Off-target Regions Research Articles

CRISPRoffT: comprehensive database of CRISPR/Cas off-targets.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) programmable nuclease system continues to evolve, with in vivo therapeutic gene editing increasingly applied in clinical settings. However, off-target effects remain a significant challenge, hindering its broader clinical application. To enhance the development of gene-editing therapies and the accuracy of prediction algorithms, we developed CRISPRoffT (https://ccsm.uth.edu/CRISPRoffT/). Users can access a comprehensive repository of off-target regions predicted and validated by a diverse range of technologies across various cell lines, Cas enzyme variants, engineered sgRNAs (single guide RNAs) and CRISPR editing systems. CRISPRoffT integrates results of off-target analysis from 74 studies, encompassing 29 experimental prediction techniques, 368 guide sequences, 226164 potential guide and off-target pairs and 8840 validated off-targets. CRISPRoffT features off-target data from different CRISPR approaches (knockout, base editing and prime editing) applied under diverse experimental conditions, including 85 different Cas/guide RNA (gRNA) combinations used across 34 different human and mouse cell lines. CRISPRoffT provides results of comparative analyses for individual guide sequences, genes, cell types, techniques and Cas/gRNA combinations under different conditions. CRISPRoffT is a unique resource providing valuable insights that facilitate the safety-driven design of CRISPR-based therapeutics, inform experimental design, advance the development of computational off-target prediction algorithms and guide RNA design algorithms.

Cell-specific effects of temporal interference stimulation on cortical function

Temporal interference (TI) stimulation is a popular non-invasive neurostimulation technique that utilizes the following salient neural behavior: pure sinusoid (generated in off-target brain regions) appears to cause no stimulation, whereas modulated sinusoid (generated in target brain regions) does. To understand its effects and mechanisms, we examine responses of different cell types, excitatory pyramidal (Pyr) and inhibitory parvalbumin-expressing (PV) neurons, to pure and modulated sinusoids, in intact network as well as in isolation. In intact network, we present data showing that PV neurons are much less likely than Pyr neurons to exhibit TI stimulation. Remarkably, in isolation, our data shows that almost all Pyr neurons stop exhibiting TI stimulation. We conclude that TI stimulation is largely a network phenomenon. Indeed, PV neurons actively inhibit Pyr neurons in the off-target regions due to pure sinusoids (in off-target regions) generating much higher PV firing rates than modulated sinusoids in the target regions. Additionally, we use computational studies to support and extend our experimental observations.

Precision genome profiling for pediatric leukemia with a software-controlled enrichment method using nanopore sequencer.

10027 Background: The real-time nature of nanopore sequencing allows for simultaneous basecalling of DNA sequences during sequencing. This unique capability enables adaptive sampling (AS), a software-controlled targeted sequencing method. AS is notable for its ability to rapidly and flexibly enrich multiple genes with long fragment reads and detect various modalities of genetic aberrations in a single run. However, the feasibility and significance of AS for cancer have not been previously reported. Methods: We performed AS on a GridION sequencer using samples from 28 pediatric leukemia patients (10 acute myeloid leukemia, 13 B-cell acute lymphoblastic leukemia, and 5 T-cell acute lymphoblastic leukemia). Target regions were comprised of 466 genes associated with hematologic malignancies and included 30-kilo base pairs of flanking regions. After 3 days of sequencing, single-nucleotide variants (SNVs), structural variations (SVs), and copy number variations (CNVs) were determined. In 21 samples, variant calling accuracy was evaluated using short-read-based whole genome sequencing (WGS). Results: In the on-target regions, mean depth was 21.0× and N50 was 11,191 bps at the median. Of the 13 samples with genetic alterations previously detected through clinical testing for diagnostic classifications, all were reconfirmed by AS. Detecting DUX4 rearrangement needed an additional analysis pipeline because of its structural complexity. Among the 15 remaining samples whose genetic alterations had not been determined by clinical diagnostic testing, AS identified putative driver aberrations in 14 samples. All of these genomic abnormalities were structural variations (SVs) or copy number variations (CNVs), most of which were not encompassed within the genes or regions targeted in clinical diagnostic testing. Detected SVs were described with accurate genomic breakpoints, enabling the efficient detection of gene rearrangements and deletions of the entire region of an exon or a gene. Regarding CNVs, both chromosomal-level CNVs, such as high-hyperdiploid, and focal CNVs, such as CDKN2A deletion, were detectable using genome-wide low-coverage reads in the off-target regions. In the other case with acute myeloid leukemia in which genetic abnormalities were not detected either by clinical testing or AS, WGS identified an NPM1 frameshift deletion located outside of known hotspots. Among variants identified by WGS, AS detected 60.9% of the SNVs, 17.6% of the small indels, and 89.2% of the SVs, with a tendency of poor detection efficiency for variants with low variant allele frequency. Conclusions: AS provides a rapid and precise description of the genomic profile of pediatric leukemia, particularly advantageous for identifying SVs and CNVs, which are difficult to detect by capture-based short-read sequencing.

