Combining Genome Editing Research Articles

Study of functional features of plant root systems using CRISPR/Cas-mediated genome editing

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function [1, 2].
 A common construct for efficient genome editing and selection of hairy roots is comprised of three components, i.e., a cassette carrying the gene encoding the Cas nuclease, a cassette expressing the guide RNA (gRNA), and a cassette encoding a screenable or selectable marker [2]. After design and construction, the resulting vector is used to transform plant using appropriateRhizobium rhizogenesstrain.
 Over 26 plant species have been used in experiments combining genome editing and hairy root transformation to date [2]. Possible applications of CRISPR/Cas9 genome editing using hairy root transformation include different directions like test the efficiency of the CRISPR/Cas9 genome editing; obtaining whole genome-edited plants regenerated from individual edited hairy roots; investigation of root development or root function, root nodule symbiosis, resistance to biotic or abiotic stresses, or metabolic engineering [2].
 The basic principles of plant CRISPR/Cas genome editing like the different components of CRISPR/Cas vectors, the types of Cas nuclease, design principles of gRNAs, as well as the possible applications of CRISPR/Cas genome editing in hairy roots will discuss. The application of this method for multigene editing strategy will also be demonstrated onDEEPER ROOTING1genes of cucumber.
 The study was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 075-15-2021-1056).

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 μmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Organoids: The current status and biomedical applications.

Organoids are three-dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self-organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts. Organoid culture is an emerging 3D culture technology, and organoids derived from various organs and tissues, such as the brain, lung, heart, liver, and kidney, have been generated. Compared with traditional bidimensional culture, organoid culture systems have the unique advantage of conserving parental gene expression and mutation characteristics, as well as long-term maintenance of the function and biological characteristics of the parental cells in vitro. All these features of organoids open up new opportunities for drug discovery, large-scale drug screening, and precision medicine. Another major application of organoids is disease modeling, and especially various hereditary diseases that are difficult to model in vitro have been modeled with organoids by combining genome editing technologies. Herein, we introduce the development and current advances in the organoid technology field. We focus on the applications of organoids in basic biology and clinical research, and also highlight their limitations and future perspectives. We hope that this review can provide a valuable reference for the developments and applications of organoids.

Mapping the SLP76 interactome in T cells lacking each of the GRB2-family adaptors reveals molecular plasticity of the TCR signaling pathway.

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Characterization of the Functional Interplay between the BRD7 and BRD9 Homologues in mSWI/SNF Complexes.

Bromodomains (BRDs) are a family of evolutionarily conserved domains that are the main readers of acetylated lysine (Kac) residues on proteins. Recently, numerous BRD-containing proteins have been proven essential for transcriptional regulation in numerous contexts. This is exemplified by the multi-subunit mSWI/SNF chromatin remodeling complexes, which incorporate up to 10 BRDs within five distinct subunits, allowing for extensive integration of Kac signaling to inform transcriptional regulation. As dysregulated transcription promotes oncogenesis, we sought to characterize how BRD-containing subunits contribute molecularly to mSWI/SNF functions. By combining genome editing, functional proteomics, and cellular biology, we found that loss of any single BRD-containing mSWI/SNF subunit altered but did not fully disrupt the various mSWI/SNF complexes. In addition, we report that the downregulation of BRD7 is common in invasive lobular carcinoma and modulates the interactome of its homologue, BRD9. We show that these alterations exacerbate sensitivities to inhibitors targeting epigenetic regulators─notably, inhibitors targeting the BRDs of non-mSWI/SNF proteins. Our results highlight the interconnections between distinct mSWI/SNF complexes and their far-reaching impacts on transcriptional regulation in human health and disease. The mass spectrometry data generated have been deposited to MassIVE and ProteomeXchange and assigned the identifiers MSV000089357, MSV000089362, and PXD033572.

High-throughput Tcell receptor engineering by functional screening identifies candidates with enhanced potency and specificity.

A major challenge in adoptive Tcell immunotherapy is the discovery of natural Tcell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human Tcell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple invitro and invivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications.

Direct Observation of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels.

Brain enriched voltage-gated sodium channel (VGSC) Nav1.2 and Nav1.6 are critical for electrical signaling in the CNS. Previous studies have extensively characterized cell-type-specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, because of a lack of reliable labeling and imaging methods, the subcellular localization and dynamics of these homologous Nav1.2 and Nav1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution, and live-cell single-molecule imaging to probe subcellular composition, relative abundances, and trafficking dynamics of Nav1.2 and Nav1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Nav1.2 to the distal axon of unmyelinated neurons. This pathway uses distinct signals residing in the intracellular loop 1 between transmembrane domain I and II to suppress the retention of Nav1.2 in the axon initial segment and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Nav1.2 is gradually excluded from the distal axon as Nav1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Nav1.2 and Nav1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.SIGNIFICANCE STATEMENT Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.

