Editing Efficiency Research Articles

Targeting glutamine synthetase with AS1411-modified exosome-liposome hybrid nanoparticles for inhibition of choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of visual impairment in wet age-related macular degeneration (wAMD). Recent investigations have validated the potential of reducing glutamine synthetase (GS) to inhibit neovascularization formation, offering prospects for treating various neovascularization-related diseases. In this study, we devised a CRISPR/Cas9 delivery system employing the nucleic acid aptamer AS1411 as a targeting moiety and exosome-liposome hybrid nanoparticles as carriers (CAELN). Exploiting the binding affinity between AS1411 and nucleolin on endothelial cell surfaces, the delivery system was engineered to specifically target the glutamine synthetase gene (GLUL), thereby attenuating GS levels and continuously suppressing CNV. CAELN exhibited spherical and uniform dispersion. In vitro cellular investigations demonstrated gene editing efficiencies of CAELN ranging from 42.05 to 55.02% and its capacity to inhibit neovascularization in HUVEC cells. Moreover, in vivo pharmacodynamic studies conducted in CNV rabbits revealed efficacy of CAELN in restoring the thickness of intra- and extranuclear tissues. The findings suggest that GS is a novel target for the inhibition of pathological CNV, while the development of AS1411-modified exosome-liposome hybrid nanoparticles represents a novel delivery method for the treatment of neovascular-related diseases.

CRISPRepi: a multi-omic atlas for CRISPR-based epigenome editing.

CRISPR-based epigenome editing integrates the precision of CRISPR with the capability of epigenetic mark rewriting, offering a tunable and reversible gene regulation strategy without altering the DNA sequences. Various epigenome editing systems have been developed and applied in different organisms and cell types; however, the detailed information is discrete, making it challenging to evaluate the precision of different editing systems and design the optimal sgRNAs for further functional studies. Herein, we developed CRISPRepi (http://crisprepi.maolab.org/ or http://crisprepi.lilab-pkuhsc.org/), a pioneering platform that consolidates extensive sequencing data from 671 meticulously curated RNA-seq, ChIP-seq, Bisulfite-seq and ATAC-seq datasets in 87 cell types manipulated by 74 epigenome editing systems. In total, we have curated 5962 sgRNAs associated with 283 target genes from 2277 samples across six species. CRISPRepi incorporates tools for analyzing editing outcomes and assessing off-target effects by analyzing gene expression changes pre- and post-editing, along with the details of multi-omic epigenetic landscapes. Moreover, CRISPRepi supports the investigation of editing potentials for newly designed sgRNA sequences in a cell/tissue-specific context. By providing a user-friendly interface for searching and selecting optimal editing designs across multiple organisms, CRISPRepi serves as an integrated resource for researchers to evaluate editing efficiency and off-target effects among diverse CRISPR-based epigenome editing systems.

Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications.

Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis

Background Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.ResultsHighly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.ConclusionsHere, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient’s dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient’s fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient’s skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Reducing competition between msd and genomic DNA improves retron editing efficiency.

Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. Here, we identify the msd gene in Escherichia coli, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited, as a critical bottleneck. Sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves very high retron editing efficiency, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis.

Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.

Cytosine base editors with increased PAM and deaminase motif flexibility for gene editing in zebrafish

Cytosine base editing is a powerful tool for making precise single nucleotide changes in cells and model organisms like zebrafish, which are valuable for studying human diseases. However, current base editors struggle to edit cytosines in certain DNA contexts, particularly those with GC and CC pairs, limiting their use in modelling disease-related mutations. Here we show the development of zevoCDA1, an optimized cytosine base editor for zebrafish that improves editing efficiency across various DNA contexts and reduces restrictions imposed by the protospacer adjacent motif. We also create zevoCDA1-198, a more precise editor with a narrower editing window of five nucleotides, minimizing off-target effects. Using these advanced tools, we successfully generate zebrafish models of diseases that were previously challenging to create due to sequence limitations. This work enhances the ability to introduce human pathogenic mutations in zebrafish, broadening the scope for genomic research with improved precision and efficiency.

PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.

Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

Allele-Specific Editing of a Dominant SCN8A Epilepsy Variant Protects against Seizures and Lethality in a Murine Model.

Developmental and epileptic encephalopathies (DEEs) can result from dominant, gain of function variants of neuronal ion channels. More than 450 de novo missense variants of the sodium channel gene SCN8A have been identified in individuals with DEE. We studied a mouse model carrying the patient Scn8a variant p.Asn1768Asp. An AAV-PHP.eB virus carrying an allele-specific single guide RNA (sgRNA) was administered by intracerebroventricular injection. Cas9 was provided by an inherited transgene. Allele-specific disruption of the reading frame of the pathogenic transcript generated out-of-frame indels in 1/4 to 1/3 of transcripts throughout the brain. This editing efficiency was sufficient to rescue lethality and seizures. Neuronal hyperexcitability was reduced in cells expressing the virus. The data demonstrate efficient allele-specific editing of a dominant missense variant and support the feasibility of allele-specific therapy for DEE epilepsy. ANN NEUROL 2024;96:958-969.

Redactable blockchain from Accountable Weight Threshold Chameleon Hash

The redactable blockchain provides the editability of blocks, which guarantees the data immutability of blocks while removing illegal content on the blockchain. However, the existing redactable blockchain relies on trusted assumptions regarding a single editing authority. Ateniese et al. (EuroS&P 2017) and Li et al. (TIFS 2023) proposed solutions by using threshold chameleon hash functions, but these lack accountability for malicious editing. This paper delves into this problem and proposes an accountability weight threshold blockchain editing scheme. Specifically, we first formalize the model of a redactable blockchain with accountability. Then, we introduce the novel concept of the Accountable Weight Threshold Chameleon Hash Function (AWTCH). This function collaboratively generates a chameleon hash trapdoor through a weight committee protocol, where only sets of committees meeting the weight threshold can edit data. Additionally, it incorporates a tracer to identify and hold accountable any disputing editors, thus enabling supervision of editing rights. We propose a generic construction for AWTCH. Then, we introduce an efficient construction of AWTCH and develop a redactable blockchain scheme by leveraging AWTCH. Finally, we demonstrate our scheme’s practicality. The editing efficiency of our scheme is twice that of Tian et al. (TIFS 2023) with the same number of editing blocks.

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells.

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site Basic Protocol 3: Cloning the sgRNA dual guide construct Basic Protocol 4: Repair template design and cloning Basic Protocol 5: Electroporation and selection of engineered B cells Basic Protocol 6: Single-cell cloning of engineered B cells.

CRISPR/Cas System in Human Genetic Diseases

Gene editing involves altering a particular sequence within an organism’s genome using gene-editing techniques. Efficiently and accurately insert, delete or replace genes to alter their genetic information and phenotypic characteristics. DNA nuclease-based gene editing technology has advanced rapidly, from the first-generation editing system ZFNs, the second-generation TALENs to the third-generation CRISPR/Cas9 system, the efficiency of gene editing has been continuously improved, the cost has been gradually reduced, and the application scope has been expanding. The new classification system categorizes CRISPR-Cas proteins into Class I and II as the two main classes. Class I encompasses several subtypes, including Type I, III, and IV, all form complexes through the coordinated function of multiple Cas proteins. While Class II includes Type II, Type V, and Type VI, which all rely on single large Cas protein to perform all response. By designing single guide RNA (sgRNA), CRISPR can be used to target any gene sequence intended to be edited, achieving the wanted therapeutic outcome in treatment of inherited genetic disease. Although potential for off-target leads to undesired genetic alterations even result in oncogenesis, CRISPR-Cas still works as powerful therapy and gene editing tool in all aspects. Future research requires us to solve off-target issue. This review systematically introduces CRISPR-Cas systems as promising therapeutic strategy towards various genetic disease, explaining molecular mechanism and classification of CRISPR and differences among them.

Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.

CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene in melon. The constructed vectors were delivered to host plants using Agrobacterium-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a PDS editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.

Repeated High-Temperature Treatment Can Increase Prime Editing Efficiency in Dicot Model Species

Repeated High-Temperature Treatment Can Increase Prime Editing Efficiency in Dicot Model Species

Efficient gene disruption in polyploid genome by Cas9-Trex2 fusion protein.