Structural variant calling and clinical interpretation in 6224 unsolved rare disease exomes

Structural variants (SVs), including large deletions, duplications, inversions, translocations, and more complex events have the potential to disrupt gene function resulting in rare disease. Nevertheless, current pipelines and clinical decision support systems for exome sequencing (ES) tend to focus on small alterations such as single nucleotide variants (SNVs) and insertions-deletions shorter than 50 base pairs (indels). Additionally, detection and interpretation of large copy-number variants (CNVs) are frequently performed. However, detection of other types of SVs in ES data is hampered by the difficulty of identifying breakpoints in off-target (intergenic or intronic) regions, which makes robust identification of SVs challenging. In this paper, we demonstrate the utility of SV calling in ES resulting in a diagnostic yield of 0.4% (23 out of 5825 probands) for a large cohort of unsolved patients collected by the Solve-RD consortium. Remarkably, 8 out of 23 pathogenic SV were not found by comprehensive read-depth-based CNV analysis, resulting in a 0.13% increased diagnostic value.

Partial volume correction in longitudinal tau PET studies: is it really needed?

Background[18F]flortaucipir (FTP) tau PET quantification is known to be affected by non-specific binding in off-target regions. Although partial volume correction (PVC) techniques partially account for this effect, their inclusion may also introduce noise and variability into the quantification process. While the impact of these effects has been studied in cross-sectional designs, the benefits and drawbacks of PVC on longitudinal FTP studies is still under scrutiny. The aim of this work was to study the performance of the most common PVC techniques for longitudinal FTP imaging. MethodsA cohort of 247 individuals from the Alzheimer's Disease Neuroimaging Initiative with concurrent baseline FTP-PET, amyloid-beta (Aβ) PET and structural MRI, as well as with follow-up FTP-PET and MRI were included in the study. FTP-PET scans were corrected for partial volume effects using Meltzer's, a simple and popular analytical PVC, and both the region-based voxel-wise (RBV) and the iterative Yang (iY) corrections. FTP SUVR values and their longitudinal rates of change were calculated for regions of interest (ROI) corresponding to Braak Areas I-VI, for a temporal meta-ROI and for regions typically displaying off-target FTP binding (caudate, putamen, pallidum, thalamus, choroid plexus, hemispheric white matter, cerebellar white matter, and cerebrospinal fluid). The longitudinal correlation between binding in off-target and target ROIs was analysed for the different PVCs. Additionally, group differences in longitudinal FTP SUVR rates of change between Aβ-negative (A-) and Aβ-positive (A+), and between cognitively unimpaired (CU) and cognitively impaired (CI) individuals, were studied. Finally, we compared the ability of different partial-volume-corrected baseline FTP SUVRs to predict longitudinal brain atrophy and cognitive decline. ResultsAmong off-target ROIs, hemispheric white matter showed the highest correlation with longitudinal FTP SUVR rates from cortical target ROIs (R2=0.28–0.82), with CSF coming in second (R2=0.28–0.42). Application of voxel-wise PVC techniques minimized this correlation, with RBV performing best (R2=0.00–0.07 for hemispheric white matter). PVC also increased group differences between CU and CI individuals in FTP SUVR rates of change across all target regions, with RBV again performing best (No PVC: Cohen's d = 0.26–0.66; RBV: Cohen's d = 0.43–0.74). These improvements were not observed for differentiating A- from A+ groups. Additionally, voxel-wise PVC techniques strengthened the correlation between baseline FTP SUVR and longitudinal grey matter atrophy and cognitive decline. ConclusionQuantification of longitudinal FTP SUVR rates of change is affected by signal from off-target regions, especially the hemispheric white matter and the CSF. Voxel-wise PVC techniques significantly reduce this effect. PVC provided a significant but modest benefit for tasks involving the measurement of group-level longitudinal differences. These findings are particularly relevant for the estimations of sample sizes and analysis methodologies of longitudinal group studies.

