gRNA Target Research Articles

Be-dataHIVE: a base editing database

Base editing is an enhanced gene editing approach that enables the precise transformation of single nucleotides and has the potential to cure rare diseases. The design process of base editors is labour-intensive and outcomes are not easily predictable. For any clinical use, base editing has to be accurate and efficient. Thus, any bystander mutations have to be minimized. In recent years, computational models to predict base editing outcomes have been developed. However, the overall robustness and performance of those models is limited. One way to improve the performance is to train models on a diverse, feature-rich, and large dataset, which does not exist for the base editing field. Hence, we develop BE-dataHIVE, a mySQL database that covers over 460,000 gRNA target combinations. The current version of BE-dataHIVE consists of data from five studies and is enriched with melting temperatures and energy terms. Furthermore, multiple different data structures for machine learning were computed and are directly available. The database can be accessed via our website https://be-datahive.com/ or API and is therefore suitable for practitioners and machine learning researchers.

Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.

Diffuse large B cell lymphomas (DLBCL) are highly aggressive tumors. Their genetic complexity and heterogeneity have hampered the development of novel approaches for precision medicine. Our study aimed to develop a personalized therapy for DLBCL by utilizing the CRISPR/Cas system to induce knockouts (KO) of driver genes, thereby causing cancer cell death while minimizing side effects. We focused on OCI-LY3 cells, modeling DLBCL, and compared them with BJAB cells as controls. Analysis of whole exome sequencing revealed significant mutations in genes like PAX5, CD79B, and MYC in OCI-LY3 cells. CRISPR/Cas9-mediated KO of these genes resulted in reduced cancer cell viability. Subsequent single and dual gRNA targeting of PAX5 mutations inhibited proliferation specifically in OCI-LY3 cells. Moreover, dual gRNA targeting of PAX5 and MYC induced chromosomal rearrangements, reducing cell proliferation substantially. However, targeting single intronic mutations did not affect cell viability, highlighting the importance of disrupting protein function. Targeting multiple mutations simultaneously addresses intra-tumoral heterogeneity, and the transient delivery of CRISPR/Cas9 allows for permanent gene disruption. While challenges such as incomplete editing efficiency and delivery limitations exist, further optimization may enhance therapeutic efficacy. Overall, our findings demonstrate the efficacy of CRISPR/Cas9 in targeting oncogenic mutations, opening avenues for precision medicine in DLBCL treatment.

Sequence-Structure Comparative and Network-Based Prediction of Drought Gene Candidate Regulator in <i>Elaeis guineensis</i>

Drought poses a significant threat to global food security, particularly impacting crops like oil palm. Selecting genes for genome editing to enhance drought tolerance presents formidable challenges. To ensure that the target gene is chosen correctly and results in the desired character, a pilot study is necessary to determine the target gene for knockout. Two genes drought-related, AtBRL3 and AtOST2, were scrutinized in this context. Aligned with the Elaeis guineensis genome, their neighbouring proteins and gene ontology were analysed to identify potential targets for genome editing. AtBRL3, identified as BRL1 (XP_010913986.1) in E. guineensis, exhibited 58.48% identity and 100% coverage. It interacts with 12 nodes, including BIR1, BRI1, and AT2G20050, crucial for signalling pathways and cellular responses. Molecular function analysis revealed kinase activity. AtOST2 showed high similarity to plasma membrane ATPase/HA1 (XP_010913679.1) in E. guineensis, with 87.46% identity and 100% query cover. It correlated with 14 genes associated with ABA stimulus, stomatal movement, and hormone response. EgBRL1 and EgHA1, resembling AtBRL3 and AtOST2, respectively, emerge as promising targets for developing drought-tolerant oil palm cultivars through gene editing. Nonetheless, further validation through in vitro gRNA target selection and in vivo conversion of OST2/BRL3-containing plasmids in oil palm calluses is indispensable to demonstrate their efficacy in conferring novel drought resistance traits.

Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition.

Delineating a protein's essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding, and that its interaction with the SpCas9 catalytic domain is inessential.

