Short Deletions Research Articles

The Role of Microhomology-Mediated End Joining (MMEJ) at Dysfunctional Telomeres.

DNA double-strand break (DSB) repair pathways are crucial for maintaining genome stability and cell viability. However, these pathways can mistakenly recognize chromosome ends as DNA breaks, leading to adverse outcomes such as telomere fusions and malignant transformation. The shelterin complex protects telomeres from activation of DNA repair pathways by inhibiting nonhomologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). The focus of this paper is on MMEJ, an error-prone DSB repair pathway characterized by short insertions and deletions flanked by sequence homology. MMEJ is critical in mediating telomere fusions in cells lacking the shelterin complex and at critically short telomeres. Furthermore, studies suggest that MMEJ is the preferred pathway for repairing intratelomeric DSBs and facilitates escape from telomere crisis. Targeting MMEJ to prevent telomere fusions in hematologic malignancies is of potential therapeutic value.

Assembly of Mitochondrial Genomes Using Nanopore Long-Read Technology in Three Sea Chubs (Teleostei: Kyphosidae).

Complete mitochondrial genomes have become markers of choice to explore phylogenetic relationships at multiple taxonomic levels and they are often assembled using whole genome short-read sequencing. Herein, using three species of sea chubs as an example, we explored the accuracy of mitochondrial chromosomes assembled using Oxford Nanopore Technology (ONT) Kit 14 R10.4.1 long reads at different sequencing depths (high, low and very low or genome skimming) by comparing them to 'gold' standard reference mitochondrial genomes assembled using Illumina NovaSeq short reads. In two species of sea chubs, Girella nigricans and Kyphosus azureus, ONT long-read assembled mitochondrial genomes at high sequencing depths (> 25× whole [nuclear] genome) were identical to their respective short-read assembled mitochondrial genomes. Not a single 'homopolymer insertion', 'homopolymer deletion', 'simple substitution', 'single insertion', 'short insertion', 'single deletion' or 'short deletion' were detected in the long-read assembled mitochondrial genomes after aligning each one of them to their short-read counterparts. In turn, in a third species, Medialuna californiensis, a 25× sequencing depth long-read assembled mitochondrial genome was 14 nucleotides longer than its short-read counterpart. The difference in total length between the latter two assemblies was due to the presence of a short motif 14 bp long that was repeated (twice) in the long read but not in the short-read assembly. Read subsampling at a sequencing depth of 1× resulted in the assembly of partial or complete mitochondrial genomes with numerous errors, including, among others, simple indels, and indels at homopolymer regions. At 3× and 5× subsampling, genomes were identical (perfect) or almost identical (quasiperfect, 99.5% over 16,500 bp) to their respective Illumina assemblies. The newly assembled mitochondrial genomes exhibit identical gene composition and organisation compared with cofamilial species and a phylomitogenomic analysis based on translated protein-coding genes suggested that the family Kyphosidae is not monophyletic. The same analysis detected possible cases of misidentification of mitochondrial genomes deposited in GenBank. This study demonstrates that perfect (complete and fully accurate) or quasiperfect (complete but with a single or a very few errors) mitochondrial genomes can be assembled at high (> 25×) and low (3-5×) but not very low (1×, genome skimming) sequencing depths using ONT long reads and the latest ONT chemistries (Kit 14 and R10.4.1 flowcells with SUP basecalling). The newly assembled and annotated mitochondrial genomes can be used as a reference in environmental DNA studies focusing on bioprospecting and biomonitoring of these and other coastal species experiencing environmental insult. Given the small size of the sequencing device and low cost, we argue that ONT technology has the potential to improve access to high-throughput sequencing technologies in low- and moderate-income countries.