Abstract C004: Assessing local ancestry inference using different sequencing assays and depth of coverage

Abstract Introduction: Several studies have reported differences in mutational frequencies between different ancestral populations. For example, PTEN alterations and TMPRSS2-ERG fusions are less common in men with African ancestry. However, these studies have primarily relied on measurements of "global ancestry" and have not been able to ascertain which genomic regions contribute to the observed differences in mutational frequencies. Recent advancements in computational algorithms now enable the estimation of “local ancestry” whereby individual chromosomal segments can be assigned to a particular ancestral background. These tools have been primarily developed for deep whole-genome sequencing (WGS) and have not been optimized for different sequencing assays including ultra-low pass WGS (ULP-WGS), whole exome sequencing (WES), and SNP genotyping arrays. The goal of our study is to establish best practices for local ancestry inference across a diverse set of sequencing and genotyping assays. Methods: We procured WGS data from four ancestries through the 1000 Genomes Project (n=1,067) for local ancestry inference. ULP-WGS and WES (with on- and off-target coverage) were simulated from the full WGS data. Pseudo-array data was emulated by restricting to SNP sites on Affymetrix Genome-Wide Human SNP array 6.0. Each genotype was imputed by Eagle-Beagle (EB) or GLIMPSE2 (GL2), and local ancestry inference was performed using G-nomix using window sizes spanning from 0.2 to 16.0 centimorgans (cM). Accuracy was assessed by comparing local ancestry calls for each assay in a training set (n=1007) to a validation set (n=62). Results: Local ancestry estimation in ULP-WGS data achieved an accuracy exceeding 98% when utilizing GL2, even with the smallest window size of 0.2 cM and a minimum sequencing depth of 0.1x. To achieve similar accuracy of 98% in ULP-WGS with EB, a window size of 1.5 cM was needed for sequencing depths from 2 to 0.5x, with a larger window size of 4 cM required for a depth of 0.25x. Interestingly, such accuracy did not obtain at a depth of 0.1x. For WES with on-target sites and pseudo-array data, we utilized EB for imputation as it was more computationally efficient and accurate with larger window sizes compared to GL2. The accuracy of WES with on-target sites exceeded 90% utilizing 4.0 cM or larger window sizes, but no window size achieved 98% accuracy. Incorporating off-target regions in WES and employing GL2 enhanced the accuracy to 98% at a window size of 1.5 cM. Pseudo-array data with EB achieved an accuracy of 98% at 0.5 cM and thus was better than WES. Conclusions: Our results show that local ancestry can be accurately estimated in different sequencing and genotyping assays when the window size and imputation tool are appropriately selected. This work will facilitate the use of local ancestry inference in studies utilizing different genotyping assays. Ultimately, researchers understand the influence of local ancestral haplotypes on molecular features of cancer. Citation Format: Tomoki Motegi, Joshua D. Campbell. Assessing local ancestry inference using different sequencing assays and depth of coverage [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C004.

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

PurposeWithin the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. MethodsData from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. ResultsWe identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). ConclusionThe “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.

A nuclear target sequence capture probe set for phylogeny reconstruction of the charismatic plant family Bignoniaceae.

The plant family Bignoniaceae is a conspicuous and charismatic element of the tropical flora. The family has a complex taxonomic history, with substantial changes in the classification of the group during the past two centuries. Recent re-classifications at the tribal and generic levels have been largely possible by the availability of molecular phylogenies reconstructed using Sanger sequencing data. However, our complete understanding of the systematics, evolution, and biogeography of the family remains incomplete, especially due to the low resolution and support of different portions of the Bignoniaceae phylogeny. To overcome these limitations and increase the amount of molecular data available for phylogeny reconstruction within this plant family, we developed a bait kit targeting 762 nuclear genes, including 329 genes selected specifically for the Bignoniaceae; 348 genes obtained from the Angiosperms353 with baits designed specifically for the family; and, 85 low-copy genes of known function. On average, 77.4% of the reads mapped to the targets, and 755 genes were obtained per species. After removing genes with putative paralogs, 677 loci were used for phylogenetic analyses. On-target genes were compared and combined in the Exon-Only dataset, and on-target + off-target regions were combined in the Supercontig dataset. We tested the performance of the bait kit at different taxonomic levels, from family to species-level, using 38 specimens of 36 different species of Bignoniaceae, representing: 1) six (out of eight) tribal level-clades (e.g., Bignonieae, Oroxyleae, Tabebuia Alliance, Paleotropical Clade, Tecomeae, and Jacarandeae), only Tourrettieae and Catalpeae were not sampled; 2) all 20 genera of Bignonieae; 3) seven (out of nine) species of Dolichandra (e.g., D. chodatii, D. cynanchoides, D. dentata, D. hispida, D. quadrivalvis, D. uncata, and D. uniguis-cati), only D. steyermarkii and D. unguiculata were not sampled; and 4) three individuals of Dolichandra unguis-cati. Our data reconstructed a well-supported phylogeny of the Bignoniaceae at different taxonomic scales, opening new perspectives for a comprehensive phylogenetic framework for the family as a whole.