MEDB-22. iPSC-derived cerebellar organoid model for hereditary genetic predisposition in SHH-medulloblastoma

Medulloblastoma is one of the most common malignant embryonal brain tumors in children. Medulloblastomas of the Sonic Hedgehog (SHH) group arise from excessive proliferation of granule neuron progenitor (GNP) cells during cerebellar development. Genetic predisposition accounts for nearly 40% of all pediatric SHH-medulloblastomas. Recently, ELP1, a novel predisposition gene, was shown to be germline mutated in 15% of SHH-medulloblastoma patients. ELP1 encodes the scaffolding member of the Elongator complex and is required for efficient translation. Heterozygous mutations in ELP1 have been associated with the neural disorder Familial Dysautonomia, but not cancer. ELP1-associated medulloblastomas frequently harbor somatic PTCH1 co-mutations. It remains unclear how ELP1 affects the GNP lineage during normal cerebellar development and tumorigenesis in pediatric SHH-medulloblastoma patients. To characterize ELP1 mutations in the GNP lineage in vitro, we established a cerebellar organoid model from human induced pluripotent stem cells (iPSCs). We genetically inserted an EGFP reporter downstream of the endogenous GNP-specific ATOH1 locus in control iPSCs and generated cerebellar organoids according to published protocols. Marker gene and protein expression levels confirmed the cerebellar identity of the 3D model. Furthermore, activation of the EGFP reporter in single cells within the organoid highlighted the specification of putative GNPs. Next, we will determine the specific cell state of putative iGNPs and compare to human GNPs identified in our scRNAseq cerebellum atlas. To analyze tumorigenesis through ELP1 loss, we will introduce patient-specific ELP1mutations into ATOH1-EGFP iPSCs. Cerebellar organoids derived from ELP1-, PTCH1-deficient and control iPSCs will serve as models to study GNP proliferation, differentiation, apoptosis and tumor formation. Combining genome editing, in vitro 3D differentiation and functional studies, we will characterize the novel predisposition gene ELP1in GNPs during cerebellar development. In addition, we will determine the interplay of ELP1and PTCH1 co-mutations, predisposing SHH-medulloblastoma formation.

Functional and electrophysiological characterization of C. elegans PEZO-1

The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo mechanosensitive ion channel family, pezo-1. PEZO-1 is expressed in the worm’s pharynx. This myogenic pump is an essential component of the worm alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive channel and contributes to pharyngeal function remains unknown. We combined genome editing, genetics, microfluidics, and electropharyngeogram recordings to establish that pezo-1 is expressed in a proprioceptive-like neuron in the pharynx and regulates pharyngeal function.

CRISPR approach in environmental chemical screening focusing on population variability.

A significant barrier to include population variability in risk assessment is our incomplete understanding of inter-individual variability and the differential susceptibility to environmental exposures induced adverse outcomes. By combining genome editing tools with the population diversity model, this article intended to highlight a potential strategy to identify and characterize the inter-individual variability factors, the determinant gene anchoring to a particular phenotype. The goal could be achieved by integrating the perturbed CRISPR-based unbiased functional genomics screening, genome-wide or a focused subset of genes, in a population-based in vitro model system (such as the lymphoblastoid cell lines, LCL, available from HapMap and 1000 Genomes project). Then data can be translated to genetic variability and individual (or subpopulation) susceptibility by incorporating ethnicity and corresponding genome-wide association studies (GWAS) with functional genomics screening results. This approach can provide complementary data for next-generation risk assessment, in particular, for environmental stressors. The current paper outlined the previous work conducted with a population-based in vitro model system, perturbed CRISPR-based functional toxicogenomic screening of environmental chemicals, and finally, the potential strategies to combine these two platforms with their opportunities and challenges to achieve a mechanistic understanding of population variability.

Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.

To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively. Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200mgL-1 than that of around 150mgL-1 produced by the wild-type strain. This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.

Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a.

Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex.

Kinetochore localized Mad1 is essential for generating a "wait anaphase" signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod-Zw10-Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1-Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1-Bub1 complex.

Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.

A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analyzed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrate that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, analysis of epistasis indicates that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programs during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

Real-Time De Novo Deposition of Centromeric Histone-Associated Proteins Using the Auxin-Inducible Degradation System.

Measuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the auxin-inducible degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and reexpression in the same experimental setup. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and reactivation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.