CRISPR-Cas9 System Research Articles

Biotechnology Patent Law Top Ten of 2022: Inducement, Clear Error, and Interferences Galore

Five-year anniversaries are symbolized by a product of natural biotechnology: wood. This article marks the wood anniversary of the “Top Ten Biotechnology Patent Cases” series that began in 2018. Imagining the world in 2018 is challenging, in part because it was, indeed, a different world. There had not been a major pandemic in one hundred years. Inflation was low. The economy hummed along. No individual war appeared to threaten more than regional stability. O tempora, o mores! The year 2022 was quite different. SARS-CoV-2 continued to stalk the land, having had a monumentally mortiferous effect for several years. High inflation was rampant. The economy was still recovering from one of the deepest declines in history, with imbalances across many sectors. Moreover, eastern Europe had let slip the dogs of war, threatening peace worldwide. Biotechnology also has seen changes of significant magnitude. Venture capital investment in biotechnology was small compared to what it is now. Efficient genome editing was restricted to first-generation CRISPR-Cas9 systems, while now it may be accomplished using more powerful and accurate methods, like base editing and prime editing (the latter offering hope for treatment in almost 90% of genetic diseases). Courts had declared that diagnostic methods did not constitute patentable subject matter, which remains the case today, although clever draftswomen continue their efforts to obtain claims protecting such methods to some extent. Over the course of 2022, courts decided a generous selection of cases covering a wide variety of biotechnology patent law issues. These cases ran the gamut, from patent doctrines concerning satisfaction of the written description requirement for antibody claims to mechanisms for augmenting patent terms using either Patent Term Extension, or Patent Term Adjustment, or both. This article has chosen ten of the most important, though, as is the case every year, our choice of only ten was difficult, and, by necessity, left worthy cases on the cutting room floor.

Type I-D CRISPR System-Mediated Genome Editing in Plants.

Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.

Application of CRISPR-Cas System in the Treatment of Human Viral Disease

CRISPR-Cas systems, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), are the latest generation of gene editing technology and have been widely used in molecular biology research. CRISPR-Cas systems also have unlimited potential in the field of medicine, especially in the treatment of human viral diseases, such as blocking virus invasion, interfering with virus replication, and eliminating viral genome and sequelae of virus infection. In this article, the latest research progress of CRISPR-Cas9 system and other CRISPR systems in treatments of several viral diseases are reviewed. In addition, the advantages and potential problems of CRISPR systems as treatment options are analyzed to provide ideas for subsequent related research.

Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9.

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiaegenome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressingguide RNAs(gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.

In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration.

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2-60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.

SgRNA-2wPSM: Identify sgRNAs on-target activity by combining two-window-based position specific mismatch and synthetic minority oversampling technique

MotivationsgRNAs on-target activity prediction is a critical step in the CRISPR-Cas9 system. Due to its importance to RNA function research and genome editing application, some computational methods were introduced, treating it as a binary classification task or a regression task. Among these methods, sgRNA-PSM is a state-of-the-art method. In this work, we improved this method by proposing a new feature extraction method called two-window-based PSM, which divides the DNA sequences into two non-overlapping segments so as to extract different patterns in the two different segments. The two-window-based PSM were fed into Support Vector Machines (SVMs), and a new method called sgRNA-2wPSM was proposed. Furthermore, a new oversampling method called SCORE-SVM-SMOTE was proposed to solve the imbalanced training set problem based on the SVM-SMOTE algorithm. Results on the benchmark datasets indicated that sgRNA-2wPSM is superior to other methods.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.

RNF213 Loss-of-Function Promotes Angiogenesis of Cerebral Microvascular Endothelial Cells in a Cellular State Dependent Manner.

Enhanced and aberrant angiogenesis is one of the main features of Moyamoya disease (MMD) pathogenesis. The ring finger protein 213 (RNF213) and the variant p.R4810K have been linked with higher risks of MMD and intracranial arterial occlusion development in east Asian populations. The role of RNF213 in diverse aspects of the angiogenic process, such as proliferation, migration and capillary-like formation, is well-known but has been difficult to model in vitro. To evaluate the effect of the RNF213 MMD-associated gene on the angiogenic activity, we have generated RNF213 knockout in human cerebral microvascular endothelial cells (hCMEC/D3-RNF213-/-) using the CRISPR-Cas9 system. Matrigel-based assay and a tri-dimensional (3D) vascularized model using the self-assembly approach of tissue engineering were used to assess the formation of capillary-like structures. Quite interestingly, this innovative in vitro model of MMD recapitulated, for the first time, disease-associated pathophysiological features such as significant increase in angiogenesis in confluent endothelial cells devoid of RNF213 expression. These cells, grown to confluence, also showed a pro-angiogenic signature, i.e., increased secretion of soluble pro-angiogenic factors, that could be eventually used as biomarkers. Interestingly, we demonstrated that that these MMD-associated phenotypes are dependent of the cellular state, as only noted in confluent cells and not in proliferative RNF213-deficient cells.

