed a major obstacle in enhancing prediction performance. Despite several prediction models have been developed to address this issue, there remains a lack of systematic research on handling data imbalance in off-target prediction. This article systematically investigates the data imbalance issue in off-target datasets and explores numerous methods to process data imbalance from a novel perspective. First, we highlight the impact of the imbalance problem on off-target prediction tasks by determining the imbalance ratios present in these datasets. Then, we provide a comprehensive review of various sampling techniques and cost-sensitive methods to mitigate class imbalance in off-target datasets. Finally, systematic experiments are conducted on several state-of-the-art prediction models to illustrate the impact of applying data imbalance solutions. The results show that class imbalance processing methods significantly improve the off-target prediction capabilities of the models across multiple testing datasets. The code and datasets used in this study are available at https://github.com/gzrgzx/CRISPR_Data_Imbalance.

Levosimendan's Effects on Length-Dependent Activation in Murine Fast-Twitch Skeletal Muscle.

Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently determined. Despite controversial results, levosimendan is still expected to interact with skeletal muscle through off-target sites (further than troponin C). Adding to this debate, we investigated levosimendan's acute impact on fast-twitch skeletal muscle biomechanics in a length-dependent activation study by submersing single muscle fibres in a levosimendan-supplemented solution. We employed our MyoRobot technology to investigate the calcium sensitivity of skinned single muscle fibres alongside their stress-strain response in the presence or absence of levosimendan (100 µM). While control data are in agreement with the theory of length-dependent activation, levosimendan appears to shift the onset of the 'descending limb' of active force generation to longer sarcomere lengths without notably improving myofibrillar calcium sensitivity. Passive stretches in the presence of levosimendan yielded over twice the amount of enlarged restoration stress and Young's modulus in comparison to control single fibres. Both effects have not been described before and may point towards potential off-target sites of levosimendan.

A bioinformatic analysis of gene editing off-target loci altered by common polymorphisms, using ‘PopOff’

ABSTRACT Gene editing therapies are designed to minimise off-target editing. However, it is not widespread practice for common polymorphisms to be considered when identifying potential off-target sites in silico. Nevertheless, genetic variants should be included as they have the potential to alter existing, or to generate new, off-target sites. To facilitate the consideration of common polymorphisms when designing targeted gene therapies we developed PopOff, a web-based tool that integrates minor allele frequencies from the gnomAD variant database into an off-target analysis. We used PopOff to analyse predicted off-target loci from guide RNAs used in four clinical trials and thirty-four research publications. From an analysis of sixty guides, we identified that approximately 20% of off-target loci overlap with a common polymorphism. Of these sites, 6.93% contained variants that reduce the level of mismatch between the off-target locus and guide, and therefore may increase off-target cleavage. In addition, we identified that 0.34% of common polymorphisms generated novel PAM sites, resulting in off-target loci that standard workflows would miss. Our findings demonstrate that common polymorphisms should be considered when designing guides to maximise the safety of CRISPR-based gene therapies. However, this may be problematic in populations where the breadth of genetic diversity remains uncharacterised.

Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Advancing genome editing with artificial intelligence: opportunities, challenges, and future directions.

Clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing (GED) technologies have unlocked exciting possibilities for understanding genes and improving medical treatments. On the other hand, Artificial intelligence (AI) helps genome editing achieve more precision, efficiency, and affordability in tackling various diseases, like Sickle cell anemia or Thalassemia. AI models have been in use for designing guide RNAs (gRNAs) for CRISPR-Cas systems. Tools like DeepCRISPR, CRISTA, and DeepHF have the capability to predict optimal guide RNAs (gRNAs) for a specified target sequence. These predictions take into account multiple factors, including genomic context, Cas protein type, desired mutation type, on-target/off-target scores, potential off-target sites, and the potential impacts of genome editing on gene function and cell phenotype. These models aid in optimizing different genome editing technologies, such as base, prime, and epigenome editing, which are advanced techniques to introduce precise and programmable changes to DNA sequences without relying on the homology-directed repair pathway or donor DNA templates. Furthermore, AI, in collaboration with genome editing and precision medicine, enables personalized treatments based on genetic profiles. AI analyzes patients' genomic data to identify mutations, variations, and biomarkers associated with different diseases like Cancer, Diabetes, Alzheimer's, etc. However, several challenges persist, including high costs, off-target editing, suitable delivery methods for CRISPR cargoes, improving editing efficiency, and ensuring safety in clinical applications. This review explores AI's contribution to improving CRISPR-based genome editing technologies and addresses existing challenges. It also discusses potential areas for future research in AI-driven CRISPR-based genome editing technologies. The integration of AI and genome editing opens up new possibilities for genetics, biomedicine, and healthcare, with significant implications for human health.

Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro

BackgroundHemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. MethodsInitially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. ResultsWe identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. ConclusionsKnock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.

Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.

