Knock-in Method Research Articles

Abstract Tu082: Novel Structural and Biochemical Effects of Nexilin Associated Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is primarily a genetic disease that results in abnormal cardiac muscle phenotypes characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Both sarcomere structural and signaling genes are associated with HCM. Among various genes, one of the implicated genes for HCM is Nexilin (NEXN). Nexilin is an F-actin binding protein widely expressed in muscle cells and localized at sarcomere and T-tubules. It is known to have a surprising role in insulin signaling. The role of Nexilin mutations is poorly studied. We hypothesized that Nexilin missense variants in genetically engineered human pluripotent stem cells (hPSCs) perturb essential cardiac metabolic signaling pathways leading to HCM. In our Indian HCM cohort, we identified seven Nexilin mutations through exome sequencing and categorized them as pathogenic per ACMG/AMP classification. A representative novel mutant in Nexilin p.Q131T was introduced in hPSC with the CRISPR-Cas9 knock-in method and was differentiated into cardiomyocytes, recapitulating patient-specific heterozygous mutation. Our data shows that Nexilin p.Q131T causes an increase in cell size and activation of fetal gene transcriptional programs in hPSC-derived cardiomyocytes. Global transcriptomics was performed to understand transcriptional changes and investigate further. We observe the significant activation of insulin-related signaling pathways, including AKT/MTOR and MAPK, confirmed by immunoblotting. Akt-mTOR and MAPK cascades are well-known to be regulated by IRS-1 signaling. Our studies show hyperactivation of these cascades, suggesting IRS-1 involvement in the pathogenesis. Nexilin and IRS-1 interaction were probed and found to be perturbed, hence causing higher activation of IRS-1 and its downstream targets. In addition, Nexilin wild type and mutant protein structure-functional analysis were performed with purified proteins. Using cryo-EM structural analysis, we identified unique F-actin binding sites for Nexilin for the first time, and the mutant p.Q131T showed decreased binding affinity to F-actin. We report Nexilin's high-resolution cryo-EM structure and a novel metabolic signaling mechanism where mutations in Nexilin cause dysregulation of IRS-1 signaling in the gene-edited cardiomyocytes, leading to HCM pathogenesis.

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

Total Biosynthesis of Melleolides from Basidiomycota Fungi: Mechanistic Analysis of the Multifunctional GMC Oxidase Mld7.

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock-in method to elucidate the biosynthetic pathway leading to 1α-hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation of the Δ6 -protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short-chain dehydrogenase/reductase family proteins. The highlight of the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation-triggered double-bond shift accompanying dehydration and acyl-group-assisted substitution with two different nucleophiles at the C6 position to afford the Δ7 -protoilludene derivatives, such as melleolide and armillarivin. The complex reaction mechanism was proposed by DFT calculations. Of particular importance is that product distribution is regulated by interaction with the cell membrane.

Construction of lipopeptide mono-producing Bacillus strains and comparison of their antimicrobial activity

Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides, including iturin, surfactin, and plipastatin, and it is challenging to discriminate each cyclic lipopeptide's antimicrobial activity directly. In this study, a chassis strain Bacillus subtilis 1A751 WR without cyclic lipopeptides operons was constructed by CRISPR/Cas9. The iturin gene cluster of B. amyloliquefaciens HYM-12 was captured and assembled with sfp and degQ genes using transformation-associated recombination (TAR). It was then integrated into the chassis strain to obtain an iturin mono-producing strain that could synthesize six iturin isoforms. The frameshift sfp gene of strain 1A751 Δpps was repaired by a scarless gene knock-in method to generate a surfactin mono-producing strain. Antimicrobial activity experiments indicated that the extracts of iturin, surfactin, and our previously constructed plipastatin mono-producing strains exhibited powerful inhibitory effects toward pathogenic bacteria. Interestingly, iturin showed significant antifungal activity, but surfactin and plipastatin exhibited weak antifungal activity. Compared with amphotericin B, iturin has a solid and durable antifungal activity. This study demonstrates that TAR cloning is efficient for cloning large gene clusters, proves the differences in the antimicrobial activity of three cyclic lipopeptides, and lays a theoretical basis for further elucidating their antimicrobial mechanism.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

Efficient knock-in method enabling lineage tracing in zebrafish.

Here, we devised a cloning-free 3' knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5' AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92, nkx6.1, krt4, and id2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested that nkx6.1 + cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas id2a + cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic id2a + duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.

Cre/lox-Mediated CRISPRi Library Reveals Core Genome of a Type I Methanotroph Methylotuvimicrobium buryatense 5GB1C.