Responses of model cortical neurons to temporal interference stimulation and related transcranial alternating current stimulation modalities

Objective. Temporal interference stimulation (TIS) was proposed as a non-invasive, focal, and steerable deep brain stimulation method. However, the mechanisms underlying experimentally-observed suprathreshold TIS effects are unknown, and prior simulation studies had limitations in the representations of the TIS electric field (E-field) and cerebral neurons. We examined the E-field and neural response characteristics for TIS and related transcranial alternating current stimulation modalities. Approach. Using the uniform-field approximation, we simulated a range of stimulation parameters in biophysically realistic model cortical neurons, including different orientations, frequencies, amplitude ratios, amplitude modulation, and phase difference of the E-fields, and obtained thresholds for both activation and conduction block. Main results. For two E-fields with similar amplitudes (representative of E-field distributions at the target region), TIS generated an amplitude-modulated (AM) total E-field. Due to the phase difference of the individual E-fields, the total TIS E-field vector also exhibited rotation where the orientations of the two E-fields were not aligned (generally also at the target region). TIS activation thresholds (75–230 V m−1) were similar to those of high-frequency stimulation with or without modulation and/or rotation. For E-field dominated by the high-frequency carrier and with minimal amplitude modulation and/or rotation (typically outside the target region), TIS was less effective at activation and more effective at block. Unlike AM high-frequency stimulation, TIS generated conduction block with some orientations and amplitude ratios of individual E-fields at very high amplitudes of the total E-field (>1700 V m−1). Significance. The complex 3D properties of the TIS E-fields should be accounted for in computational and experimental studies. The mechanisms of suprathreshold cortical TIS appear to involve neural activity block and periodic activation or onset response, consistent with computational studies of peripheral axons. These phenomena occur at E-field strengths too high to be delivered tolerably through scalp electrodes and may inhibit endogenous activity in off-target regions, suggesting limited significance of suprathreshold TIS.

Optimized APPS-tDCS electrode position, size, and distance doubles the on-target stimulation magnitude in 3000 electric field models

Transcranial direct current stimulation (tDCS) is a widely used noninvasive brain stimulation technique with mixed results to date. A potential solution is to apply more efficient stimulation to ensure that each participant receives sufficient cortical activation. In this four-part study, we used electric field (E-field) modeling to systematically investigate the cortical effects of conventional and novel tDCS electrode montages, with the goal of creating a new easily adoptable form of tDCS that induces higher and more focal E-fields. We computed 3000 anatomically accurate, MRI-based E-field models using 2 mA tDCS to target the left primary motor cortex in 200 Human Connectome Project (HCP) participants and tested the effects of: 1. Novel Electrode Position, 2. Electrode Size, and 3. Inter-Electrode Distance on E-field magnitude and focality. In particular, we examined the effects of placing electrodes surrounding the corticomotor target in the anterior and posterior direction (anterior posterior pad surround tDCS; APPS-tDCS). We found that electrode position, electrode size, and inter-electrode distance all significantly impact the cortical E-field magnitude and focality of stimulation (all p < 0.0001). At the same 2 mA scalp stimulation intensity, APPS-tDCS with smaller than conventional 1 × 1 cm electrodes surrounding the neural target deliver more than double the on-target cortical E-field (APPS-tDCS: average of 0.55 V/m from 2 mA; M1-SO and bilateral M1: both 0.27 V/m from 2 mA) while stimulating only a fraction of the off-target brain regions; 2 mA optimized APPS-tDCS produces 4.08 mA-like cortical E-fields. In sum, this new optimized APPS-tDCS method produces much stronger cortical stimulation intensities at the same 2 mA scalp intensity. APPS-tDCS also more focally stimulates the cortex at the intended target, using simple EEG coordinate locations and without MRI scans. This APPS-tDCS method is adoptable to any existing, commercially available tDCS device and can be used to ensure sufficient cortical activation in each person. Future directions include testing whether APPS-tDCS produces larger and more consistent therapeutic tDCS effects.

CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR-Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: 'San Marzano' (SM) and 'Oxheart' (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a -7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (-7 bp and -2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.

The Association of Age-Related and Off-Target Retention with Longitudinal Quantification of [18F]MK6240 Tau PET in Target Regions.

6-(fluoro-18F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18F]MK6240) tau PET tracer quantifies the brain tau neurofibrillary tangle load in Alzheimer disease. The aims of our study were to test the stability of common reference region estimates in the cerebellum over time and across diagnoses and evaluate the effects of age-related and off-target retention on the longitudinal quantification of [18F]MK6240 in target regions. Methods: We assessed reference, target, age-related, and off-target regions in 125 individuals across the aging and Alzheimer disease spectrum with longitudinal [18F]MK6240 SUVs and SUV ratios (SUVRs) (mean ± SD, 2.25 ± 0.40 y of follow-up). We obtained SUVR from meninges, exhibiting frequent off-target retention with [18F]MK6240. Additionally, we compared tracer uptake between 37 cognitively unimpaired young (CUY) (mean age, 23.41 ± 3.33 y) and 27 cognitively unimpaired older (CU) adults (amyloid-β-negative and tau-negative, 58.50 ± 9.01 y) to identify possible nonvisually apparent, age-related signal. Two-tailed t testing and Pearson correlation testing were used to determine the difference between groups and associations between changes in region uptake, respectively. Results: Inferior cerebellar gray matter SUV did not differ on the basis of diagnosis and amyloid-β status, cross-sectionally and over time. [18F]MK6240 uptake significantly differed between CUY and CU adults in the putamen or pallidum (affecting ∼75% of the region) and in the Braak II region (affecting ∼35%). Changes in meningeal and putamen or pallidum SUVRs did not significantly differ from zero, nor did they vary across diagnostic groups. We did not observe significant correlations between longitudinal changes in age-related or meningeal off-target retention and changes in target regions, whereas changes in all target regions were strongly correlated. Conclusion: Inferior cerebellar gray matter was similar across diagnostic groups cross-sectionally and stable over time and thus was deemed a suitable reference region for quantification. Despite not being visually perceptible, [18F]MK6240 has age-related retention in subcortical regions, at a much lower magnitude but topographically colocalized with significant off-target signal of the first-generation tau tracers. The lack of correlation between changes in age-related or meningeal and target retention suggests little influence of possible off-target signals on longitudinal tracer quantification. Nevertheless, the age-related retention in the Braak II region needs to be further investigated. Future postmortem studies should elucidate the source of the newly reported age-related [18F]MK6240 signal, and invivo studies should further explore its impact on tracer quantification.

Investigating Tau and Amyloid Tracer Skull Binding in Studies of Alzheimer Disease.

Off-target binding of [18F]flortaucipir (FTP) can complicate quantitative PET analyses. An underdiscussed off-target region is the skull. Here, we characterize how often FTP skull binding occurs, its influence on estimates of Alzheimer disease pathology, its potential drivers, and whether skull uptake is a stable feature across time and tracers. Methods: In 313 cognitively normal and mildly impaired participants, CT scans were used to define a skull mask. This mask was used to quantify FTP skull uptake. Skull uptake of the amyloid-β PET tracers [18F]florbetapir and [11C]Pittsburgh compound B (n = 152) was also assessed. Gaussian mixture modeling defined abnormal levels of skull binding for each tracer. We examined the relationship of continuous bone uptake to known off-target binding in the basal ganglia and choroid plexus as well as skull density measured from the CT. Finally, we examined the confounding effect of skull binding on pathologic quantification. Results: We found that 50 of 313 (∼16%) FTP scans had high levels of skull signal. Most were female (n = 41, 82%), and in women, lower skull density was related to higher FTP skull signal. Visual reads by a neuroradiologist revealed a significant relationship with hyperostosis; however, only 21% of women with high skull binding were diagnosed with hyperostosis. FTP skull signal did not substantially correlate with other known off-target regions. Skull uptake was consistent over longitudinal FTP scans and across tracers. In amyloid-β-negative, but not -positive, individuals, FTP skull binding impacted quantitative estimates in temporal regions. Conclusion: FTP skull binding is a stable, participant-specific phenomenon and is unrelated to known off-target regions. Effects were found primarily in women and were partially related to lower bone density. The presence of [11C]Pittsburgh compound B skull binding suggests that defluorination does not fully explain FTP skull signal. As signal in skull bone can impact quantitative analyses and differs across sex, it should be explicitly addressed in studies of aging and Alzheimer disease.

