CRISPR Research Articles

Pharmaceutical Potential of Remedial Plants and Helminths for Treating Inflammatory Bowel Disease.

Research is increasingly revealing that inflammation significantly contributes to various diseases, particularly inflammatory bowel disease (IBD). IBD is a major medical challenge due to its chronic nature, affecting at least one in a thousand individuals in many Western countries, with rising incidence in developing nations. Historically, indigenous people have used natural products to treat ailments, including IBD. Ethnobotanically guided studies have shown that plant-derived extracts and compounds effectively modulate immune responses and reduce inflammation. Similarly, helminths and their products offer unique mechanisms to modulate host immunity and alleviate inflammatory responses. This review explored the pharmaceutical potential of Aboriginal remedial plants and helminths for treating IBD, emphasizing recent advances in discovering anti-inflammatory small-molecule drug leads. The literature from Scopus, MEDLINE Ovid, PubMed, Google Scholar, and Web of Science was retrieved using keywords such as natural product, small molecule, cytokines, remedial plants, and helminths. This review identified 55 important Aboriginal medicinal plants and 9 helminth species that have been studied for their anti-inflammatory properties using animal models and in vitro cell assays. For example, curcumin, berberine, and triptolide, which have been isolated from plants; and the excretory-secretory products and their protein, which have been collected from helminths, have demonstrated anti-inflammatory activity with lower toxicity and fewer side effects. High-throughput screening, molecular docking, artificial intelligence, and machine learning have been engaged in compound identification, while clustered regularly interspaced short palindromic repeats (CRISPR) gene editing and RNA sequencing have been employed to understand molecular interactions and regulations. While there is potential for pharmaceutical application of Aboriginal medicinal plants and gastrointestinal parasites in treating IBD, there is an urgent need to qualify these plant and helminth therapies through reproducible clinical and mechanistic studies.

Ultrasensitive detection of MMP-8 in saliva for monitoring periodontitis using an Immuno-CRISPR/Cas12a assay

Periodontitis is one of the most prevalent oral diseases, and the diagnosis and monitoring of periodontitis rely on clinical and imaging examinations. Matrix metalloproteinase-8 (MMP-8) in saliva is a reliable indicator of periodontitis. The traditional detection method is enzyme-linked immunosorbent assay (ELISA), which requires professional expertise and is not sensitive enough to low-abundance protein detection. In this study, we developed a nanoimmunoassay combined with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas)12a for ultrasensitive detection of MMP-8 in saliva. Antibody-DNA-gold nanoparticles (AuNPs) and trans-cleavage activity of CRISPR/Cas12a were employed for signal amplification. With this protocol, the limit of detection (LoD) for MMP-8 was reduced to the fg/mL, indicating 100-fold higher sensitivity than that of the commercial ELISA kit and its amenability for ultrasensitive detection of trace proteins. The diagnostic sensitivity of the assay was 100 %, and the specificity was 83.3 % based on the testing of 23 clinical saliva samples. The sensitivity and specificity were considerably higher than those for ELISA. Thus, this immuno-CRISPR/Cas12a assay holds promise for both diagnosing and monitoring periodontitis in clinical settings.

Mott-Schottky junction mediated photothermal-pyroelectric synergy for effective collection of waste heat in flexible sensing platform

Solar–energy–induced photothermal–pyroelectric synergy sensing platform provides a win-win route to harvest waste heat and convert energy. However, the increase of carrier collision probability from high temperature inevitably leads to the loss of quantum efficiency. Herein, a flexible photothermal-pyroelectric electrode platform with Schottky junction was successfully constructed for maximum utilization of carrier. Under solar-simulated irradiation, the electron-rich and electron-depletion region formed by the Bi13S18Br2-S/alloy rectifier interface in flexible polyvinylidene difluoride-hexafluoropropylene film can increase the accumulation of photogenerated electrons. Meanwhile, the surface bound charges released by dipole oscillation in photothermal-pyroelectric field are continuously supplemented by the emerged high concentration of photogenerated electrons from the Schottky junction, and this synergistic effect prolonged their lifetimes and significantly improves the photoelectric conversion efficiency. To reasonably realize the accurate quantification of the simulated target, the cleavage activity of the CRISPR–Cas system can be specifically restored with the assistance of a synergistic dual-activator, releasing silica as a padlock to produce a target concentration-dependent photoelectric signal. Besides, the temperature variation on the electrode interface was simulated, revealing the synergistic effect between Schottky junction and photothermal–pyroelectric field under photoexcitation. This work broadens a new perspective for upgrading photoelectron utilization and overall performance of flexible sensing platform.