Expression and Purification of the Full Length and N-Terminal Truncated Variants of Insect CYP6Z2 in the Cytosol of Escherichia Coli for Potential 3D Experimental Studies

Cytochrome P450 enzymes (P450s) offer innate resistance defence for malaria vectors against the insecticides permitted by WHO to be used in vector control tools. P450s can detoxify broad substrates and simultaneously metabolise them, thus the availability of experimental three-dimensional structures of these key insecticide detoxifiers is vital to improving our knowledge of their enzyme activities. Despite the importance of this family of proteins in insecticide resistance, there are no available experimental three-dimensional structures of insect P450 yet. For this investigation, a carboxy-terminal Histidine-tagged recombinant CYP6Z2 was heterologously expressed in E. coli to generate a soluble holoprotein suitable for an experimental three-dimensional structure. The expressed enzyme was purified from the cytosol of E. coli via the combination of various purification techniques and cholic acid sodium salt. Two truncated N-terminal signal peptides: short deletion of 11 amino acids and long deletion of 23 amino acids of the hydrophobic domain, were created to prevent aggregation, improve solubility, and facilitate crystallisation. The CYP6Z2 (full length) produced a holoprotein with a P450 protein concentration of 0.60 nmol/mL, whereas the two truncated CYP6Z2 isoforms produced only the inactive species with no peak at 450 nm. We conclude that the hydrophobic signal peptide region of the insect Cytochrome P450s seems sensitive and indispensable to ensuring 3-D folding and stability.

Distinct functions of PAXX and MRI during chromosomal end joining.

A key step of Canonical Nonhomologous End Joining (C-NHEJ) is synapsis of DNA double strand break (DSB) ends for ligation. The DNA-PKcs dimer mediates synapsis in a long-range complex with DSB ends remaining apart, whereas the XLF homodimer can mediate synapsis in both long-range and short-range complexes. Recent structural studies found the PAXX homodimer may also facilitate synapsis in long-range complexes with DNA-PKcs via its interactions with Ku70. Thus, we examined the influence of PAXX in C-NHEJ of chromosomal DSBs, which we compared to another Ku-binding factor, MRI. Using EJ of blunt DSBs with Cas9 reporters as a readout for C-NHEJ, we found that PAXX and/or MRI are dispensable. However, when combined with disruption of DNA-PKcs, particularly with DNA-PKcs kinase inhibition, PAXX becomes important for blunt DSB EJ. In contrast, while DNA-PKcs is also important to suppress short deletion mutations with microhomology, this effect is not magnified with PAXX loss. MRI loss had no effect combined with DNA-PKcs disruption, but becomes important for blunt DSB EJ when combined with disruption of XLF, as is PAXX. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with combined loss of MRI. Altogether, we suggest that PAXX promotes DSB end synapsis during C-NHEJ in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI appears to be mainly important in the context of XLF disruption.

Hetero-antagonism of avibactam and sulbactam with cefiderocol in carbapenem-resistant Acinetobacter spp.

Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii, combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.IMPORTANCEThe emergence of carbapenem-resistant Acinetobacter strains as a serious global health threat underscores the urgent need for effective treatment options. Although few drugs show promise against CR Acinetobacter infections, resistance to both drugs has been reported. In this study, the molecular characterization of spontaneous cefiderocol-resistant variants, a CR A. baumannii strain with antagonism to sulbactam, and an A. lwoffii strain with antagonism to avibactam, provides valuable insights into the mechanisms of resistance to cefiderocol. Some mechanisms observed are associated with mutations affecting efflux pumps, regulators, and iron homeostasis genes. These findings highlight the importance of understanding resistance mechanisms to optimize treatment options. They also emphasize the importance of early evaluation of drug synergies to address the challenges of antimicrobial resistance in Acinetobacter infections.

Crossing a CRISPR/Cas9 transgenic tomato plant with a wild-type plant yields diverse mutations in the F1 progeny.

