Editing Platform Research Articles

Cas-Clover Editing Efficiency and Off-Target Activity in Human Hepatocytes at the KLKB1 Locus

Cutting-edge gene editing holds enormous promise for tackling devastating genetic diseases like hereditary angioedema (HAE). Here, we describe the efficient inactivation of the gene encoding pre-kallikrein, KLKB1, using our proprietary Cas-CLOVER™ high-fidelity nuclease with our non-viral, lipid nanoparticle (LNP) delivery system. Genetic inactivation of KLKB1 is an alternative clinical approach that provides durable relief to both Type I and II HAE. HAE is a rare genetic disease characterized by subcutaneous and submucosal edema, with swelling of the upper respiratory tract posing a life-threatening situation. Type I and II HAE are the most common types and are caused by mutations in the SERPING1 gene, which leads to compromised production or function of the C1 protease inhibitor. Strategies for treatment and prophylaxis include the restoration of C1 inhibitor function, or downstream antagonism of active plasma kallikrein. Safe and effective gene editing of KLKB1 could be a viable alternative for patients not adequately responding to the current standard of care. However, gene editing approaches must demonstrate an exquisitely high level of fidelity for optimal safety. To demonstrate such an approach with Cas-CLOVER, multiple guide RNAs (gRNA) targeting the human KLKB1 gene were screened in human hepatoma cell lines to identify gRNA pairs with optimal editing. Next, we evaluated KLKB1 protein reduction in primary human hepatocytes (PHH) that were incubated with LNPs encapsulating Cas-CLOVER mRNA along with each gRNA pair. Lead candidate gRNAs showed robust KLKB1 editing in a dose-responsive manner, achieving >65% editing and >85% reduction in KLKB1 protein secreted into culture medium at 0.5 ug/mL (EC90). To evaluate Cas-CLOVER off-target activity, oligo incorporation by iGUIDE was carried out by a licensed contract research organization. In this assay, double-stranded oligodeoxyribonucleotides (dsODNs) were co-electroporated with Cas-CLOVER mRNA, along with our lead KLKB1 gRNA pair, in the Huh7 cell line, and candidate off-target sites were identified by Illumina next-generation sequencing. Off-target activity was assessed by amplicon-seq at the eight top sites nominated by iGUIDE. In PHHs treated with 0.5 ug/mL of Cas-CLOVER LNPs, off-target editing was detected in 3/8 sites at very low levels (<0.25%). Remarkably, this low level of off-target editing remained unchanged when PHHs were treated with 10-fold higher concentrations of Cas-CLOVER LNP. For further evaluation of our platform, we sought to determine KLKB1 editing efficiency and fidelity in a mouse model of liver humanization. TK-Nog mice engrafted with PHHs were treated with a single intravenous injection of an LNP formulation co-encapsulating Cas-CLOVER mRNA and our lead KLKB1 gRNA pair. Amplicon-seq analysis demonstrated that 60% of KLKB1 alleles in the liver were edited. Importantly, no off-target editing was detected among the top eight sites identified by iGUIDE, including the three off-target sites validated in cultured PHHs. Next, we evaluated efficacy and tissue specificity of our platform in wild type mice. C57BL/6 male and female mice were dosed with LNP encapsulating Cas-CLOVER mRNA and mouse Klkb1-targeting gRNAs. A single intravenous LNP injection achieved high Klkb1 editing (>50% of haploid genomes) in the liver and >80% reduction in serum pre-kallikrein levels. No Klkb1 editing was detected in gonads. In summary, these results highlight the efficacy and specificity of our high-fidelity Cas-CLOVER gene editing platform that enables targeted and therapeutically relevant kallikrein reduction in a fully non-viral manner. These data provide a promising foundation for the development of a highly specific gene editing therapy for HAE.

Multiplex Prime Editing and PASSIGE TM for Non-Viral Generation of an Allogeneic CAR-T Cell Product

While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system. Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells. Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product. To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality. CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 + tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 + tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.