High-throughput characterization of functional variants highlights heterogeneity and polygenicity underlying lung cancer susceptibility

Genome-wide association studies (GWASs) have identified numerous lung cancer risk-associated loci. However, decoding molecular mechanisms of these associations is challenging since most of these genetic variants are non-protein-coding with unknown function. Here, we implemented massively parallel reporter assays (MPRAs) to simultaneously measure the allelic transcriptional activity of risk-associated variants. We tested 2,245 variants at 42 loci from 3 recent GWASs in East Asian and European populations in the context of two major lung cancer histological types and exposure to benzo(a)pyrene. This MPRA approach identified one or more variants (median 11 variants) with significant effects on transcriptional activity at 88% of GWAS loci. Multimodal integration of lung-specific epigenomic data demonstrated that 63% of the loci harbored multiple potentially functional variants in linkage disequilibrium. While 22% of the significant variants showed allelic effects in both A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cell lines, a subset of the functional variants displayed a significant cell-type interaction. Transcription factor analyses nominated potential regulators of the functional variants, including those with cell-type-specific expression and those predicted to bind multiple potentially functional variants across the GWAS loci. Linking functional variants to target genes based on four complementary approaches identified candidate susceptibility genes, including those affecting lung cancer cell growth. CRISPR interference of the top functional variant at 20q13.33 validated variant-to-gene connections, including RTEL1, SOX18, and ARFRP1. Our data provide a comprehensive functional analysis of lung cancer GWAS loci and help elucidate the molecular basis of heterogeneity and polygenicity underlying lung cancer susceptibility.

CRISPR-Cas system in multi drugs resistant Klebsiella pneumoniae from different clinical samples and its correlation with antibiotic-resistant genes in Mosul city / Iraq

CRISPR-Cas system in multi drugs resistant Klebsiella pneumoniae from different clinical samples and its correlation with antibiotic-resistant genes in Mosul city / Iraq

A new anti-CRISPR gene promotes the spread of drug-resistance plasmids in Klebsiella pneumoniae.

The Klebsiella pneumoniae (K. pneumoniae, Kp) populations carrying both resistance-encoding and virulence-encoding mobile genetic elements (MGEs) significantly threaten global health. In this study, we identified a new anti-CRISPR gene (acrIE10) on a conjugative plasmid with self-target sequence in K. pneumoniae with type I-E* CRISPR-Cas system. AcrIE10 interacts with the Cas7* subunit of K. pneumoniae I-E* CRISPR-Cas system. The crystal structure of the AcrIE10-KpCas7* complex suggests that AcrIE10 suppresses the I-E* CRISPR-Cas by binding directly to Cas7 to prevent its hexamerization, thereby preventing the surveillance complex assembly and crRNA loading. Bioinformatic and functional analyses revealed that AcrIE10 is functionally widespread across diverse species. Our study reports a novel anti-CRISPR and highlights its potential role in spreading resistance and virulence among pathogens.

Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain’s Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Reelin marks cocaine-activated striatal ensembles, promotes neuronal excitability, and regulates cocaine reward.

Drugs of abuse activate defined neuronal ensembles in brain reward structures such as the nucleus accumbens (NAc), which are thought to promote the enduring synaptic, circuit, and behavioral consequences of drug exposure. While the molecular and cellular effects arising from experience with drugs like cocaine are increasingly well understood, the mechanisms that sculpt NAc ensemble participation are largely unknown. Here, we leveraged unbiased single-nucleus transcriptional profiling to identify expression of the secreted glycoprotein Reelin (encoded by the Reln gene) as a marker of cocaine-activated neuronal ensembles within the rat NAc. Multiplexed in situ detection confirmed selective expression of the immediate early gene Fos in Reln+ neurons after cocaine experience, and also revealed enrichment of Reln mRNA in Drd1 + medium spiny neurons (MSNs) in both the rat and human brain. Using a novel CRISPR interference strategy enabling selective Reln knockdown in the adult NAc, we observed altered expression of genes linked to calcium signaling, emergence of a transcriptional trajectory consistent with loss of cocaine sensitivity, and a striking decrease in MSN intrinsic excitability. At the behavioral level, loss of Reln prevented cocaine locomotor sensitization, abolished cocaine place preference memory, and decreased cocaine self-administration behavior. Together, these results identify Reelin as a critical mechanistic link between ensemble participation and cocaine-induced behavioral adaptations.