Impact of spill-in counts from off-target regions on [18F]Flortaucipir PET quantification

Background[18F]Flortaucipir (FTP) PET quantification is usually hindered by spill-in counts from off-target binding (OFF) regions. The present work aims to provide an in-depth analysis of the impact of OFF in FTP PET quantification, as well as to identify optimal partial volume correction (PVC) strategies to minimize this problem. Methods309 amyloid-beta (Aβ) negative cognitively normal subjects were included in the study. Additionally, 510 realistic FTP images with different levels of OFF were generated using Monte Carlo simulation (MC). Images were corrected for PVC using both a simple two-compartment and a multi-region method including OFF regions. FTP standardized uptake value ratio (SUVR) was quantified in Braak Areas (BA), the hippocampus (which was not included in Braak I/II) and different OFF regions (caudate, putamen, pallidum, thalamus, choroid plexus (ChPlex), cerebellar white matter (cerebWM), hemispheric white matter (hemisWM) and cerebrospinal fluid (CSF)) using the lower portion of the cerebellum as a reference region. The correlations between OFF and cortical SUVRs were studied both in real and in simulated PET images, with and without PVC. ResultsIn-vivo, we found correlations between all OFF and target regions, especially strong for the hemisWM (slope>0.63, R2>0.4). All the correlations were attenuated but remained significant after applying PVC, except for the ChPlex. In MC simulations, the hemisWM and CSF were the main contributors to PVE in all BA (slopes 0.15-0.26 and 0.13-0.21 respectively). The hemisWM (slope=0.2), as well as the ChPlex (slope=0.02), influenced SUVRs in the hippocampus. The CerebWM was negatively correlated with all target regions (slope<-0.02, R2>0.8). While no other correlations between OFF and target regions were found, hemisWM was correlated with all OFF regions but the cerebWM (slopes 0.06-0.33). HemisWM correlations attenuated (slopes<0.06) when applying two-compartment PVC, but the hippocampus-ChPlex and the cerebWM correlations required more complex PVC with dedicated compartments for these regions. In-vivo, PVC removed a notably higher fraction of the correlation between OFF regions found to be affected by PVE in the simulation studies and BA (≈50%) than for OFF regions not affected by PVE (16%). ConclusionHemisWM is the main driver of spill-in effects in FTP PET, affecting both target regions and the rest of OFF regions. PVC successfully reduces PVE, even when using a simple two-compartment method. Despite PVC, non-zero correlations were still observed between target and OFF regions in vivo, which suggests the existence of biological or tracer-related contributions to these correlations.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Ultrafast and Multiplexed Bacteriophage Susceptibility Testing by Surface Plasmon Resonance and Phase Imaging of Immobilized Phage Microarrays

In the context of bacteriophage (phage) therapy, there is an urgent need for a method permitting multiplexed, parallel phage susceptibility testing (PST) prior to the formulation of personalized phage cocktails for administration to patients suffering from antimicrobial-resistant bacterial infections. Methods based on surface plasmon resonance imaging (SPRi) and phase imaging were demonstrated as candidates for very rapid (&lt;2 h) PST in the broth phase. Biosensing layers composed of arrays of phages 44AHJD, P68, and gh-1 were covalently immobilized on the surface of an SPRi prism and exposed to liquid culture of either Pseudomonas putida or methicillin-resistant Staphylococcus aureus (i.e., either the phages’ host or non-host bacteria). Monitoring of reflectivity reveals susceptibility of the challenge bacteria to the immobilized phage strains. Investigation of phase imaging of lytic replication of gh-1 demonstrates PST at the single-cell scale, without requiring phage immobilization. SPRi sensorgrams show that on-target regions increase in reflectivity more slowly, stabilizing later and to a lower level compared to off-target regions. Phage susceptibility can be revealed in as little as 30 min in both the SPRi and phase imaging methods.