Generating CRISPR/Cas9-mediated mutants in tomato (Solanum lycopersicum L.) involves screening shoots regenerated from cultured cells transformed with a T-DNA harboring sequences encoding Cas9 and single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbers may be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR (RIN) gene, and the resulting F1 seedlings were classified using a kanamycin resistance marker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F1 seedlings, which do carry the T-DNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. This method was also successfully applied to produce mutations in Geranylgeranyl pyrophosphate synthase 2 (GGPS2). Because this method involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants.

Unveiling novel genetic variants in 370 challenging medically relevant genes using the long read sequencing data of 41 samples from 19 global populations.

A large number of challenging medically relevant genes (CMRGs) are situated in complex or highly repetitive regions of the human genome, hindering comprehensive characterization of genetic variants using next-generation sequencing technologies. In this study, we employed long-read sequencing technology, extensively utilized in studying complex genomic regions, to characterize genetic alterations, including short variants (single nucleotide variants and short insertions and deletions) and copy number variations, in 370 CMRGs across 41 individuals from 19 global populations. Our analysis revealed high levels of genetic variants in CMRGs, with 68.73% exhibiting copy number variations and 65.20% containing short variants that may disrupt protein function across individuals. Such variants can influence pharmacogenomics, genetic disease susceptibility, and other clinical outcomes. We observed significant differences in CMRG variation across populations, with individuals of African ancestry harboring the highest number of copy number variants and short variants compared to samples from other continents. Notably, 15.79% to 33.96% of short variants were exclusively detectable through long-read sequencing. While the T2T-CHM13 reference genome significantly improved the assembly of CMRG regions, thereby facilitating variant detection in these regions, some regions still lacked resolution. Our results provide an important reference for future clinical and pharmacogenetic studies, highlighting the need for a comprehensive representation of global genetic diversity in the reference genome and improved variant calling techniques to fully resolve medically relevant genes.

A Subset of Microsatellite Unstable Cancer Genomes Prone to Short Insertions over Deletions Is Associated with Elevated Anticancer Immunity.

Deficiencies in DNA mismatch repair (MMRd) leave characteristic footprints of microsatellite instability (MSI) in cancer genomes. We used data from the Cancer Genome Atlas and International Cancer Genome Consortium to conduct a comprehensive analysis of MSI-associated cancers, focusing on indel mutational signatures. We classified MSI-high genomes into two subtypes based on their indel profiles: deletion-dominant (MMRd-del) and insertion-dominant (MMRd-ins). Compared with MMRd-del genomes, MMRd-ins genomes exhibit distinct mutational and transcriptomic features, including a higher prevalence of T>C substitutions and related mutation signatures. Short insertions and deletions in MMRd-ins and MMRd-del genomes target different sets of genes, resulting in distinct indel profiles between the two subtypes. In addition, indels in the MMRd-ins genomes are enriched with subclonal alterations that provide clues about a distinct evolutionary relationship between the MMRd-ins and MMRd-del genomes. Notably, the transcriptome analysis indicated that MMRd-ins cancers upregulate immune-related genes, show a high level of immune cell infiltration, and display an elevated neoantigen burden. The genomic and transcriptomic distinctions between the two types of MMRd genomes highlight the heterogeneity of genetic mechanisms and resulting genomic footprints and transcriptomic changes in cancers, which has potential clinical implications.

Characterization of Japanese Encephalitis Virus Isolated from Persistently Infected Mouse Embryo Cells.

Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus Flavivirus of the family Flaviviridae. Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3'NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3'NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency.

DNA Checkpoint Gene Mutation as a Biomarker for Immune Checkpoint Inhibitor Therapy in Advanced Biliary Tract Cancer.