Hepatocyte-confined CRISPR/Cas9-based nanocleaver precisely eliminates viral DNA for efficient and safe treatment of hepatitis B virus infection

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9)-based therapies are attractive to achieve precise and targeted disruption of the hepatitis B virus (HBV) genome for HBV infection treatment. However, unspecific in vivo distribution of the CRISPR/Cas9 vector after systemic administration not only impedes CRISPR/Cas9 expression in the liver, but also causes undesired gene editing in off-site organs, decreasing the therapeutic efficacy and leading to potential side effects. Herein, we designed a hepatocyte-confined CRISPR/Cas9-based nanocleaver (HepCCCleaver) to realize precise and efficient cleavage of HBV DNA, specifically in hepatocytes, for safe and effective HBV treatment. This HepCCCleaver was formed by encapsulating the hepatocyte-specific CRISPR/Cas9 plasmid into a galactose-modified membrane-fusogenic carrier. Our galactose-modified membrane-fusogenic carrier allowed the HepCCCleaver to specifically accumulate in the liver and enter hepatocytes through membrane fusion, bypassing lysosome entrapment to avoid degradation for superior delivery efficiency. The hepatocyte-specific CRISPR/Cas9 plasmid released in the cytoplasm could switch on the gene expression only in hepatocytes, ensuring precise cleavage on viral DNA in HBV-infected but not other types of cells. By integrating the liver-targeting membrane-fusogenic delivery system with hepatocyte-specific CRISPR/Cas9 plasmid, this HepCCCleaver could precisely and efficiently confine over 95% events of viral DNA gene editing in hepatocytes. This HepCCCleaver is a universal gene editing platform enabling safe and efficient disease treatment, specifically in the cell type of interest.

Usefulness of current sgRNA design guidelines and in vitro cleavage assays for plant CRISPR/Cas genome editing: a case targeting the polyphenol oxidase gene family in eggplant (Solanum melongena L.).

The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.

Tilapia, a good model for studying reproductive endocrinology

The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2, cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1, cyp11a1, cyp17a2, cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a/esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy, dmrt1, gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish.

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3×1011 vg donor template and 1×1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1×1012 vg SP-B-donor template and 5×1011 vg nuclease significantly extended median survival (p= 0.0034) from 5days in the untreated off doxycycline group to 16days in the donor AAV and nuclease group, with one gene-edited mouse living 243days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

Engineering Nucleotidoproteins for Base‐Pairing‐Assisted Cytosolic Delivery and Genome Editing

AbstractProtein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super‐negatively charged, nucleotide‐enriched structure of nucleic acids, adenylated pro‐proteins (A‐proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine‐modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A‐proteins to form salt‐resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A‐proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR‐Cas9 ribonucleoproteins targeting fusion oncogene EWSR1‐FLI1, leading to pronounced anti‐tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing.

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

Site-Specific m6 A Erasing via Conditionally Stabilized CRISPR-Cas13b Editor.

N6-methyladenosine (m6 A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m6 A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m6 A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m6 A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m6 A eraser fusion protein and site-specific m6 A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m6 A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m6 A function with minimal steric interference from bulky dCas13b fusion protein.

Site‐Specific m6A Erasing via Conditionally Stabilized CRISPR‐Cas13b Editor

AbstractN6‐methyladenosine (m6A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m6A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m6A editing platform (FKBP*‐dCas13b‐ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m6A editing system was achieved by adding or removing the Shield‐1 molecule. We further demonstrated that the targeted recruitment of dCas13b‐m6A eraser fusion protein and site‐specific m6A erasing were achieved under the control of Shield‐1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m6A on the target RNAs after Shield‐1 removal, which provides an ideal opportunity to study the m6A function with minimal steric interference from bulky dCas13b fusion protein.

Analisa Proses Editing Video Berita Menggunakan Software Edius Dan Adobe Premiere Pada Pasca Produksi Untuk Program Televisi Di PT. Indrasakti Media Televisi

The analysis of this study is to provide a good overview of the advantages and disadvantages of the two video editing platforms, namely Edius and Adobe Premiere, in the context of the news video editing process in post-production. The analysis also highlights that the decision to choose between the two comes down to user preference, whether they prefer a broad feature set or a more seamless editing experience. The last comments have provided clear recommendations to users with different needs. Users who need a lot of features tend to prefer Adobe Premiere, while those who prefer agility and fluency in the editing process may prefer Edius.