Plcz1 Deficiency Decreased Fertility in Male Mice Which Is Associated with Sperm Quality Decline and Abnormal Cytoskeleton in Epididymis.

Phospholipase C zeta1 (Plcz1) was known to be a physiological factor in sperm that activates oocytes to complete meiosis by triggering Ca2+ oscillations after fertilisation. However, the role of male Plcz1 in spermatogenesis and early embryo development in progeny has been controversial. Plcz1 knockout (Plcz1-/-) mouse model (Plcz1m3 and Plcz1m5) was generated by using the CRISPR-Cas9 system. The fertility of Plcz1-/- mice was evaluated by analysing the number of offsprings, sperm quality, pathological changes in the testis and epididymis. RNA-seq and RT-PCR were performed to screen differentially expressed genes and signalling pathways related to fertility in Plcz1-/- mice. Further mechanism was explored by using Plcz1-/- cells. Plcz1 knockout led to hypofertility in male mice. In particular, a significant time delay in development and polyspermy was found in eggs fertilized by both Plcz1m3 and Plcz1m5 sperm. Interestingly, a decline in sperm quality combined with pathological changes in epididymis was found in Plcz1m3 mice but not in Plcz1m5 mice. Notably, abnormal cytoskeleton appears in epididymis of Plcz1m3 mice and Plcz1-/- cells. Cytoskeleton damage of epididymis is involved in fertility decline of males upon Plcz1 deficiency in this model.

A Toolkit for Effective and Successive Genome Engineering of Escherichia coli

The bacterium Escherichia coli has been well-justified as an effective workhorse for industrial applications. In this study, we developed a toolkit for flexible genome engineering of this microorganism, including site-specific insertion of heterologous genes and inactivation of endogenous genes, such that bacterial hosts can be effectively engineered for biomanufacturing. We first constructed a base strain by genomic implementation of the cas9 and λRed recombineering genes. Then, we constructed plasmids for expressing gRNA, DNA cargo, and the Vibrio cholerae Tn6677 transposon and type I-F CRISPR-Cas machinery. Genomic insertion of a DNA cargo up to 5.5 kb was conducted using a transposon-associated CRISPR-Cas system, whereas gene inactivation was mediated by a classic CRISPR-Cas9 system coupled with λRed recombineering. With this toolkit, we can exploit the synergistic functions of CRISPR-Cas, λRed recombineering, and Tn6677 transposon for successive genomic manipulations. As a demonstration, we used the developed toolkit to derive a plasmid-free strain for heterologous production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by genomic knock-in and knockout of several key genes with high editing efficiencies.

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.

