High-throughput Genome-wide Translocation Sequencing Research Articles

Orientation Regulation of Class-switch Recombination in Human B Cells.

We developed a linear amplification-mediated high-throughput genome-wide translocation sequencing method to profile Ig class-switch recombination (CSR) in human B cells in an unbiased and quantitative manner. This enables us to characterize CSR junctions resulting from either deletional recombination or inversion for each Ig class/subclass. Our data showed that more than 90% of CSR junctions detected in peripheral blood in healthy control subjects were due to deletional recombination. We further identified two major CSR junction signatures/patterns in human B cells. Signature 1 consists of recombination junctions resulting from both IgG and IgA switching, with a dominance of Sµ-Sγ junctions (72%) and deletional recombination (87%). Signature 2 is contributed mainly by Sµ-Sα junctions (96%), and these junctions were almost all due to deletional recombination (99%) and were characterized by longer microhomologies. CSR junctions identified in healthy individuals can be assigned to both signatures but with a dominance of signature 1, whereas almost all CSR junctions found in patients with defects in DNA-PKcs or Artemis, two classical nonhomologous end joining (c-NHEJ) factors, align with signature 2. Thus, signature 1 may represent c-NHEJ activity during CSR, whereas signature 2 is associated with microhomology-mediated alternative end joining in the absence of the studied c-NHEJ factors. Our findings suggest that in human B cells, the efficiency of the c-NHEJ machinery and the features of switch regions are crucial for the regulation of CSR orientation. Finally, our high-throughput method can also be applied to study the mechanism of rare types of recombination, such as switching to IgD and locus suicide switching.

SMC3 contributes to heart development by regulating super-enhancer associated genes

Abnormal cardiac development has been observed in individuals with Cornelia de Lange syndrome (CdLS) due to mutations in genes encoding members of the cohesin complex. However, the precise role of cohesin in heart development remains elusive. In this study, we aimed to elucidate the indispensable role of SMC3, a component of the cohesin complex, in cardiac development and its underlying mechanism. Our investigation revealed that CdLS patients with SMC3 mutations have high rates of congenital heart disease (CHD). We utilized heart-specific Smc3-knockout (SMC3-cKO) mice, which exhibit varying degrees of outflow tract (OFT) abnormalities, to further explore this relationship. Additionally, we identified 16 rare SMC3 variants with potential pathogenicity in individuals with isolated CHD. By employing single-nucleus RNA sequencing and chromosome conformation capture high-throughput genome-wide translocation sequencing, we revealed that Smc3 deletion downregulates the expression of key genes, including Ets2, in OFT cardiac muscle cells by specifically decreasing interactions between super-enhancers (SEs) and promoters. Notably, Ets2-SE-null mice also exhibit delayed OFT development in the heart. Our research revealed a novel role for SMC3 in heart development via the regulation of SE-associated genes, suggesting its potential relevance as a CHD-related gene and providing crucial insights into the molecular basis of cardiac development.

Abstract 404: LINE-1 retrotransposition promotes chromosomal translocations through RNA-templated DNA repair in human cancers

Abstract Introduction: Approximately 50% of human cancers have somatic integrations of long interspersed element-1 (LINE-1; also known as L1). L1 is a retrotransposon that copies itself through RNA and integrates into new genomic loci. Although L1 sequences comprise approximately 17% of the human genome, only 100-150 L1 loci have retained the ability to retrotranspose. These full-length, retrotransposition-competent L1s are typically repressed in human somatic cells, but there is evidence of their significant reactivation in human cancers. Therefore, L1 overexpression is a hallmark of many human cancers, particularly in highly malignant tumor types such as breast, ovarian, pancreatic, esophageal, lung, head-and-neck, colon, prostate, and liver cancers. Results: In many cancers, chromosomal translocations are fundamental pathogenetic events initiated by the generation of DNA double-strand breaks (DSBs). Although LINE-1 (L1) retrotransposition events have been implicated in chromosomal rearrangements associated with most human cancers, the frequency and mechanisms by which L1 is involved in these rearrangements, including chromosomal translocations, remain poorly characterized. By combining high-throughput genome-wide translocation sequencing (HTGTS) and two sequencing techniques to detect L1 insertion sites (PolyA-seq and CELTICS-seq), we demonstrated that the presence of an active human L1 retrotransposition significantly increases the frequency of chromosomal translocations. By identifying L1-dependent translocation hotspots in HEK293T cells we highlight the requirement for the L1 reverse transcriptase (RT) activity and the crucial role of pre-existing DNA DSBs in promoting L1-mediated chromosomal translocations. L1-dependent translocation hotspots showed a marked preference for transcription end sites (TES) of active genes, early replication timing regions, and accessible chromatin regions. Therefore, while cancer genome analysis provides a static view of chromosomal rearrangements based on canonical L1 integrations, our functional studies reveal an unanticipated predominance of chromosomal translocations facilitated by non-canonical L1 retrotransposition via RNA-templated repair of existing DSBs, largely expanding our knowledge on the mechanism by which L1 retrotransposition induces genomic instability in human cancers. Citation Format: Jianli Tao, Luca Alessandri, Carlos Mendez-Dorantes, Kathleen H. Burns, Qi Wang, Roberto Chiarle. LINE-1 retrotransposition promotes chromosomal translocations through RNA-templated DNA repair in human cancers [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 404.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid 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 403.