Karyosequencing: Integrating Genome-Wide and Targeted Sequencing for Comprehensive Diagnosis of Lymphoproliferative Disorders

Introduction: Molecular diagnostic testing for lymphoproliferative disorders (LPDs) includes detection of clonal immunoglobulin (IG) and/or T cell receptor (TCR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. To date, laboratories still rely on subjective and labour-intensive technologies, e.g. karyotyping/FISH to detect complex genomic alterations. Whole Genome Sequencing (WGS) can detect all genomic alterations, but factors such as cost, computation/storage, DNA requirements and poor detection of clinically relevant subclonal mutations limits the routine implementation of WGS in clinical diagnostics. We have developed “KaryoSequencing” (KS), a novel approach that combines targeted deep-sequencing, using a targeted hybridisation capture NGS panel, for rare mutation and translocation detection with shallow WGS (sWGS) for genome wide copy number analysis, in a single test.Methods: KS was validated using 138 clinical samples from patients with acute lymphoblastic leukaemia (ALL) (n=46), chronic lymphocytic leukaemia (CLL) (n=46) and plasma cell myeloma (PCM) (n=46) samples from 3 UK laboratories. Samples underwent library preparation and hybridisation using the EuroClonality-NDC assay. A KS library for each sample was prepared by combining the pre-capture and post-capture libraries at an optimised ratio to enable high coverage (>500 x) at regions covered by the targeted panel while providing 0.5-1x coverage genome-wide. Forty-six KS libraries were pooled and sequenced per NovaSeq 6000 run, using a 200-cycle SP flow cell and a 100bp paired-end strategy. For analysis of targeted regions, somatic mutation calling was performed using VarDict and structural variants (rearrangements and translocations) were detected using ARResT/Interrogate. For analysis of large chromosomal copy number variation using sWGS, a modified version of QDNASeq/ACE with a window size of 50kb was performed. For sWGS analysis, bioinformatic exclusion of all target capture regions and panel-specific off-target regions was performed using a panel of 48 DNA samples from healthy individuals ran on the same KS protocol.Results:Analysis of genome-wide copy number by sWGS was concordant in 477 of 503 (95%) evaluable FISH tests, including precise detection of hyper and hypo-diploidy and other complex karyotypes. The performance of the targeted deep-sequencing component of the KS approach was assessed to ensure comparable performance to previously validated results for the EuroClonality-NDC. The EuroClonality-NDC detected a clonal IG rearrangement in 46/46 (100%) CLL cases and in 42/46 (91.3%) PCM cases. Clonality was detected in 43/46 (93.5%) and 44/46 (95.7%) cases at an IG and TCR locus respectively in ALL, a disease entity known to exhibit cross-lineage rearrangements. Overall, 44 translocations were detected in the 138 samples by the EuroClonality-NDC. FISH results were available for 32 of the 44 translocations detected by NGS and were concordant in 29/32 (91%). In two PCM samples NGS and FISH reported a different translocation; in the ALL sample, the FISH pattern showed an additional copy but not a split signal. Across the entire cohort of 138 samples, KaryoSequencing detected 190 mutations with a variant allele frequency ranging from 4.0 - 97.6%. Of the detected mutations, 69/190 (36.3%) had a VAF <20% which can be difficult to detect reliably with typical coverage depths observed with conventional WGS. The most frequently mutated genes were KRAS [30.4%], NRAS [13.0%], KMT2D [10.9%] in ALL; SF3B1 [23.9%], NOTCH1 [19.6%], ATM [15.2%] and TP53[13.0%] in CLL and KRAS [19.6%], NRAS [15.2%] and TP53 [10.9%] in PCM, consistent with expected frequencies in each entity.Conclusions:KaryoSequencing demonstrated >95% sensitivity and specificity for detection of gross genome-wide copy number alterations while retaining the analytical performance for detection of rearrangements, translocations, and mutations for the lymphoid-specific targeted regions of the EuroClonality-NDC assay. KS is a cost-effective and high-throughput integrated alternative to current diagnostic strategies in haematological malignancies and can be implemented in routine clinical practice. DisclosuresJenner: Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy; Takeda: Consultancy; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau. Gonzalez: Univ8 Genomics Ltd: Current holder of individual stocks in a privately-held company.