The DNA checkpoint (DNACHK) pathway is engaged in signaling the need for cell cycle arrest. This pathway is being actively researched to assess its role in cancer immunotherapy. A total of 62 patients participated in this study. These patients were treated with immune checkpoint inhibitors (ICIs) for advanced biliary tract cancers (BTCs) from March 2020 to August 2022 at Samsung Medical Center. DNACHK mutated were defined as genomic alterations, such as single nucleotide variants, multi-nucleotide variants, and short insertion and deletions in seven genes; checkpoint kinase 1 (CHEK1), checkpoint kinase 2 (CHEK2), BRCA1, DNA repair-associated (BRCA1), the serine/threonine kinase ATM, the serine/threonine kinase ATR, mediator of DNA damage checkpoint 1 (MDC1) and tumor protein p53 binding protein 1 (TP53BP1). We analyzed the effect of DNACHK mutations on the efficacy of ICIs in advanced BTCs. Patient median age at diagnosis was 68.0 years. 10 patients (16.1%) had GB cancer; the remaining patients (n=52, 83.9%) were diagnosed with cholangiocarcinoma. Thirty-seven (59.7%) patients were categorized into the DNACHK wild-type (WT) group and 25 (40.3%) into the DNACHK mutated (MT) group. The most observed DNA checkpoint mutations were ATM mutations (n=14). Patients in the DNACHK MT group had better disease control rate (DCR) than patients in the DNACHK WT (60.0% vs. 48.6%, p=0.53). Median overall survival (OS) was 8.1 months (95% CI 5.1-22.8) in the MT group and 5.6 months (95%CI 3.1-11.0) in the WT group (p=0.33). The DNACHK pathway is expected to serve as a potential biomarker for ICI treatment.

Pharmacogenomic Predictors of Antibiotic-Associated Drug-Induced Liver Injury in Critically Ill Children: Observational Study Results

INTRODUCTION. The pathogenesis of antibiotic-associated drug-induced liver injury (DILI) in children has not been fully elucidated to date. Certain genotypes in patients increase the probability of developing DILI. Therefore, the identification of pharmacogenetic markers associated with DILI in children is essential.АIM. This study aimed to identify pharmacogenetic biomarkers of new-onset DILI associated with tigecycline and meropenem in children.MATERIALS AND METHODS. This prospective observational study was conducted in the Morozov Children’s City Clinical Hospital from 1 February 2020 to 1 September 2021. The study analysed the incidence and types of antibiotic-associated adverse drug reactions (ADRs) in 100 critically ill children aged 0 to 17 years (44 boys and 56 girls). Pharmacogenetic testing was performed in children with ADRs (n=30) to identify potential mechanisms involved in the development of their ADRs. The authors isolated and tested DNA from buccal epithelium swabs using the Agena Bioscience iPLEX® PGx Pro-based VeriDose® Core Panel covering 68 single nucleotide polymorphisms (SNPs) or short insertions and deletions (INDELs) and 5 copy number variants (CNVs).RESULTS. The odds of developing DILI associated with meropenem and tigecycline were higher in carriers of the homozygous cytochrome genotype CYP3A5*3/*3 (OR: 12.6; 95% CI: 1.9–79.4, r=6.54, p=0.011) than in patients with the heterozygous genotype CYP3A5*1A/*3. The odds were even higher in patients not carrying the CYP3A5*1A/*3 genotype (OR: 17.14; 95% CI: 1.79–16.3, r=6.24, p=0.013). The detection of the CYP3A5*3/*3 ge­notype had a prognostic accuracy of 76.7%, a sensitivity of 82%, and a specificity of 74% in predicting the risk of DILI associated with meropenem and tigecycline. Moreover, children with DILI carried the heterozygous ge­notype SLCO1B1*1/*5 (rs4149056 polymorphism) more often than children with other adverse reactions ­associated with meropenem and tigecycline (r=9.8, p=0.002).CONCLUSION. The results of this study prove the prognostic significance of the homozygous cytochrome genotype CYP3A*3/*3 as an indicator of a potential risk for developing DILI associated with meropenem and tigecycline in children in critical conditions.The study was registered at ClinicalTrials.gov under No. NCT04141657 on 24 October 2019.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Abstract 7624: DNA checkpoint gene mutation as a biomarker of immune checkpoint inhibitor in advanced biliary tract cancer