Abstract 313: Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers

Abstract Introduction: Tyrosine kinase (TK) fusions are common events in the development of resistance to targeted therapy for non-small cell lung cancer (NSCLC) and others. The mechanisms by which recurrent breakpoints are generated in solid tumors and by which they are selected are unclear. APOBEC3 proteins are the main factors for mutations in solid cancers and the mutagenic activity of APOBEC3 can activate the DNA damage response by contributing to double-strand break (DSB) formation. However, the possibility that APOBEC3 proteins could induce chromosomal translocations in solid tumors has not been investigated. In addition, mechanistic explanations on how recurrent chromosomal translocation occur in lung cancer and solid tumors in precise genomic breakpoints and with specific partners are lacking. In this work, we present a newly developed functionally active chromosomal translocation sequencing (FACTS) approach to provide insights on the mechanisms that determine the selection of oncogenic fusions in human cancers. Methods: We applied FACTS to lung cells to map genome-wide translocations when DNA DSB is introduced in TK genes by the CRISPR/Cas9 system. We generated genomic maps of DNA breakpoints joining to anaplastic lymphoma kinase (ALK) before and after selection by high-throughput genome-wide translocation sequencing (HTGTS). We tested the effect of APOBEC3 proteins in an overexpression system or in an all seven APOBEC3 gene knockout lung cells by FACTS. Results: Overexpression of APOBEC3 proteins significantly increased the formation of functional fusions compared to control cells. In addition, deletion of APOBEC3 genes significantly decreased the formation of functional fusions compared to APOBEC3 wild-type cells. With the FACTS data and bioinformatics analyses, we found that gene expression rather than locus accessibility dictates the selection of TK fusions and that TK fusions are likely selected from a pool of translocations occurring at the right position in transcribe genes. In addition, we found that active transcription in silent genes was sufficient to introduce the spontaneous formation of functional fusions. By using HTGTS approach, we found that breakpoints before selection did not show a preferential strand bias, but after selection the breakpoints showed a strong bias. By comparing oncogenic potential of EML4-ALK fusion variants, we found that protein stability, but not subcellular localization, is key to determining the exon usage of EML-ALK fusions. In addition, we revealed that the selection of fusion partners for each TK is more likely dictated by protein stability rather than by mRNA expression. Conclusion: FACTS allows rapid and comprehensive mapping of functional chromosomal translocations and provides insights into the mechanisms of their formation and selection. The FACTS approach could be used to predict the oncogenic activity of fusion proteins to guide clinical decisions. Citation Format: Taek-Chin Cheong, Ahram Jang, Qi Wang, Giulia C. Leonardi, Maria K. Lehtinen, Marian H. Harris, Roberto Chiarle. Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 313.

Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing

In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction. Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus. Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning. Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.

C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency.

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

TET Deficiency Is Associated with Accumulation of G-Quadruplex and R-Loop Structures during Oncogenesis

The three members of TET family of Fe(II) and alpha-ketoglutarate-dependent dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5-hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development, and loss of TET function due to mutations, metabolic perturbations and hypoxia, among other mechanisms, occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins (TET2, most commonly) and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, are not fully-understood.To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, we generated mice with B-cell-specific deletion of TET2 and TET3, the major TET homologs expressed in mature B cells. TET deficiency in B cells perturbed mature B cell homeostasis resulting in spontaneous development of Germinal Center-derived B cell lymphomas. Moreover, B cells with TET deficiency demonstrated increased genomic instability, a feature previously associated with TET loss-of-function in other hematopoietic lineages. Transcriptional profiling of TET-deficient expanded B cells revealed altered expression of genes and proteins involved in modulating the levels of secondary DNA structures, G-quadruplexes and DNA:RNA hybrids (R-loops) which have been linked to genomic instability and transcriptional perturbations in many different cancers. Using previously described methods and newer approaches, we observed a substantial increase in the levels of G-quadruplex and R-loop structures in TET-deficient B cells compared with control B cells. The increase in G-quadruplex and R-loop structures was evident in naïve, activated and GC B cells following acute TET deletion as well as in TET-deficient myeloid cells and T cells. Genome-wide mapping studies and high-throughput genome-wide translocation sequencing (HTGTS) showed a strong correlation of increased G-quadruplex and R-loop structures with increased DNA DSBs in switch regions of immunoglobulin heavy chain locus in TET-deficient B cells. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented the expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and caused a notable delay in lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell-derived and other malignancies, and highlight novel therapeutic avenues that could be further explored. DisclosuresRao: Cambridge Epigenetix: Membership on an entity's Board of Directors or advisory committees.