A Universal CRISPR/Cas9n-Mediated Genome Editing Approach for ELANE-Related Severe Congenital Neutropenia

Severe congenital neutropenia (CN) is an inherited bone marrow failure syndrome characterized by impaired maturation of neutrophil granulocytes. Due to the lack or very low levels of neutrophils in the peripheral blood, patients experience severe, life-threatening bacterial infections as early as birth. Autosomal-dominant ELANE mutations are the most common cause of CN. Although most patients respond to daily treatment with recombinant human granulocyte colony-stimulating factor, about 15% of patients do not respond to this cytokine, and approximately 20% of patients develop myelodysplasia or acute myeloid leukemia. The only curative treatment available thus far is allogenic hematopoietic stem cell transplantation, which is associated with serious side effects. Autologous transplantation of gene-edited patients' HSPCs offers a novel curative therapy. Considering the wide range of mutations distributed throughout the ELANE gene, A universal, high-safety-profile approach could be advantageous for all ELANE-CN patients. Our group has already published a universal CRISPR/Cas9-mediated knockout approach for the ELANE gene; however, targeting the coding sequence region (CDS) of the ELANE gene could lead to the introduction of new disease-causing variants, so it may be harmful for patients. In order to address this concern, we reformatted our previously published knockout concept into a clinical-grade gene therapy strategy. We have screened cis-regulatory regions in the ELANE core promoter and, using CRISPR/Cas9n, introduced two nicks on opposing strands of the Goldberg-Hognes box region (TATA-box) to make the transcription starting process inefficient, as transcription factors and RNApol II cannot recognize the modified sequence. Theproposed strategy targets the non-coding region of the ELANE gene and thus does not generate new unwanted variants. Also, by replacing Cas9 with Cas9-ncikase, the off-target activity should decrease by up to 1000-fold. We have observed a markedly elevated neutrophil differentiation in gene-edited ELANE-CN patients HSPCs (n = 2), as assessed by the percentage of CD45 +CD11b +CD15 +, CD45 +CD15 +CD16 +, and CD45 +CD16 +CD66b + myeloid/granulocytic cells, compared to the mock electroporated ELANE-CN group, while no disruption of granulopoiesis in healthy donor HSPCs (n = 2) was detected. Results validated by the CFU assay. To have nucleotide-level resolution of on-target events, we performed targeted next-generation sequencing of edited loci on gene-edited healthy donors and ELANE-CN HSPCs. Next-generation sequencing results showed >90% on-target efficiency. CRISPR/Cas9n-mediated targeting of the TATA box rescued defective granulopoiesis in vivo, as gene-edited ELANE-CN HSPCs (n = 3) were transplanted in NSG mice and assessed by the percentage of neutrophils (hCD19 -hCD3 -hCD66b Int/lowhCD33 +hCD16 high) after 16 weeks. We have also observed efficient engraftment and preserved the multilineage potential of gene edited HSPCs in immunodeficient NSG mice. The strategy also depicted a safe profile upon applying gene editing to healthy donor CD34 + HSPCs and performing GUIDE-seq and CAST-seq. GUIDE-seq highlighted two potential off-target sites after >=6 mismatches, while no chromosomal translocations were detected by CAST-seq. RNA-sequencing of in vitro-generated neutrophils from gene-edited HSPCs of healthy donors (n = 2) confirmed a >9-fold reduction of ELANE gene expression level. Important to note, none of the other serine proteases or neighboring genes were down-regulated. Taken together, ex vivo CRISPR-Cas9n-based gene editing of ELANE's gene promoter in the setting of autologous stem cell transplantation could be a safe therapeutic approach for all ELANE-CN patients.

Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato.

CRISPR/Cas9-mediated genome editing technology has been widely used for the study of gene function in crops, but the differences between species have led to widely varying genome editing efficiencies. The present study utilized a potato hairy root genetic transformation system and incorporated a rapid assay with GFP as a screening marker. The results clearly demonstrated that salt and osmotic stress induced by NaCl (10 to 50 mM) and mannitol (50 to 200 mM) treatments significantly increased the positive rates of genetic transformation mediated by A. rhizogenes and the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato. However, it was observed that the regeneration of potato roots was partially inhibited as a result. The analysis of CRISPR/Cas9-mediated mutation types revealed that chimeras accounted for the largest proportion, ranging from 62.50% to 100%. Moreover, the application of salt and osmotic stress resulted in an increased probability of null mutations in potato. Notably, the highest rate of null mutations, reaching 37.5%, was observed at a NaCl concentration of 10 mM. Three potential off-target sites were sequenced and no off-targeting was found. In conclusion, the application of appropriate salt and osmotic stress significantly improved the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato, with no observed off-target effects. However, there was a trade-off as the regeneration of potato roots was partially inhibited. Overall, these findings present a new and convenient approach to enhance the genome editing efficiency of the CRISPR/Cas9-mediated gene editing system in potato.

Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum)

Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.

Design and off-target prediction for antisense oligomers targeting bacterial mRNAs with the MASON web server.

Antisense oligomers (ASOs), such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (make antisense oligomers now), a web server for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON's off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10-mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq data set from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed that our findings are not unique to Salmonella We believe that MASON's off-target assessment will improve the design of specific PNAs and other ASOs.

CRISPR/Cas9 editing of the polygalacturonase FaPG1 gene improves strawberry fruit firmness.

Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Ten independent lines, cv. "Chandler", were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.

Prime editing: A potential treatment option for β-thalassemia.

The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.

Preliminary Safety and Efficacy Results of EDI001: An Investigator Initiated Trial on CRISPR/Cas9-Modified Autologous CD34+ Hematopoietic Stem and Progenitor Cells for Patients with Transfusion Dependent β-Thalassemia

Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1). Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints. Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation. One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion. The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B). The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively. Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unbiased prediction of off-target sites in genome-edited rice using SITE-Seq analysis on a web-based platform.

Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.