Abstract Introduction: The DNA checkpoint (DNACHK) pathway is engaged in signaling the need for cell cycle arrest. The G1 and S phase checkpoint allows for repair of damaged DNA, preventing damaged DNA from being copied. The G2 and M phase checkpoint prevents transmission of mutated DNA to the next generation. This pathway, along with mismatch repair (MMR), homologous recombination (HR), and base excision repair (BER), is being actively researched to assess its role in cancer immunotherapy. Methods: Sixty-two patients participated in this study. These patients were treated with immune checkpoint inhibitors (ICIs) for advanced biliary tract cancers (BTCs) from Mar. 2020 to Aug. 2022 at Samsung Medical Center. DNACHK mutations were defined as genomic alterations such as SNVs, MNVs, and short insertion and deletions in seven genes These genes include checkpoint kinase 1 (CHEK1), checkpoint kinase 2 (CHEK2), BRCA1, DNA repair associated (BRCA1), the serine/threonine kinase ATM, the serine/threonine kinase ATR, and mediator of DNA damage checkpoint 1 (MDC1). We analyzed the effect of DNACHK mutations on the efficacy of ICIs in advanced BTCs. Results: Patient median age at diagnosis was 68.0 years. Ten patients (16.1%) had gall bladder (GB) cancer; the remaining patients (n = 52, 83.9%) were diagnosed with cholangiocarcinoma. Among the 62 patients with advanced BTCs treated with ICIs, 37 (59.7%) were categorized into the DNACHK wild-type (WT) group and 25 (40.3%) into the DNACHK mutated (MT) group. The most observed DNA checkpoint mutations were ATM mutations (n=14). MDC1 mutations were found in seven patients, ATR mutations in three, BRCA1 mutations in four, and CHEK2 mutations in three. Patients in the DNACHK MT group had better disease control rates than patients in the DNACHK WT group (60.0% vs. 48.6%, p=0.53). Median overall survival (OS) was 8.1 months (95% CI, 5.1, 22.8) in the MT group and 5.6 months (95% CI, 3.1, 11.0) in the WT group (p=0.33). Conclusions: The results of this study suggest that the DNACHK pathway may be a valuable source of biomarkers for ICI and worthy of further study. Citation Format: Ji Eun Shin, Seung Tae Kim, Dae-Ho Choi, Junho Lee, Jiyeon Hyeon. DNA checkpoint gene mutation as a biomarker of immune checkpoint inhibitor in advanced biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7624.

Real-world analysis of patients with advanced gastrointestinal (GI) cancers harboring inactivating TSC1 and TSC2 alterations using the Foundation Medicine genomic database.

