Editing Platform Research Articles

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5244.

Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.

The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

Near‐Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR‐Cas9 Ribonucleoprotein for Brain Gene Editing

AbstractThe CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene‐editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near‐infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene‐editing potential of the formulation was demonstrated in vitro at the single‐cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

HyCas9-12aGEP: an efficient genome editing platform for Corynebacterium glutamicum.

Corynebacterium glutamicum plays a crucial role as a significant industrial producer of metabolites. Despite the successful development of CRISPR-Cas9 and CRISPR-Cas12a-assisted genome editing technologies in C. glutamicum, their editing resolution and efficiency are hampered by the diverse on-target activities of guide RNAs (gRNAs). To address this problem, a hybrid CRISPR-Cas9-Cas12a genome editing platform (HyCas9-12aGEP) was developed in C. glutamicum in this study to co-express sgRNA (corresponding to SpCas9 guide RNA), crRNA (corresponding to FnCas12a guide RNA), or hfgRNA (formed by the fusion of sgRNA and crRNA). HyCas9-12aGEP improves the efficiency of mapping active gRNAs and outperforms both CRISPR-Cas9 and CRISPR-Cas12a in genome editing resolution and efficiency. In the experiment involving the deletion of the cg0697-0740 gene segment, an unexpected phenotype was observed, and HyCas9-12aGEP efficiently identified the responsible genotype from more than 40 genes. Here, HyCas9-12aGEP greatly improve our capability in terms of genome reprogramming in C. glutamicum.

Metamaterial-based passive analog processor for wireless vibration sensing

Real-time, low-cost, and wireless mechanical vibration monitoring is necessary for industrial applications to track the operation status of equipment, environmental applications to proactively predict natural disasters, as well as day-to-day applications such as vital sign monitoring. Despite this urgent need, existing solutions, such as laser vibrometers, commercial Wi-Fi devices, and cameras, lack wide practical deployment due to their limited sensitivity and functionality. Here we proposed a fully passive, metamaterial-based vibration processing device, fabricated prototypes working at different frequencies ranging from 5 Hz to 285 Hz, and verified that the device can improve the sensitivity of wireless vibration measurement methods by more than ten times when attached to vibrating surfaces. Additionally, the device realizes an analog real-time vibration filtering/labeling effect, and the device also provides a platform for surface editing, which adds more functionalities to the current non-contact sensing systems. Finally, the working frequency of the device is widely adjustable over orders of magnitudes, broadening its applicability to different applications, such as structural health diagnosis, disaster warning, and vital signal monitoring.

Health and economic cost estimates of short-term total and wildfire PM2.5 exposure on work loss: using the consecutive California Health Interview Survey (CHIS) data 2015–2018

InstructionTo help determine the health protectiveness of government regulations and policies for air pollutant control for Americans, our study aimed to investigate the health and economic impacts of work loss...

Engineering self-deliverable ribonucleoproteins for genome editing in the brain

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

TALE-based organellar genome editing and gene expression in plants.

TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity

During the past twenty years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector mediated gene addition, inborn errors of immunity (IEIs) are excellent candidate diseases for a corrective autologous haematopoietic stem cell gene editing strategy. Research on gene editing for IEIs is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of IEIs. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

CRISPR/Cas systems combined with DNA nanostructures for biomedical applications.

DNA nanostructures are easy to design and construct, have good biocompatibility, and show great potential in biosensing and drug delivery. Numerous distinctive and versatile DNA nanostructures have been developed and explored for biomedical applications. In addition to DNA nanostructures that are completely assembled from DNA, composite DNA nanostructures obtained by combining DNA with other organic or inorganic materials are also widely used in related research. The CRISPR/Cas system has attracted great attention as a powerful gene editing technology and is also widely used in biomedical diagnosis. Many researchers are committed to exploring new possibilities by combining DNA nanostructures with CRISPR/Cas systems. These explorations provide support for the development of new detection methods and cargo delivery pathways, provide inspiration for improving relevant gene editing platforms, and further expand the application scope of DNA nanostructures and CRISPR/Cas systems. This paper mainly reviews the design principles and biomedical applications of CRISPR/Cas combined with DNA nanostructures based on the types of DNA nanostructures. Finally, the application status, challenges and development prospects of CRISPR/Cas combined with DNA nanostructures in detection and delivery are summarized. It is expected that this review will enable researchers to better understand the current state of the field and provide insights into the application of CRISPR/Cas systems and the development of DNA nanostructures.

Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.

A taxonomic backbone for the Plumbaginaceae (Caryophyllales).

A taxonomic backbone of the Plumbaginaceae is presented and the current state of knowledge on phylogenetic relationships and taxon limits is reviewed as a basis for the accepted taxon concepts. In total, 4,476 scientific names and designations are treated of which 30 are not in the family Plumbaginaceae. The Plumbaginaceae are subdivided in three tribes with 26 genera and 1,179 accepted species. Two subgenera, 17 sections, two subsections and 187 infraspecific taxa are accepted. At the species and infraspecific level 2,782 synonyms were assigned to accepted taxa, whereas 194 names were excluded from the core checklist (i.e., unplaced taxa, infrageneric subdivisions with still uncertain application, names of verified uncertain application, invalid horticultural names, excluded names from other families, other excluded designations, and unresolved names). The EDIT Platform for Cybertaxonomy was utilized as the tool to compile and manage the names and further taxonomic data under explicit taxon concepts. Secundum references are given in case taxon concepts were taken from the literature, whereas this study serves as reference for newly circumscribed taxa. The family's division into the tribes Aegialitideae, Limonieae, and Plumbagineae departs from earlier two-subfamily classifications, prompted by recent phylogenetic findings that challenge the subfamilial affinity of Aegialitis. The genus Acantholimon was extended to include Gladiolimon, as currently available phylogenetic and morphological data support this merger. In Limonium, all accepted species could be assigned to sections and subsections or the "Mediterranean lineage", respectively, making use of the phylogenetic distribution of their morphological characters and states. A new combination and/or status is proposed for Dyerophytumsocotranum, Limoniumthymoides, Limonium×fraternum, Limonium×rossmaessleri, and Limoniumsect.Jovibarba. Special attention is given to nomenclatural issues, particularly for Staticenomenambiguum to resolve the names under accepted names. The use of artificial groupings like "aggregates", "complexes" and "species groups" in alpha-taxonomic treatments is discussed. The taxonomic backbone will receive continued updates and through the Caryophyllales Taxonomic Expert Network, it contributes the treatment of the Plumbaginaceae for the World Flora Online.

Prime editing in mice with an engineered pegRNA

CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3′-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.

Breaking Osteoclast-Acid Vicious Cycle to Rescue Osteoporosis via an Acid Responsive Organic Framework-Based Neutralizing and Gene Editing Platform.

In the osteoporotic microenvironment, the acidic microenvironment generated by excessive osteoclasts not only causes irreversible bone mineral dissolution, but also promotes reactive oxygen species (ROS) production to induce osteoblast senescence and excessive receptor activator of nuclear factor kappa-B ligand (RANKL) production, which help to generate more osteoclasts. Hence, targeting the acidic microenvironment and RANKL production may break this vicious cycle to rescue osteoporosis. To achieve this, an acid-responsive and neutralizing system with high in vivo gene editing capacity is developed by loading sodium bicarbonate (NaHCO3) and RANKL-CRISPR/Cas9(RC) plasmid in a metal-organic framework. This results showed ZIF8-NaHCO3@Cas9 (ZNC) effective neutralized acidic microenvironment and inhibited ROS production . Surprisingly, nanoparticles loaded with NaHCO3 and plasmids show higher transfection efficiency in the acidic environments as compared to the ones loaded with plasmid only. Finally, micro-CT proves complete reversal of bone volume in ovariectomized mice after ZNC injection into the bone remodeling site. Overall, the newly developed nanoparticles show strong effect in neutralizing the acidic microenvironment to achieve bone protection through promoting osteogenesis and inhibiting osteolysis in a bidirectional manner. This study provides new insights into the treatment of osteoporosis for biomedical and clinical therapies.

