IMMU-37. DISRUPTION OF PD-L1 BY ENHANCED TWO-SGRNAS CRISPR/CAS9 IN TREATMENT OF GLIOBLASTOMA

Anti-glioblastoma multiform (GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) plays a key role in GBM immunosuppression, vitality, proliferation, and migration. Targeting PD-L1 for immunotherapy is a promising new avenue for treating GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in PD-L1 genomic sequence. Two sgRNAs combination with a homology-directed repair template (HDR) was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into one CRISPR/Cas9 plasmid, and was co-transfected with HDR. GFP tagged CRISPR/Cas9 plasmid containing of g82 and g165 (Cas9-g82/165) was loaded into Rhodamine labeled nanoparticles (Cas9-g82/165-NPs) and then treated to GBM U87 cells. The enhanced intracellular uptake and transfection of Cas9-g82/165-NPs were detected by a fluorescence microscopy. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, two-sgRNAs CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.

Phage-encoded ribosomal protein S21 expression is linked to late stage phage replication

The ribosomal protein S21 (bS21) gene has been detected in diverse viruses with a large range of genome sizes, yet its in situ expression and potential significance have not been investigated. Here, we report five closely related clades of bacteriophages (phages) represented by 47 genomes (8 curated to completion and up to 331 kbp in length) that encode a bS21 gene. The bS21 gene is on the reverse strand within a conserved region that encodes the large terminase, major capsid protein, prohead protease, portal vertex proteins and some hypothetical proteins. These phages are predicted to infect Bacteroidetes species that inhabit a range of depths in freshwater lakes. Transcriptionally active bS21-encoding phages were sampled in the late-stage of replication, when core structural genes, bS21 and a neighboring gene of unknown function were highly expressed. Thus, our analyses suggest that bS21, which is involved in translation initiation, substitutes into the Bacteroidetes ribosomes and selects for phage transcripts during the late-stage replication when large-scale phage protein production is required for assembly of phage particles.

NGMA-2. Dual sgRNA-directed PD-L1 knockout in human glioblastoma cells using the CRISPR/Cas9 system

Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor. It induces an immunosuppressive microenvironment by exploiting immune checkpoints such as the PD-1/PD-L1 pathway. Targeting the PD-1/PD-L1 pathway for immunotherapy is a promising new avenue for treating GBM, but more work is needed to develop a safe and effective method for clinical applications. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in the genomic sequence that can lead to the production of a non-functional PD-L1 protein. A homology-directed repair template (HDR) containing an in-frame stop codon was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into the CRISPR/Cas9 plasmid, and was co-transfected with the added HDR template. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, a dual sgRNA CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.

Increased yields of duplex sequencing data by a series of quality control tools

Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences (DCS), and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics toolset that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain polymerase chain reaction and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which likely reflect barcode collisions. Finally, we also developed a tool that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. With this tool, we can include reads without a family and check the reliability of the call, that increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

Complete Genome Sequence of Rahel, a C1 Cluster Mycobacteriophage

ABSTRACT Rahel is a lytic Myoviridae bacteriophage that infects Mycobacterium smegmatis mc2155. It has 1,555,955 bp and 64.7% G+C content. Rahel has a circularly permuted genome with 270 genes, 53 of them of known function, 33 tRNAs, and 1 transfer-messenger RNA (tmRNA). Only five genes are coded on the reverse strand.

Increased yields of duplex sequencing data by a series of quality control tools

Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences, and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics tool-set that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain PCR and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which result in unpaired families that do not form DCS. Finally, we also developed a tool called Variant Analyzer that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. We demonstrate that this tool identified false positive variants tagged by the tier-based classification. Furthermore, with this tool we can include reads without a family and check the reliability of the call, which increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

An exploration of ambigrammatic sequences in narnaviruses

Narnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of ~3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are ‘ambigrammatic’ and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The >3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development.

Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay enzymes

ABSTRACTIn animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Similarly, active enhancer regions in animals initiate transcription of exosome-sensitive enhancer RNAs (eRNAs). Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs and eRNAs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs and 113 loci producing eRNA-like transcripts were identified. In addition, we found ∼200 transcription start sites within 3’-UTR-encoding regions that produce unspliced exosome-sensitive antisense RNAs covering much of the cognate pre-mRNA. A typical representative of this class of RNAs is the previously characterized non-coding RNA controlling the expression of the key seed dormancy regulator, DELAY OF GERMINATION1. Exosome-sensitive antisense RNAs are overrepresented in transcription factor genes, suggesting a potential for widespread control of gene expression. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.

An exploration of ambigrammatic sequences in narnaviruses

ABSTRACTNarnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of ∼ 3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are ‘ambigrammatic’ and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The > 3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development.

Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.