558 Background: Genomic profiling of GI tumors can identify potentially actionable genetic alterations, ultimately leading to better outcomes in patients who currently have few treatment options. In exploratory biomarker analyses of patients with perivascular epithelioid cell tumors treated with the mTOR inhibitor nab-sirolimus (AMPECT, NCT02494570), those with inactivating alterations in the tumor suppressor genes TSC1 or TSC2 (critical negative regulators of mTOR activity) had confirmed responses (8/9 patients with inactivating TSC2 alterations and 1/5 patients with inactivating TSC1 alterations). PRECISION 1, a currently enrolling tumor-agnostic study, will evaluate nab-sirolimus in patients with TSC1 or TSC2 alterations. To enumerate the frequency of these alterations in GI cancers, we analyzed data from a real-world genomic database. Methods: Next-generation sequencing (NGS) data from Foundation Medicine’s genomic database of patients with advanced cancer were analyzed using the FoundationInsights web-based platform to identify patients with GI cancers who had inactivating TSC1 or TSC2 alterations (short variants [base substitutions, insertions and deletions], rearrangements, and copy number deletions). Tumor mutational burden (TMB), microsatellite instability status and other mutations were also characterized in this group of patients. Results: As of 29 March 2022, tumor tissue NGS data were available for 5816 patients with GI cancers bearing alterations in TSC1 or TSC2. Of these, 1642 patients had at least one inactivating alteration. The GI tumor types that most frequently expressed inactivating alterations in TSC1 or TSC2 were hepatocellular carcinoma (alterations in 6.5%); colorectal adenocarcinoma (1.7%); gastrointestinal stromal tumors (1.7%); and cholangiocarcinoma (1.6%). Of all 1642 patients, 688 were female, and most (74%) were 51–80 years of age when the sample was obtained. Of patients with inactivating TSC1 and TSC2 alterations, 37.6% were in TSC1 and 62.9% were in TSC2 and included short variants (31.3% and 48.4%), rearrangements (2.7% and 6.6%), and copy number deletions (3.5% and 7.4%), respectively. TMB was low (<6 mutations/megabase) in 58% of patients, and most patients had microsatellite stable (MSS; 71.3%) tumors. Other commonly mutated genes in this cohort were TP53 (62.7%), APC (38.5%) and KRAS (30.4%). Conclusions: TSC1 and/or TSC2 alterations were commonly observed in GI cancers. These cancers were frequently microsatellite stable and of low TMB suggesting that these are actionable alterations that may be candidates for targeted therapy. This hypothesis is being tested in the PRECISION 1 (NCT05103358) trial which is open for enrollment or just-in-time clinical trial sites and available to patients with GI cancers harboring TSC1 or TSC2 alterations.

MotifbreakR v2: expanded variant analysis including indels and integrated evidence from transcription factor binding databases.

motifbreakR scans genetic variants against position weight matrices of transcription factors (TFs) to determine the potential for the disruption of binding at the site of the variant. It leverages the Bioconductor suite of software packages and annotations to query a diverse array of genomes and motif databases. Initially developed to interrogate the effect of single-nucleotide variants on TF binding sites, in motifbreakR v2, we have updated the functionality. New features include the ability to query other types of complex genetic variants, such as short insertions and deletions. This capability allows modeling a more extensive array of variants that may have significant effects on TF binding. Additionally, predictions based on sequence preference alone can indicate many more potential binding events than observed. Adding information from DNA-binding sequencing datasets lends confidence to motif disruption prediction by demonstrating TF binding in cell lines and tissue types. Therefore, motifbreakR can directly query the ReMap2022 database for evidence that a TF matching the disrupted motif binds over the disrupting variant. Finally, in motifbreakR, in addition to the existing interface, we implemented an R/Shiny graphical user interface to simplify and enhance access to researchers with different skill sets. motifbreakR is implemented in R. Source code, documentation, and tutorials are available on Bioconductor at https://bioconductor.org/packages/release/bioc/html/motifbreakR.html and GitHub at https://github.com/Simon-Coetzee/motifBreakR.

Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates.

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines. Key features • Versatile approach combining AAV-DJ donors and CRISPR ribonucleoproteins, providing an efficient method for long and short edits, insertions, and deletions in human pluripotent stem cells. • One-step cloning method for rapid generation of customized AAV donor plasmids. • Simplified AAV purification pipeline for ready-to-infect virion preparations.

CRISPR/Cas9-mediated editing of the phytoene desaturase gene in Vanilla planifolia enabling targeted domestication

ABSTRACT Vanilla is an undomesticated species and the botanical source of vanilla extract. There has been increasing demand to expand vanilla cultivation in the Americas to strengthen the vanilla supply chain. Recent development in vanilla genomics resources has opened opportunities for interrogation of vanilla diversity and discovery of candidate genes responsible for key traits. The ability to precisely change genomic sequences could be a valuable tool for domesticating vanilla while enabling functional genomics. Here we report the successful biolistic-mediated transformation of seed-derived callus and CRISPR/Cas9 gene editing of the phytoene desaturase (PDS) gene in vanilla. Sequencing of leaves from regenerated plantlets displaying the expected albino phenotype showed short deletions from 1 to 163 base pairs in the phytoene desaturase gene at each of three target loci. The ability to apply gene editing to vanilla can now be used in combination with the vanilla genome to create targeted knockouts to support the domestication of vanilla.