Altered H3 histone acetylation impairs high-fidelity DNA repair to promote cerebellar degeneration in spinocerebellar ataxia type 7.

SUMMARYA common mechanism in inherited ataxia is a vulnerability of DNA damage. Spinocerebellar ataxia type 7 (SCA7) is a CAG-polyglutamine-repeat disorder characterized by cerebellar and retinal degeneration. Polyglutamine-expanded ataxin-7 protein incorporates into STAGA co-activator complex and interferes with transcription by altering histone acetylation. We performed chromatic immunoprecipitation sequencing ChIP-seq on cerebellum from SCA7 mice and observed increased H3K9-promoter acetylation in DNA repair genes, resulting in increased expression. After detecting increased DNA damage in SCA7 cells, mouse primary cerebellar neurons, and patient stem-cell-derived neurons, we documented reduced homology-directed repair (HDR) and single-strand annealing (SSA). To evaluate repair at endogenous DNA in native chromosome context, we modified linear amplification-mediated high-throughput genome-wide translocation sequencing and found that DNA translocations are less frequent in SCA7 models, consistent with decreased HDR and SSA. Altered DNA repair function in SCA7 may predispose the subject to excessive DNA damage, leading to neuron demise and highlights DNA repair as a therapy target.

Abstract PO-012: Ultra-high dose rate (FLASH) irradiation does not alter microhomology mediated recombination under varying oxygen tension when compared to standard clinical dose rates

Abstract The mechanism(s) driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV) ionizing radiation (IR) dose-rate are not known. However, attenuated DNA damage and transient oxygen depletion are among a number of proposed models. In this study, we employ high-throughput genome-wide translocation sequencing (LAM-HTGTS), a highly-sensitive assay used to identify “prey” DNA double strand breaks (DSBs) via their translocation to a single CRISPR-Cas9 “bait” DSB break, to investigate the potential differences in DSBs and repair generated by 10 Gy delivered at CONV (0.13 Gy/s) or FLASH (5.106 Gy/s) dose rates with the eRT6 (PMB-Alcen, CHUV). Investigations were conducted under different oxygen tensions. IR exposure in HEK293T cells at normoxic (21% O2) conditions increased both the proportion and total number of translocations from the junctions recovered, as expected, but were indistinguishable between CONV-RT and FLASH-RT dose-rates. Although, increased proportions of translocations were observed as cells were acutely transitioned to either physioxic (4%) or hypoxic (<2%) conditions alone, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal overt differences in the level of increased translocations nor in junction structures, which were predominantly increased in microhomology utilization. We discuss these findings with other related LAM-HTGTS studies. Citation Format: Paul Barghouth, Jonathan Ollivier, Pierre Montay-Gruel, Billy W. Loo Jr., Marie-Catherine Vozenin, Charles Limoli, Richard Frock. Ultra-high dose rate (FLASH) irradiation does not alter microhomology mediated recombination under varying oxygen tension when compared to standard clinical dose rates [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-012.

Abstract 2393: APOBEC3 enzymes induce chromosomal translocations in solid cancers