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. We report characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, raising the possibility that only two fusion peptides per trimer are required for viral–host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

Abstract 571: Detection of somatic copy number alterations from on-target and off-target sequencing data

Abstract Background: Several computational methods have been developed to identify copy number alterations (CNA) leading to or associated with cancer development and shown in recent studies to precede cancer diagnosis by many years. Current methods involving cell-free DNA (cfDNA) targeted sequencing data are based on the depth of coverage of on-target or off-target regions, whereas computational methods incorporating germline SNP information for making inferences on copy number alterations and tumor fraction remain underdeveloped. Methods: Using sequencing data from a large database of more than 100k clinical cell-free DNA (cfDNA) patient samples (Guardant Health, CA), we developed a probabilistic model to simultaneously normalize molecular coverage, segment the genome, predict copy number alterations, and estimate the tumor content in cfDNA samples, while accounting for mixtures of cell populations. The model is using off-target and on-target coverage. Copy number status, including loss of heterozygosity (LoH), is inferred in order to predict gene level somatic CNAs or genome wide instability/LoH. Results: We demonstrated the improvement from the off-target incorporation in three aspects. First, to estimate sensitivity improvement in detections of CNAs, we simulated deletions and amplifications of regions exceeding 40 Mb, using coverage and MAF variability observed in existing data. Combining coverage of on-target and off-target regions is expected to improve the LoD for detection of CNAs by 20%, when compared to CNA detection from on-target coverage. Next, we obtained samples from 15,618 cancer patients of different cancer types processed on GuardantOMNI® RUO and determined human leukocyte antigen (HLA) allele-specific copy number using this off-target assisted method. We observed a high prevalence (more than 15%) of LoH in HLA in bladder cancer, prostate cancer, NSCLC and HNSC, consistent with previous studies that HLA LOH is a common feature of several cancer types and diminishes immunotherapy efficacy. Finally, tumor fraction (TF) estimate was validated by comparing the TF against the maximum variant allele fraction of known oncogenic driver mutations in 6,000 cancer cases of various types. High concordance was observed in CRC samples (R2=0.75), gastric cancer (R2=0.63) and bladder cancer (R2=0.6), which suggest the use of this metric to better estimate tumor shedding levels in cfDNA in cases when driver mutations are not represented on a targeting panel. Conclusion: Our results show that probabilistic modeling of coverage data generated from both on-target and off-target cfDNA sequencing can detect gene specific or whole genome level somatic copy number alterations and LoH. This method may enable improvements in CNA detection accuracy, sensitivity, and specificity in plasma and provides more precise interrogation of LoH status and tumor fraction. Citation Format: Catalin Barbacioru, Han-Yu Chuang, Rebecca Nagy, Darya Chudova, AmirAli Talasaz. Detection of somatic copy number alterations from on-target and off-target sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 571.

IN SILICO ANALYSIS OF POTENTIAL OFF-TARGET SITES TO GENE EDITING FOR MUCOPOLYSACCHARIDOSIS TYPE I BY USING THE CRISPR/CAS9 SYSTEM: IMPLICATIONS FOR POPULATIONS-SPECIFIC TREATMENTS

<h3>Background</h3> The CRISPR/Cas9 system is an accessible and less onerous editing system to treat human genetic diseases, but off-target prediction is paramount to ensure the safety and efficiency of this system in clinical applications. The aim of this study is to evaluate off-target regions for a guide RNA (sgRNA) designed to correct the most common variant found in Mucopolysaccharidosis type I (MPS I) patients and also possible implications for population-specific treatment to treat genetic diseases. <h3>Methods</h3> The most employed in silico tools for off-targets prediction were used: CHOPCHOP, COSMID, Cas-OFFinder, CCTop and CRISPOR. Moreover, new PAM sequences (NNG or NGN) were also evaluated by using Burrows-Wheeler Aligner for this sgRNA against human genome version hg19. The potential off-target regions were selected from sequences selected by the tools' score and with up to 6 mismatches and up to 2 indels, and the conservation of 5 nucleotides upstream PAM (SEED). The polymorphic site evaluation was made using 1000 Genomes and Latin America databases (ABraOM and LOVD 3-Argentina). Polymorphic sites that have equal or greater 1% in at least one of the populations were selected. <h3>Results</h3> From a total of 13,422 off-targets, 272 potential off-targets were selected for this sgRNA. A total of 84 polymorphic sites were recognized in the off-target sequences. The 1000 Genomes had the highest amount of polymorphic sites, whereas ABraOM reported only six. In most cases polymorphic sites decrease the probability of off-target cleavage by increasing the number of mismatches and/or indels and also disrupt PAM and SEED regions. On the other hand, one off-target sequence not previously recognized by all the five predictors was created by a polymorphic variant with frequencies of 42% in African populations and of 16% in European and Latin American populations. <h3>Conclusion</h3> Here we show the importance of identifying potential off-target sites considering the analysis of frequent polymorphic sites in different populations. Thus, this analysis contributes to a better evaluation of safety aspects of gene editing to treat MPS I patients and other genetic diseases in a population-specific context by using the CRISPR/Cas9 system.