Deep learning enhancing guide RNA design for CRISPR/Cas12a-based diagnostics.

Rapid and accurate diagnostic tests are fundamental for improving patient outcomes and combating infectious diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas12a-based detection system has emerged as a promising solution for on-site nucleic acid testing. Nonetheless, the effective design of CRISPR RNA (crRNA) for Cas12a-based detection remains challenging and time-consuming. In this study, we propose an enhanced crRNA design system with deep learning for Cas12a-mediated diagnostics, referred to as EasyDesign. This system employs an optimized convolutional neural network (CNN) prediction model, trained on a comprehensive data set comprising 11,496 experimentally validated Cas12a-based detection cases, encompassing a wide spectrum of prevalent pathogens, achieving Spearman's ρ = 0.812. We further assessed the model performance in crRNA design for four pathogens not included in the training data: Monkeypox Virus, Enterovirus 71, Coxsackievirus A16, and Listeria monocytogenes. The results demonstrated superior prediction performance compared to the traditional experiment screening. Furthermore, we have developed an interactive web server (https://crispr.zhejianglab.com/) that integrates EasyDesign with recombinase polymerase amplification (RPA) primer design, enhancing user accessibility. Through this web-based platform, we successfully designed optimal Cas12a crRNAs for six human papillomavirus (HPV) subtypes. Remarkably, all the top five predicted crRNAs for each HPV subtype exhibited robust fluorescent signals in CRISPR assays, thereby suggesting that the platform could effectively facilitate clinical sample testing. In conclusion, EasyDesign offers a rapid and reliable solution for crRNA design in Cas12a-based detection, which could serve as a valuable tool for clinical diagnostics and research applications.

HTA81 Assessing Evidence Packages of CRISPR Technologies: Will They Meet Payer Needs?

HTA81 Assessing Evidence Packages of CRISPR Technologies: Will They Meet Payer Needs?

Efficient CRISPR-Cas12f1-Mediated Multiplex Bacterial Genome Editing via Low-Temperature Recovery.

CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.

Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.

Multiplexed Detection Strategies for Biosensors Based on the CRISPR-Cas System.

A growing number of applications require simultaneous detection of multiplexed nucleic acid targets in a single reaction, which enables higher information density in combination with reduced assay time and cost. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-Cas system have broad applications for the detection of nucleic acids due to their strong specificity, high sensitivity, and excellent programmability. However, realizing multiplexed detection is still challenging for the CRISPR-Cas system due to the nonspecific collateral cleavage activity, limited signal reporting strategies, and possible cross-reactions. In this review, we summarize the principles, strategies, and features of multiplexed detection based on the CRISPR-Cas system and further discuss the challenges and perspective.

Clustered Regularly Interspaced Short Palindromic Repeat-1 (CRISPR-1) Locus as a Tool for Tracing the Zoonotic History of Salmonella enterica Strains.

Background Salmonella enterica is a significant foodborne pathogen that causes considerable illness and death in humans and animals. The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system in bacteria acts as an adaptive immune defense against invasive genetic elements by incorporating short intergenic spacers (IGSs) into CRISPR loci. These loci serve as molecular records of past interactions with phages and plasmids, providing insights into the transmission and evolution of bacterial strains across different hosts. Aim This study aimed to investigate the diversity of IGSs in the CRISPR-1 locus of S. enterica isolates from humans and camels. The objective was to assess the potential of IGSs to distinguish strains, track sources, and understand patterns of zoonotic transmission. Materials and methods Genomic DNA was extracted from multiple strains of S. enterica, and the CRISPR-1 locus was polymerase chain reaction (PCR) amplified and sequenced. The sequences were compared to identify distinct patterns of IGSs and potential host-specific characteristics. Sanger sequencing and bioinformatics tools were used to classify the IGSs and determine their similarity to known sequences in the National Center for Biotechnology Information (NCBI) database. Results Sequence analysis revealed five distinct CRISPR-1 types among S. enterica isolates from humans and three among camel isolates. The presence of shared IGSs between human and camel S. entericaisolates suggested zoonotic or reverse-zoonotic transmission events. Additionally, host-specific unknown IGSs (UIGS) were identified. Importantly, camel isolates initially identified as S. enterica subspeciesenterica serovar Enteritidis based on rrnH gene sequencing were reclassified as S. enterica serovar Enteritidis based on CRISPR-1 profiling, demonstrating the higher resolution of CRISPR-based genotyping. Conclusion The diversity of IGSs in the CRISPR-1 locus effectively differentiated S. enterica strains and provided insights into their evolutionary origins and transmission dynamics. CRISPR-based genotyping proves to be a promising tool to complement traditional serotyping methods, enhancing the molecular epidemiology of salmonellosis and potentially leading to better management and control strategies for this pathogen.

CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion.

Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

CRISPR-CAS System: Current Applications and Future Prospect

The CRISPR-Cas system is a revolutionary technology that has transformed gene editing and has the potential to revolutionize many fields. It is a bacterial immune system that can be programmed to recognize and cleave specific DNA sequences using Cas proteins and guide RNAs. This technology has numerous biotechnological and bioengineering applications and is simpler to design with high targeting efficiency and multiplex editing capability compared to older gene editing technologies. This review aims to explore the general application, impact on molecular biology and medicine, limitations, and future potential of the CRISPR-Cas system. With its potential to revolutionize medicine, agriculture, and environmental science, the CRISPR-Cas system is a powerful gene editing tool that offers hope for a wide range of applications.

Abstract A023: Determining genetic interaction from double knockout CRISPR screening

Abstract Background For decades double knockout (KO) perturbation screens were limited to model organisms such as S. pombe and S. cerevisiae. CRISPR technology has revolutionized genetic interaction discovery by allowing large scale screening in human cell lines, organoids, and mouse models. However, there remains much uncertainty regarding the optimal way to determine the presence of genetic interaction from the raw data generated from these large scale double perturbation experiments. Here we compare two different analysis methods run on the same normalized dataset to determine to what degree does the analysis method influence the determination of genetic interaction. Methods A publicly available genetic interaction dataset containing 24,908 double knock-out constructs across three cell lines (Hela, A549, 293T) in four time points (day 3, 14, 21, 28) and two replicates generated from a pair-wise CRISPR-Cas9 KO screen was used for analysis (Shen et al, Nature Methods, 2017). These data were used to measure single gene fitness scores for 73 known cancer driver genes and all 2628 pair-wise interactions using (1) the numerical Bayesian method from Shen et al, called CTG (Compositional and Time-course-aware Genetic analysis), and (2) the variational Bayesian method GEMINI (Zamanighomi et al, Genome Biology, 2019). Results Single gene KO fitness measurements from CTG and GEMINI were highly correlated for all three cell lines (pearson r 0.678, 0.604, 0.784 for HeLa, A549, and 293T, respectively; p&amp;lt; 0.1 x10-8 for each). In contrast, correlation of genetic interaction scores between the two methods was essentially random: HeLa r= -0.0143, p= 0.47, A549; A549 r= -0.0476, p=0.015; 293T r= -0.0135, p= 0.49. Of 52 synthetic lethal interactions identified by CTG in HeLa at z-score cut off -3, none were identified by GEMINI at same Z cutoff. Conversely of 4 interactions identified by GEMINI, none were identified by CTG. Similarly in A549, of 57 interactions identified by CTG none were identified by GEMINI, of 3 interactions identified by GEMINI none were identified by CTG. Restricting to genetic interactions that were validated in low-throughput drug-drug assays, of 5 synthetic lethal interactions found in HeLa by CTG (CHEK1-MAP2K1, CHEK1-TYMS, ADA-CHEK1, ATM-CHEK1, CDK9-CHEK1) all but CHEK1-TYMS were validated in low-throughput assays. However none of the 5 were scored as hits by GEMINI. Of 3 interactions scored as synthetic lethal in A549 (PRKDC-RRM2, CDK9-PRKDC, CDK4-PRKDC) all but PRKDC-RRM2 were validated, none of the 3 were scored as hits by GEMINI. Conclusions This study highlights dramatic differences in calculated genetic interaction scores from two different computational algorithms applied to the same experimental data. With only 8 of 2628 (0.3%) interactions tested in validation experiments it is not currently possible to know the ground truth in order to assess which method is most accurate. The generation of synthetic genetic interaction data will be an important step for further optimization of algorithms to detect genetic interaction. Citation Format: John Paul Shen, Yue Gu, Saikat Chowdhury. Determining genetic interaction from double knockout CRISPR screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A023.