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role inRYMVsusceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Abstract C019: Inactivating TSC1 and TSC2 alterations, co-mutations, and genomic instability in advanced cancers: Analysis of a real-world (RW) patient (pt) population using the Foundation Medicine genomic database

Abstract Background Tumor suppressor genes tuberous sclerosis complex subunit 1 and 2 (TSC1, TSC2) are critical negative regulators of mTOR activity. In the exploratory biomarker analysis (AMPECT, NCT02494570) and an Expanded Access Program (NCT03817515), pts with inactivating alterations in TSC1 or TSC2 demonstrated responses with the mTOR inhibitor, nab-sirolimus. While these alterations are observed in most cancer histologies, characterization and distribution of TSC1 and TSC2 alterations across tumor types are not well understood. PRECISION I (NCT05103358), a currently enrolling tumor-agnostic study, will assess the clinical benefit of nab-sirolimus in pts with cancer who harbor inactivating TSC1 and TSC2 alterations. To appreciate the potential for TSC1 and TSC2 as therapeutic biomarkers, we analyzed TSC1 and TSC2 mutational data from a RW genomic database. Methods Next-generation sequencing (NGS) data from Foundation Medicine’s genomic database of pts primarily with advanced cancer were analyzed using the FoundationInsights™ web-based platform. Frequency and actionability of TSC1 and TSC2 alterations were assessed. Known or likely pathogenic inactivating TSC1 and TSC2 alterations (ie, indel, rearrangement, frameshift, biallelic loss, nonsense, and some missense mutations) were further characterized for co-mutations, tumor mutational burden (TMB), and microsatellite instability status. Results As of 29 Mar 2022, 438,974 pts with advanced cancer in the Foundation Medicine genomic database had tumor tissue NGS data available. Known/likely inactivating alterations in TSC1 and TSC2 were identified in 8464 (1.9%) pts; 4.3% had variants of unknown significance, primarily missense (~89%), that were not known to be inactivating. Of the inactivating alterations, 52.1% were TSC1 and 48.6% TSC2, including short variants, defined as base substitutions and short indels (85.0%), rearrangements (10.8%), and copy number deletions (17.9%). Alterations were most frequently identified in urinary bladder (8.6%), kidney and renal pelvis (5.8%), corpus and uterus (3.4%), soft tissue (including heart; 2.4%), and liver and intrahepatic bile duct (2.3%) cancers. The most frequently co-occurring mutations included TP53 (62.2%), CDKN2A (26.2%), and TERT (22.2%). TMB was low (<6 mutations per megabase) in 51.6% of pts with inactivating alterations in TSC1 and TSC2. Samples were mostly microsatellite stable (MSS; 82.2%) across tumor types. Conclusion In a large RW database of pts with advanced cancer, inactivating TSC1 and TSC2 variants occurred in 1.9% of pts. These occurred across common tumor types at rates as high as 8.6% (urinary bladder tumors). A majority of pts had low TMB and/or MSS status, suggesting that these are actionable alterations and not passenger events (Bennett et al, Mod Pathol 2022). Common co-mutations also have limited clinical actionability. These observations suggest cancers with TSC1 and TSC2 alterations may be candidates for targeted therapy; this hypothesis is being tested in the currently enrolling PRECISION I (NCT05103358) study. Citation Format: David J. Kwiatkowski, Norma A. Palma, Willis H. Navarro, Gopa Iyer. Inactivating TSC1 and TSC2 alterations, co-mutations, and genomic instability in advanced cancers: Analysis of a real-world (RW) patient (pt) population using the Foundation Medicine genomic database [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C019.