Abstract B098: Unbiased in vitro and in vivo drug anchor screens identify mechanisms of resistance and sensitization for MTA-cooperative PRMT5 inhibitors in MTAP-deleted cancer models

Abstract Homozygous deletion of the MTAP gene is one of the most common genetic alterations in cancer, affecting 10-15% of all human cancer. Clinical MTA-cooperative PRMT5 inhibitors, including TNG908 and TNG462, were developed to leverage the well-characterized synthetic lethal interaction between PRMT5 inhibition and MTAP deletion. Indeed, the first disclosed clinical data for an MTA-cooperative PRMT5 inhibitor demonstrated the ability of TNG908 to selectively inhibit PRMT5 in MTAP-deleted tumors while sparing adjacent normal, MTAP-proficient cells in patients. To further explore the mechanism of action of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells, we employed multiple CRISPR-based editing platforms (CRISPRn, CRISPRi and CRISPRa) to conduct unbiased MTA-cooperative PRMT5 inhibitor anchor screens in vitro and in vivo. A panel of cancer models representing multiple histologies revealed potential PRMT5 inhibitor sensitization and resistance pathways that also include mechanisms that are specific to MTA-cooperative inhibitors. Specifically, the results of these screens identify 1) the effect of MTA/SAM metabolism on MTA-cooperative PRMT5 inhibitors, and 2) identification of potential sub-stratification strategies for patients with MTAP-deleted cancer, and 3) the activity of PRMT5 in anti-apoptotic pathways and synergy between PRMT5 and BCLxL inhibition. Citation Format: Steven Lombardo, Matthew R Tonini, Lauren Grove, Silvia Fenoglio, James Tepper, Binzhang Shen, Hannah Stowe, Shangtao Liu, Samuel R Meier, Ashley H Choi, Tenzing Khendu, Yi Yu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Kimberly J Briggs, Teng Teng. Unbiased in vitro and in vivo drug anchor screens identify mechanisms of resistance and sensitization for MTA-cooperative PRMT5 inhibitors in MTAP-deleted cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B098.

A modular dCas9-based recruitment platform for combinatorial epigenome editing.

Targeted epigenome editing tools allow precise manipulation and investigation of genome modifications, however they often display high context dependency and variable efficacy between target genes and cell types. While systems that simultaneously recruit multiple distinct 'effector' chromatin regulators can improve efficacy, they generally lack control over effector composition and spatial organisation. To overcome this we have created a modular combinatorial epigenome editing platform, called SSSavi. This system is an interchangeable and reconfigurable docking platform fused to dCas9 that enables simultaneous recruitment of up to four different effectors, allowing precise control of effector composition and spatial ordering. We demonstrate the activity and specificity of the SSSavi system and, by testing it against existing multi-effector targeting systems, demonstrate its comparable efficacy. Furthermore, we demonstrate the importance of the spatial ordering of the recruited effectors for effective transcriptional regulation. Together, the SSSavi system enables exploration of combinatorial effector co-recruitment to enhance manipulation of chromatin contexts previously resistant to targeted editing.

DNA-free CRISPR/Cas9 genome editing system for oil palm protoplasts using multiple ribonucleoproteins (RNPs) complexes

CRISPR/Cas9 is a powerful genome editing tool applicable to diverse plant species. However, the application of CRISPR/Cas9-ribonucleoproteins (RNPs) in oil palm (Elaeis guineensis) has not yet been reported. In this paper, the specificity, efficiency, and sensitivity of CRISPR/Cas9-RNPs in oil palm were evaluated by assessing the effect of different amounts of Cas9 and heat stress using E. guineensis phytoene desaturase (EgPDS) as the target gene. Seven gRNAs were designed and combined with Cas9 protein to form three combinations of RNPs complexes; RNP1/RNP2, RNP3/RNP4, and RNP5/RNP6/RNP7. Assessment of optimal amount of Cas9 and heat stress using RNP5/RNP6/RNP7 demonstrated that 40 µg of Cas9 and heat treatment at 39ºC increased the genome editing efficiency up to 95% in oil palm protoplasts. The use of both optimal conditions produced targeted mutations at frequencies of up to 63.6% in protoplasts transformed with RNP1/RNP2 and RNP3/RNP4 and up to 100% with RNP5/RNP6/RNP7 combinations. Multiple site mutagenesis at two to three target sites with fragment removal up to 179 bp, 69 bp, and 816 bp occurred at high frequencies producing editing efficiency and a Knockout Score (KO) of 100%. Altogether, this study demonstrates a promising, highly efficient, and precise transgene-free CRISPR/Cas9-RNPs genome editing platform in oil palm that also holds enormous potential for application in other transformation-recalcitrant species.