Methanotrophs play key roles in global methane cycling and are promising platforms for methane bioconversion. However, major gaps existing in fundamental knowledge undermines understanding of these methane-consuming microorganisms. To associate genes with a phenotype at the genome-wide level, we developed a Cre/lox-mediated method for constructing a large-scale CRISPRi library in a model methanotroph Methylotuvimicrobium buryatense 5GB1C. The efficiency of this Cre mediated integration method was up to a level of 105 CFU/μg DNA. Targeting 4,100 predicted protein-coding genes, our CRISPRi pooled screening uncovered 788 core genes for the growth of strain 5GB1C using methane. The core genes are highly consistent with the gene knockout results, indicating the reliability of the CRISPRi screen. Insights from the core genes include that annotated isozymes generally exist in metabolic pathways and many core genes are hypothetical genes. This work not only provides functional genomic data for both fundamental research and metabolic engineering of methanotrophs, but also offers a method for CRISPRi library construction. IMPORTANCE Due to their key role in methane cycling and their industrial potential, methanotrophs have drawn increasing attention. Genome-wide experimental approaches for gene-phenotype mapping accelerate our understanding and engineering of a bacterium. However, these approaches are still unavailable in methanotrophs. This work has two significant implications. First, the core genes identified here provide functional genetic basics for complete reconstruction of the metabolic network and afford more clues for knowledge gaps. Second, the Cre-mediated knock-in method developed in this work enables large-scale DNA library construction in methanotrophs; the CRISPRi library can be used to screen the genes associated with special culture conditions.

Genome Editing of Medaka.

Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.

Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.

CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.

CRISPR/Cas9 - mediated knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.

Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.

Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic “safe harbors”) using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of “euchromatization” of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.

Identification of biomarkers for acute leukemia via machine learning-based stemness index.

Traditional methods to understand leukemia stem cell (LSC)'s biological characteristics include constructing LSC-like cells and mouse models by transgenic or knock-in methods. However, there are some potential pitfalls in using this method, such as retroviral insertion mutagenesis, non-physiological level gene expression, non-physiological expansion, and difficulty to construct. The mRNAsi index for each sample of the Cancer Genome Atlas (TCGA) could avoid these potential pitfalls by machine learning. In this work, we aimed to construct a network of LSC genes utilizing the mRNAsi. First, mRNAsi value was analyzed with expressions distributions, survival analysis, age, and gender in acute myeloid leukemia (AML) samples. Then, we used the weighted gene co-expression network analysis (WGCNA) to construct modules of stemness genes. The correlation of the LSC genes transcription and interplay among LSC proteins was analyzed. We performed functional and pathway enrichment analysis to annotate stemness genes. Survival analysis further identified prognostic biomarkers by clinical data of TCGA and the Gene Expression Omnibus (GEO) database. We found that the result of mRNAsi overall survival is not significant, which may be due to the heterogeneity of AML in the stage of myeloid differentiation, French-American-British (FAB) classification systems. Enrichment analysis indicated that the stemness genes were biologically clustered as a group and mainly associated with cell cycle and mitosis. Moreover, 10 key genes (SNRNP40, RFC4, RFC5, CDC6, HSPE1, PA2G4, SNAP23P, DARS2, MIS18A, and HPRT1) were screened by survival analysis with the data from TCGA and GEO. Among them, RFC4 and RFC5 were the distinguished biomarkers for their double-validated prognostic value in both databases. Additionally, the expression of RFC4 and RFC5 had the same trend as mRNAsi score in FAB subtypes. In conclusion, our result demonstrated that mRNAsi based LSC-related genes were found to have strong interactions as a cluster. These genes, especially RFC4 and RFC5, could be the therapeutic targets for inhibiting the stemness characteristics of AML. This work is also a comprehensive pipeline for future cancer stem cell studies.

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3′ splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

35P Increased sensitivity to olaparib by BRCA1/2 knockdown using a CRISPR/Cas9-mediated knock-in method in pancreatic cancer cell lines

Pancreatic cancer is one of the most incurable disease. About 80% of pancreatic adenocarcinoma are locally advanced at diagnosis or metastatic. Therefore, only 20% of patients undergo surgery and the majority have local relapse or metastasis. Chemotherapy regimens are used in adjuvant therapy with gemcitabine or FOLFIRINOX protocol, the standard of care for metastatic pancreatic cancer. Olaparib, a PARP inhibitor, is used as maintenance treatment in patient with metastatic pancreatic adenocarcinoma bearing a germline BRCA1/2 mutation.

Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase \u03b8/DNA-PK

CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

Genetically Engineered Methanotroph as a Platform for Bioaugmentation of Chemical Pesticide Contaminated Soil.

Bioaugmentation is a promising alternative in soil remediation. One challenge of bioaugmentation is that exogenous pollutant-degrading microbes added to soil cannot establish enough biomass to eliminate pollutants. Considering that methanotrophs have a growth advantage in the presence of methane, we hypothesize that genetically engineered methanotrophs could degrade contaminants efficiently in soil with methane. Here, methanotroph Methylomonas sp. LW13, herbicide bensulfuron-methyl (BSM), and two kinds of soil were chosen to confirm this hypothesis. The unmarked gene knock-in method was first developed for strain LW13. Then, BSM hydrolase encoding gene sulE was inserted into the chromosome of strain LW13, conferring it BSM-degrading ability. After inoculation, the cell amount of strain LW13-sulE in soil raised considerably (over 100 fold in 9 days) with methane provision; meanwhile, >90% of BSM in soil was degraded. This study provides a proof of the concept that genetically engineered methanotroph is a potential platform for soil remediation.

Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis

The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.

Protocol for De Novo Gene Targeting Via In Utero Electroporation.

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.