2350. LiaF is required for LiaX mediated protection against Daptomycin and LL-37 in Enterococcus faecalis OG1RF

Abstract Background Daptomycin (DAP) resistance in enterococci is regulated by the LiaFSR three-component regulatory system. Mutations in liaF have been linked to activation of this system, with increased expression of the protein LiaX, and resistance to DAP in clinical isolates. LiaX functions by recognizing DAP in the extracellular medium, and serves as a signal transduction molecule. However, the role of LiaF in signaling and its relationship with LiaX are unknown. Methods We generated a liaF null mutant in E. faecalis OG117 by adding four stop codons at amino acid positions 11–14 (OG117liaF*11-14) using a CRISPR-Cas9 system. The mutant was complemented by restoring wild-type liaF in its chromosomal location (OG117liaF*11-14::liaF). Mutants were confirmed with whole genome sequencing (WGS). Anionic phospholipid (AP) microdomain distribution was assessed on microscopy using 10-N-nonyl-acridine-orange (NAO). LiaFSR activation was assessed by surface expression of LiaX via ELISA. DAP MICs were performed by broth microdilution in the presence/absence of exogenous LiaX (eLiaX). LL37 (50µg/ml) and DAP (1.5µg/ml) killing assays with eLiaX were also performed in triplicate. Results Truncation of liaF did not have any effect on DAP MICs, AP microdomain distribution or LiaX surface expression compared to OG117. No additional mutations were observed by WGS in the mutants. In the presence eLiaX, DAP MIC increased 8-fold in wild-type OG117 but remained the same (1 µg/mL) in OG117liaF*11-14. Complementation of liaF restored the increase in DAP MIC in the presence of eLiaX. In the LL37 killing assay, survival of OG117 was significantly increased on the addition of eLiaX (2.9% vs 11.8%, respectively, P < .002). Addition of eLiaX was unable to rescue OG117liaF*11-14 in the presence of LL-37 (2.4% vs 2.5% respectively, P=0.51), but was able to increase survival in the complemented strain (2.8% vs 7.6%, respectively, P < .002). A similar trend was seen in the DAP killing assay, with increased survival for OG117 (8.7% vs 20%) and OG117liaF*11-14::liaF (11.6% vs 17%), but not OG117liaF*11-14 (0.38% vs 1%). Conclusion LiaF is required for full tolerance to the cathelicidin LL37 and DAP in E. faecalis OG1RF. These results suggest that LiaF may interact with LiaX as a part of the LiaFSR stress response. Disclosures William R. Miller, MD, Entasis: Grant/Research Support|UpToDate: Honoraria Cesar A. Arias, MD, PhD, Entasis: Grant/Research Support|MeMed Diagnostics Ltd: Grant/Research Support|Merck: Grant/Research Support.

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains

BackgroundOwing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems.ResultsHere, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains.ConclusionThe Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.

A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.

Towards an appropriate African framework for public engagement with human genome editing: a call to synergistic action

The CRISPR-Cas9 system has revolutionised the biotechnology of human genome editing. Human germline gene editing promises exponential benefits to many in Africa and elsewhere, especially those affected by the highly prevalent monogenic disorders - for which, thanks to CRISPR, a relatively safe heritable radical therapy is now possible. Africa evidently presents a unique opportunity for empirical research in human germline gene editing because of its high prevalence of monogenic disorders. Critically, however, germline gene editing has raised serious ethical concerns especially because of the significant risks of inadvertent and intentional misuse of its transgenerational heritability. Calls for due prudence have become even more pronounced in the wake of the 2018 case of He Jiankui’s ‘CRISPR’d babies’. Meanwhile, Africa is seriously lagging in articulating its position on human genome editing. Conspicuously, there has been little to no attempt at comprehensively engaging the African public in discussions on the promises and concerns about human genome editing. Thus, the echoing key question remains as to how Africa should prudently embrace and govern this revolutionary biotechnology. In this article, therefore, I lay the groundwork for the possible development of an appropriate African framework for public engagement with human genome editing and call upon all stakeholders to urgent synergistic action. I particularly highlight the World Health Organisation’s possible leadership role in promptly establishing the requisite expert working group for this urgent need.

RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

Effect of deleting four Toxoplasma gondii calcium-binding EGF domain-containing proteins on parasite replication and virulence.

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress fromthe host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated.

CRISPR-Cas9 Genome Editing and Rapid Selection of Cell Pools.

The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.

Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses

Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing. Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment. Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors. Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

CsIAGLU Regulates the Angle of Leaf Petiole by Affecting Endogenous Content of Auxin in Cucumber (Cucumis sativus L.).

The leaf angle is an important factor determining plant shoot architecture that may boost crop yield by increasing photosynthetic efficiency and facilitating high-density planting. Auxin is an important phytohormone involved in leaf angle regulation. Here, we identified two Single-Nucleotide Polymorphisms (SNPs) in the Indoleacetic Acid (IAA) glucosyltransferase gene CsIAGLU in 80 re-sequenced cucumber lines, of which the CsIAGLU717G,1234T is the dominant allele associated with a small leaf pedicle angle (LPA), whereas CsIAGLU717C,1234A is linked with a large LPA. CsIAGLU was highly expressed in leaves and petioles. In natural cucumber populations, the expression of CsIAGLU was negatively correlated with the LPA. The mutation of CsIAGLU induced by the CRISPR-Cas9 system resulted in elevated free IAA levels and enlarged cell expansion on the adaxial side of the petiole base, thus producing a greater LPA. Consistently, exogenous IAA treatment led to increased LPA and cell size. Therefore, our findings suggest that CsIAGLU functions as a negative regulator of LPA development via auxin-mediated cell expansion in cucumber, providing a valuable strategy for cucumber breeding with small LPAs.