Sterilization of fish through adaptable gRNAs targeting dnd1 using CRISPR-Cas13d system

Sterilizing fish is a valuable technique not only for preventing genetic contamination from escaped aquaculture fish but also for enhancing fish meat quality. The clustered regularly interspaced short palindromic repeats-CRISPR-associated 13d (CRISPR-Cas13d) system is capable of RNA-targeted gene knockdown with reduced off-target effects. Unlike morpholino knockdown, which is limited to specific target sites, the CRISPR-Cas13d system utilizes the entire mRNA as a target site for gRNA. This enables the design of adaptable gRNAs by targeting the conserved sequences across fish species, leading to promising sterilization for a wide variety of aquaculture fish. In this study, we demonstrated that the effectiveness of this system in fish sterilization by the knockdown of dead end1 (dnd1) and nanos3, which are crucial for germ cell development. Initially, we demonstrated that gRNAs designed using the cas13design tool, which was developed based on studies of cultured mammalian cells, effectively induced germ cell-deficient medaka with low-cytotoxicity. Additionally, we identified the nucleotide positions of the gRNA (seed sequence) that are crucial for knockdown in teleost fish. These data will be valuable for designing adaptable gRNA in teleost fish and targeting multiple paralogous genes. To develop an adaptable sterilization technique using the CRISPR-Cas13d system, we aligned 81 dnd1 sequences from 70 fish species and identified a highly conserved region of the gRNA targets. Despite not finding a perfectly matched target region in the dnd1 alignment, we designed 15 adaptable gRNAs that potentially targeting dnd1 in 55 out of 70 fish species, leveraging sequences from representative aquaculture and aquarium fish (salmon, tilapia, puffer fish, amberjack, tuna, guppy and carp) and experimental model fish (medaka, zebrafish, and stickleback. To confirm whether adaptable gRNAs had a knockdown effect, we initially injected the gRNAs into medaka embryos. Adaptable gRNAs with a single-nucleotide mismatch against medaka dnd1 resulted in the complete loss of germ cells with little cytotoxicity, suggesting that mismatches are unlikely to induce off-target effects crucial for embryonic development. Additionally, adaptable gRNAs designed based on the salmon dnd1 sequence, which was 100% identical in the Oncorhynchus, Salvelinus, and Salmo groups, effectively generated germ cell-deficient rainbow trout. These results indicate that the knockdown of dnd1 using the CRISPR-Cas13d system is effective for sterilizing both aquaculture and experimental model fish species. Moreover, the adaptable gRNAs designed in this study can potentially sterilize a diverse range of fish species.