The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.

Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors

Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generate four versions of near-PAMless base editors and systematically evaluate their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We develop BEguider, a deep learning model, to accurately predict editing results for near-PAMless base editors. We also provide experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We make our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.

A-to-I-edited miR-1251-5p restrains tumor growth and metastasis in lung adenocarcinoma through regulating TCF7-mediated Wnt signaling pathway

BackgroundRNA editing from A (adenine nucleotide) to I (hypoxanthine nucleotide) mediated by ADARs has attracted more and more attention as an important post-transcriptional processing method. This research investigates the regulatory mechanism of A-to-I-edited miR-1251-5p in lung adenocarcinoma (LUAD).MethodsRT-qPCR, Western blot, and Immunohistochemistry assays measured miRNA and gene expression. Colony formation, CCK-8, Transwell, Wound-healing, and Animal assays were used to determine MiRNA function. Luciferase reporter assay tested miRNA downstream target.ResultsADAR1 increased the A-to-I editing efficiency of miR-1251-5p in LUAD cells. In comparison to the unedited wild-type form, edited miR-1251-5p exhibits a marked inhibition of tumor growth and metastasis in LUAD. Moreover, knockdown of TCF7 also exhibits tumor inhibitory effect in LUAD development. Mechanically, edited miR-1251-5p inactivates Wnt signaling pathway by inhibiting TCF7, MYC, and CCND1 expression.ConclusionCompared with original miR-1251-5p, edited miR-1251-5p has stronger anti-cancer effect on LUAD development through inactivating Wnt signaling pathway by inhibiting TCF7.

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

Abstract B046: Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types

Abstract Engineered immune cell therapies have great potential to revolutionize cancer therapeutics and progress has been made in developing both engineering and manufacturing methodologies. However, limitations remain in some areas including long manufacturing times, potential for cell exhaustion, and limited access to the disease site as in the case for solid tumors. In addition, some immune cell types such as monocytes, B cells, and gamma delta T cells have remained difficult to engineer.Portal has developed a novel implementation of mechanoporation technology using a silicon membrane with pores which enables deformation of the cell and poration of the cell membrane, allowing for diffusion of any cargo, charged or uncharged, into the cell. Mechanoporation is a gentle intracellular delivery technique which preserves cell viability and function in cells (DiTommaso, et al PNAS 2018) and has the potential to shorten manufacturing time as well as allow for introduction of key therapy enhancing factors via transient modifications shortly before therapy administration. Portal has established methodology for cytoplasmic introduction of many diverse cargo including mRNA, circular RNA, siRNA, proteins, peptides, and CRISPR RNPs to primary immune cells, including both unstimulated and activated T cells, B cells, NK cells, monocytes and iPSCs. The Portal technology is easily scalable, enabling both research scale (1e6 - 5e7 cells) and process scale (over 1e9 cells) deliveries. Editing and RNA expression efficiencies are above 80% with viability maintained at 70% or above. We have demonstrated sustained expression of circular RNA, and have successfully delivered an mRNA CAR. Cargo can easily be multiplexed with equivalent delivery efficiencies across cargo classes. Portal’s methodology is gentle and effective enough to be utilized multiple times within a therapeutic workflow. For example, we have demonstrated high editing efficiencies in unstimulated T cells, followed by rapid expansion for 7 days and subsequent mRNA delivery at clinical scale - yielding over 50% knockout and 90% mRNA expression. Portal’s technology and approach has the potential to further advance immune cell therapy developments, particularly in multiplexed engineering approaches involving simultaneous delivery of multiple cargos. Coupled with Portal’s compatibility with diverse immune cell types and next generation cargos, such as circRNA, we believe this simple approach to intracellular delivery can dramatically reduce manufacturing time and cost, easily integrate into existing clinical equipment and enable unique enhancements that underpin a new generation of therapeutics. Citation Format: Zhihui Song, Eleni Rogers, Sophia Hirsch, Andrew Larocque, Darby Kreienberg, Rachel Conover, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei. Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B046.