Towards a Quality Assurance and Quality Control Mechanism for Species List Building

Catalogue of Life (COL) brings together the efforts and contributions of taxonomists from around the world, and addresses the needs of researchers, policy-makers, environmental managers and the wider public for a consistent, up-to-date and authoritative listing of all the world’s known species. The names that science gives to species are fundamental tools that allow us to refer to these units of biodiversity. Knowing the name for a species unlocks everything that has been learned about its biology, distribution and relevance to humans. Every day, taxonomists continue to publish new scientific names and refine our understanding of the world’s species. Over the past 10 years, game-changing progress has been made with the Catalogue of Life and its authoritative taxonomic name indexing services. Major steps were made towards content completion (over 2.1 million species out of all described 2.4 million are now documented and validated through COL (Bánki et al. 2023). The COL data infrastructure and services were rebuilt in collaboration with the Global Biodiversity Information Facility (GBIF). This resulted in the GBIF-COL ChecklistBank, an open global repository for publishing and sharing taxonomic and nomenclatural datasets (> 48,000 datasets, including over 160 Global Species Databases). The data managing/editing process underlying the integration of data into a single checklist (index) could be more transparent with clearly documented protocols and procedures, and is today far too time consuming. In the ChecklistBank infrastructure, there are thousands of data sources at our disposal and we need a powerful, semi-automated, community-expertise based, quality assurance process to warrant the best possible product for users of taxonomic checklists. COL aspires to document, with as much detail as possible, the current quality control and quality assurance processes as they relate to activities that occur at the following levels: 1) data providers, 2) pipeline and data conversions, 3) ChecklistBank infrastructure, and 4) checklist output. Ideally, this should happen throughout the consortium of partners that help the taxonomic community with mobilising and editing authoritative taxonomic data, including initiatives like Atlas of Living Australia, Catalogue of Life China, EDIT Platform for Cybertaxonomy, Integrated Taxonomic Information System, Freshwater Animal Diversity Assessment, TaxonWorks / Species Files, World Flora Online / Rhakhis, World Register of Marine Species / LifeWatch Aphia. In recent years more attention has been given to the principles of creating an authoritative list of the world's species (Garnett et al. 2020). COL has developed and adopted new criteria for the inclusion of taxonomic data sources in the COL Checklist (Hobern et al. 2021). And ChecklistBank, certainly provides more avenues for (automated) quality checks and procedures. But to guarantee the highest confidence in data, we are in need of a transparent and consistent data quality assurance and data quality control mechanism from the creation of taxonomic data, to data publishing, to the delivery into taxonomic data products and the global list of known species. We will present the current status of quality control and quality assurance processes in Catalogue of Life, and discuss what should constitute a quality control and quality assurance mechanism for species list building.

A Strategy for Building the WFO Plant List

A Strategy for Building the WFO Plant List

Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy.

CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO2 ) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.

Bioorthogonal Reaction‐Mediated Tumor‐Selective Delivery of CRISPR/Cas9 System for Dual‐Targeted Cancer Immunotherapy

AbstractCRISPR system‐assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor‐targeting delivery strategy based on bioorthogonal reactions for dual‐targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS‐STING pathway was achieved simultaneously through tumor microenvironment (TME)‐biodegradable hollow manganese dioxide (H‐MnO2) nano‐platform. Subsequently, CRISPR/Cas9 system‐loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.

Ionizable Lipid Nanoparticles for Therapeutic Base Editing of Congenital Brain Disease.

Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.

Interrogation of human microglial phagocytosis by CRISPR genome editing.

Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aβ and glioblastoma phagocytosis assays. Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

Auto-generation of paper patterns for children’s jeans

This study aimed to address these limitations by employing a systematic approach. Initially, body measurements of 115 children were meticulously recorded and subjected to statistical analysis. Subsequently, a children’s jeans paper pattern was chosen, and a predictive model for pattern sizing was developed using BP neural networks. Additionally, a parametric mathematical model was constructed by integrating the design principles of the paper pattern. Using the Visual-LISP programming language on the Auto-CAD 2020 edition platform, an editing process was performed to create a children’s jeans automatic drawing program. The predicted pattern size data for children’s jeans were then imported into the DCL dialog box of Auto-CAD, enabling the automatic generation of paper patterns. To evaluate the effectiveness of the approach, the generated garment paper patterns were imported into the CLO-3D virtual fitting software.

Efficient and versatile multiplex prime editing in hexaploid wheat

Prime editing is limited by low efficiency in plants. Here, we develop an upgraded engineered plant prime editor in hexaploid wheat, ePPEplus, by introducing a V223A substitution into reverse transcriptase in the ePPEmax* architecture. ePPEplus enhances the efficiency by an average 33.0-fold and 6.4-fold compared to the original PPE and ePPE, respectively. Importantly, a robust multiplex prime editing platform is established for simultaneous editing of four to ten genes in protoplasts and up to eight genes in regenerated wheat plants at frequencies up to 74.5%, thus expanding the applicability of prime editors for stacking of multiple agronomic traits.