Abstract B012: BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors

Abstract Background: Bladder cancer (BC) remains a common and deadly disease, with 83,190 cases and 32,350 deaths projected in the U.S. in 2024. We previously showed bromodomain and extra-terminal protein inhibitors (BETi) are potent in multiple preclinical models of BC. Our bulk RNA-seq and RT-qPCR data showed BETi significantly reduces expression of homologous recombination (HR) genes, such as RAD51 and RBBP8, which could activate alternative DNA repair pathways including non-homologous end joining (NHEJ) and theta-mediated end joining (TMEJ). Specifically, DNA breaks are repaired through TMEJ by polymerase theta (Polθ) utilizing microhomology templates. Recently published data revealed Polθ inhibition is synthetically lethal when other DDR genes are concomitantly lost or inhibited. Thus, in this study, we sought to elucidate whether BETi-induced HR gene repression impacts TMEJ and POLQ expression, and to identify combinations with BETi that best leverage Polθ-mediated synthetic lethality. Methods: 5637 and J82 BC cells were pretreated with birabresib for 24 h and then transfected via electroporation with a Cas9 ribonucleoprotein, a gRNA targeting LBR2 and a 996 bp HR donor. Transfected cells were treated with birabresib for 24H, and DDR pathway function was assessed by digital droplet PCR. BC cells were synchronized at the G1/S transition with 2 mM thymidine incubated with birabresib alone. After 48 h, cells were harvested, and changes to target gene expression (i.e., POLQ) was assessed by RT-qPCR. Next, the same cells were treated with 8 ascending doses of the BETi birabresib (0.1–100 μM) alone or in combination with either the PARP inhibitor olaparib, CtIP inhibitor triapene, or RAD51 inhibitor RI-1 (10 nM–200 μM). After 72-96 h, CellTiter-Glo™ measured cell viability, and Compusyn v1 calculated combination index (CI) scores where <1.0 indicated synergism and >1.0 indicated antagonism. RT-qPCR was conducted to evaluate expression changes to POLQ after combination treatment. Results: After 48 h, birabresib significantly reduced TMEJ in both 5637 and J82 BC cells (49% reduction, P=0.008 and 41% reduction, P=0.004 both n=3), but did not significantly impact HR or NHEJ. POLQ mRNA expression was most reduced in synchronized 5637 and J82 BC cells after treatment with birabresib alone (46% reduction, P=0.006, n=3, and 38% reduction, P=0.001, n=3). Birabresib combined with olaparib was the most synergistic combination in both 5637 and J82 BC cells (CI = 0.83 and 0.29), but birabresib combined with triapene was also synergistic (CI = 0.91 and 0.90). In J82 cells, birabresib significantly reduced POLQ expression (27% reduction, P=0.008, n=3), but olaparib did not. In both 5637 and J82 cells combined birabresib and olaparib increased POLQ expression (41% increase and 5% increase, ns, n=3). Combined birabresib and triapene displayed a similar trend (46% increase and 17% increase, ns, n=3). Conclusions: These preliminary data support further exploration of POLQ expression increases caused by combined BETi+DDRi treatment in preclinical models of BC. Citation Format: Ryan M. Kemper, Bhavika C. Chirumamilla, Dennis A. Simpson, Manfred Meng, Gaorav P. Gupta, Daniel J. Crona. BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B012.

Abstract 3077: Identifying Regulators Of Human Adventitial Fibroblast Activation Through A Genome-wide CRISPR Screen

Background: Vascular diseases, including atherosclerosis, are the leading cause of death in the US and world-wide. A quiescent population of fibroblasts that reside in the adventitia (AdvFib) activates and expands upon different vascular insults and contribute to the progression of vascular diseases. However, what regulates AdvFib’s activation remains unknown. We designed a genome-wide CRISPRi screen on human AdvFib to identify these regulators. Methods: We generated an immortalized human coronary AdvFib line that stably expresses dCas9-KRAB, which effectively silences any genes of interest. These cells were infected with a pool of lentivirus carrying guide RNAs (gRNA) covering the entire transcriptome at 200X coverage and allowed to expand freely. Frequency of gRNAs was assessed at different cell passages to identify those that influence AdvFib activation and was followed by experimental validation. Lastly, bioinformatic analysis was performed to determine regulatory pathways and gene networks involved in AdvFib activation. Results: Comparing relative gRNA abundances from early to late passages revealed several hundred putative regulators of AdvFib activation. GO biological process analysis of the top 250 enriched gRNA targets demonstrated disproportionate representation of cyclin inhibitors, validating the effectiveness of our screening. Molecular signatures for adipogenesis and key regulators of tissue mineralization including IBSP and ENPP1 were notably enriched in our screen. Human disease association using PhenGenI revealed a significant association with atherosclerosis. Indeed, numerous coronary artery disease GWAS genes including TCF21 , ZEB2 , CDKN1A , and CDKN2A were enriched in our screen, providing additional support for AdvFib’s importance in atherosclerosis. These hits were subsequently validated individually in vitro. Conclusion: A genome-wide CRISPRi screen of AdvFib activation modulators identified numerous potential regulator genes, many of which have known genetic and observational association with human vascular diseases. This study affirms the potentially important role of AdvFib in human vascular disease and may pave the way to future therapeutics targeting this often neglected cell population.

Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange

Historically, mutagenesis in the non-model enteropathogenic bacterium Clostridioides difficile has been challenging. Developing a versatile and reliable method of generating targeted mutations in C. difficile is important to further our understanding of its pathogenesis. Some of the most common targeted mutagenesis systems rely on allelic exchange mediated by either uracil auxotrophy combined with a toxic uracil precursor, a toxin/anti-toxin system, group II introns, or CRISPR/Cas mutagenesis. However, each of these methods suffers from its own issues. Here, we develop and test an allelic exchange strategy which better facilitates screening for integration and selecting for excision than previous systems. This is achieved by controlling plasmid replication with a theophylline-dependent riboswitch cloned upstream of repA, the gene whose product controls plasmid replication. This allows efficient mutant generation, can be performed in a wild-type strain of C. difficile, does not have the off-target effects inherent to group II introns, and alleviates the problem of testing multiple gRNA targets in CRISPR mutagenesis.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9.

Introduction: CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS. Methods: Using a targeted sequence approach, we studied approximately 20,000 genomic sites with degenerate sequence homology of up to five base pairs relative to guide RNA (gRNA) target sites. We analyzed those sites in 96 individual tree samples that represented 37 independent insertion events containing one or multiples of six unique gRNAs. Results: We found low rates of off-target mutations, with rates of 1.2 × 10-9 in poplar and 3.1 × 10-10 in eucalypts, respectively, comparable to that expected due to sexual reproduction. The rates of mutation were highly idiosyncratic among sites and not predicted by sequence similarity to the target sites; a subset of two gRNAs showed off-target editing of four unique genomic sites with up to five mismatches relative to the true target sites, reaching fixation in some gene insertion events and clonal ramets. The location of off-target mutations relative to the PAM site were essentially identical to that seen with on-target CRISPR mutations. Discussion: The low rates observed support many other studies in plants that suggest that the rates of off-target mutagenesis from CRISPR/Cas9 transgenes are negligible; our study extends this conclusion to trees and other long-lived plants where CRISPR/Cas9 transgenes were present in the genome for approximately four years.

Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML

Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated epigenome/transcriptome/proteome that inhibits differentiation and induces leukemia transformation (sAML) and therapy-refractoriness in MPN with excess blasts (> 5% in PB) or MPN transformed to AML (sAML). Treatment with JAK inhibitor (JAKi), e.g., ruxolitinib, venetoclax or hypomethylating agents alone or in combination are ineffective in improving the poor survival in MPN-sAML. CDK7 is the catalytic component of the general transcription factor TFIIH recruited with RNA-pol II (RNAP2) to transcription start-sites to initiate mRNA transcription. By phosphorylating CDK9 and negative transcript elongation factors, CDK7 also enables productive transcript elongation of oncogenes involved in growth and survival. In MPN-sAML, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1, and MYC. By phosphorylating CDK1/2/4/6, CDK7 also induces cell cycle. RNA-Seq analysis in BEAT AML study showed significantly higher CDK7 expression in MPN-sAML compared to normal progenitor cells. DepMap data involving CRISPR-mediated gene-knockouts show CDK7 as a dependency in the MPN-sAML SET2 and HEL cell lines. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and clinical grade SY-5609, dose-dependently (20 to 250 nM) increased % G1 while reducing the % of cell-cycle S phase SET2 and HEL cells. CDK7i treatment induced in vitro loss of viability in these cells, as well as in patient-derived (PD), CD34+ MPN-sAML but not in normal CD34+ progenitor cells. CRISPR-mediated CDK7 depletion (over 85%) in HEL cells significantly reduced SY-5609-mediated loss of viability. Importantly, pre-treatment with SY-5609 significantly increased sensitivity to ruxolitinib-induced loss of viability in JAKi-resistant SET2 and HEL cells, suggesting that CDK7 inhibition could overcome resistance to JAKi in MPN-sAML cells. RNA-Seq analysis conducted after SY-5609 treatment (100 nM for 16 hours) showed significant negative-enrichment of the gene-sets of MYC and E2F targets, cell-cycle checkpoints and of protein translation initiation/elongation, with log2 fold-decline in the mRNA levels of MYC, MYB, PLK1/4, KIT, CDK6, AURKA, PIM1 and CCND1, but upregulation of CDKN1A and HEXIM1. Following SY-5609 treatment of HEL cells, mass spectrometry demonstrated significant log2 fold-decline in c-Myc, c-Myb, RUNX1, PLK1, PIM1, but increase in p21, CASP9, BAD, DAP and TGFβ1 protein levels. CyTOF analysis demonstrated that SY-5609 treatment markedly reduced c-Myc, RUNX1 and MCL1, while increasing TP53, p21, and cleaved PARP proteins levels in MPN-sAML stem-progenitor cells defined by high expression of CD34, CLEC12A, CD123, CD99 but low expression of CD11b. Co-treatment with SY-5609 and ruxolitinib induced synergistic loss of viability in HEL, SET2 and PD MPN-sAML cells (n = 5) (delta synergy scores > 1.0 by the ZIP method). A CRISPR screen in SET2 and HEL cells targeting epigenetic regulators revealed significant log2 fold-decline in gRNA targeting BRD4, CBP and p300, highlighting them as dependencies. Consistent with this, co-treatment with SY-5609 and the BETi OTX015 was synergistically lethal in SET2, HEL, mouse MPN (JAK2-V617F plus TP53 loss) cells and PD MPN-sAML cells (n=5). SY-5609 treatment also exerted synergistic lethality with the BETi pelabresib or BD2-selective BETi ABBV-744 or the CBP/p300 inhibitor GNE-049 in MPN-sAML cells. In the xenograft model of HEL-Luc/GFP cells in NSG mice, monotherapy with SY-5609 (1.5 or 3.0 mg/kg/day, by oral gavage), vs vehicle control, significantly reduced the MPN-sAML burden and improved survival, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with SY-5609 and OTX015 (30 mg/kg/day by oral gavage) reduced more MPN-sAML burden and significantly improved survival in a HEL-Luc/GFP xenograft model without inducing toxicity. These findings demonstrate promising preclinical activity of CDK7 inhibition against the cellular models of MPN-sAML, supporting the rationale to further evaluate in vivo efficacy of CDK7i-based combinations against advanced MPN with excess blasts or MPN-sAML.

Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive.•All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

CRISPR screen identifies CEBPB as contributor to dyskeratosis congenita fibroblast senescence via augmented inflammatory gene response.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.

1724-P: Caspase 3/7 Activation and RIPK3 Are Dispensable for Synthetic hIAPP-Induced ß-Cell Death

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck &amp; Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p&amp;lt;0.001) and when they were inhibited (n=6, p&amp;lt;0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.

Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P43. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster ispG-idi-dxs-ispD could positively affect the production of lycopene, while the cluster dxr-ispE-ispF-ispH had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. IMPORTANCE We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.

Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.

Evaluate the guide RNA effectiveness via Agrobacterium-mediated transient assays in Nicotiana benthamiana.

CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the Agrobacterium-mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in Nicotiana benthamiana and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting Nicotiana benthamiana and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting NbEDS1, NbWRKY70, GmKTI1, and GmKTI3 had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting NbNDR1 was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.

Polarity of the CRISPR roadblock to transcription.

CRISPR utility relies on stable Cas complex binding to its target site. However, a Cas complex bound to DNA may be removed by motor proteins carrying out other host processes, and the mechanism governing this removal remains unclear. Intriguingly, during CRISPR interference (CRISPRi), RNA polymerase (RNAP) progression is blocked by a bound dCas from the PAM-proximal side but not from the PAM-distal side. This observation offers an appealing opportunity to examine the mechanics of Cas binding stability. In this work, by mapping dCas-DNA interactions, we discovered that the collapse of the dCas R-loop allows RNAP read-through from the PAM-distal side. This finding holds for both dCas9 and dCas12a, despite their gRNA target sequences located at the 5’-end and 3’-end, respectively, and is not unique to RNAP as the DNA translocase Mfd encountered the same polarity. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the gRNAs. This work highlights the importance of the R-loop in dCas stability and provides valuable mechanistic insights for broad applications of CRISPR technology.