Abstract Introduction: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases provides a defense for the host genome against exogenous viruses and endogenous retro-elements, but their enzymatic activity may cause host genomic instability via a conversion of cytosine to uracil during RNA and DNA editing. While it is well established that activation-induced cytidine deaminase (AID; a B-cell specific APOBEC family enzyme) induces genomic instability by initiating mutations and oncogenic chromosomal translocations involved in the development and progression of lymphoma, it is unclear whether APOBEC3 enzymes could play a similar role in non-hematopoietic cells. Recently, APOBEC-signature mutation (C-to-T transition) has been identified in almost half of all human cancers, including breast and lung cancers. However, enzymes that trigger chromosomal translocation in solid cancers are largely underdetermined. In this study, we investigated the role of APOBEC3 proteins in chromosomal translocation formation and validated APOBEC3-induced chromosomal translocations in solid cancers. Methods: We generated genome-wide chromosomal translocation maps of human APOBEC3 enzymes in both mouse and human cells by adapting the high-throughput, genome-wide translocation sequencing (HTGTS) technique. We exploited immortalized mouse fibroblasts carrying a 25x I-SceI meganuclease cassette targeted in the c-myc locus and also human MCF10A (breast), Beas-2B (lung), and PC-9 (non-small cell lung cancer) cells by generating DNA DSBs in either the c-myc locus or the EML4 locus by the CRISPR/Cas9 system. Results: All human APOBEC3 enzymes have cytidine deaminase activity on their preferred target motifs with different potency. By overexpressing APOBEC3 proteins in AID−/− B cell, we found that APOBEC3 enzymes, in contrast to AID, did not induce IgG1 class switch recombination (CSR) indicating a different mechanism of action between AID and other APOBEC3 enzymes. By HTGTS, we demonstrated that APOBEC3s and AID induced significantly higher number of translocations in novel hotspots compared to control fibroblasts. Strikingly, bioinformatics analysis of the junctions revealed that APOBEC3 proteins induce chromosomal translocations with similar frequency, but only partially overlap with AID. In addition, by analyzing individual genes or translocation targets, we observed similar but distinct effects on translocation frequency for each member of the APOBEC3 family. Interestingly, AID preferentially targets genomic regions associated with high transcriptional activity, but APOBEC3Cs do not. The analysis of HTGTS from human breast and lung cell lines revealed the similar pattern of translocations found in mouse fibroblasts. Remarkably, APOBEC3 off-target hotspots are distinct and unique in each cell type. Conclusions: We showed that human APOBEC3 enzymes enhance chromosomal translocations in non-hematopoietic cells with similar potency to AID, suggesting that APOBEC3 enzymes could be initiating genomic instability in solid tumors where AID is not expressed. Thus, APOBEC3 expression has to be tightly controlled in non-hematopoietic cells to limit its genotoxic effects. Citation Format: Taek-Chin Cheong, Qi Wang, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2393.

Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach

Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5′ and 3′ regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.

Increased Neural Progenitor Proliferation in a hiPSC Model of Autism Induces Replication Stress-Associated Genome Instability.

The association between macrocephaly and autism spectrum disorder (ASD) suggests that the mechanisms underlying excessive neural growth could contribute to ASD pathogenesis. Consistently, neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) of ASD individuals with early developmental brain enlargement are inherently more proliferative than control NPCs. Here, we show that hiPSC-derived NPCs from ASD individuals with macrocephaly display an altered DNA replication program and increased DNA damage. When compared with the control NPCs, high-throughput genome-wide translocation sequencing (HTGTS) demonstrates that ASD-derived NPCs harbored elevated DNA double-strand breaks in replication stress-susceptible genes, some of which are associated with ASD pathogenesis. Our results provide a mechanism linking hyperproliferation of NPCs with the pathogenesis of ASD by disrupting long neural genes involved in cell-cell adhesion and migration.

Abstract B171: Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination

Abstract RAG endonuclease initiates V(D)J recombination by binding to the recombination signal sequences (RSSs), introducing DNA breaks between two V(D)J gene segments, and mediating their joining. This process is tightly regulated to generate diverse antigen receptor repertoires and prevent aberrant events that could cause genomic translocations/deletions and lymphoid cancer. RAG can track directionally over chromosomal loop domain in which they reside. Tracking is evidenced by cleavage/joining of short cryptic RSSs lying exclusively in convergent orientation to a bona fide RSS. However, the mechanism of RAG tracking and its impacts remain unknown. Here I established an unbiased assay system by utilizing allele-specific barcode and employing high throughput genome-wide translocation sequencing (HTGTS) to quantitatively evaluate RAG-mediated recombination frequency. I found that insertion of one single bona fide RSS is sufficient to initiate RAG tracking in the c-Myc domain and the recombination between the RSS and its cryptic RSSs seems to follow the 12/23 rule to some degree. When ectopically inserted bona fide RSS pair is 0.5kb apart, convergent configuration of the RSS pair is 4.8-fold more frequent than the tandem configuration. When the distance increases to 267kb, the difference increases to 14-fold, suggesting the contribution of RAG tracking over diffusion-based mechanism increases when the distance between RSS pair increases. In addition, the presence of CTCF-binding element promotes recombination by more than 10-fold. Depletion of Wapl, cohesin remover, to disrupt cohesin turnover reduces V(D)J recombination. The distal RSSs are much preferentially affected. The data suggest that RAG tracking may be driven by cohesin sliding along chromatin. Citation Format: Cheng-Sheng Lee, Jiazhi Hu, Frederick W. Alt. Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B171.

Improved HTGTS for CRISPR/Cas9 off-target detection.

Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a "bait" DNA double-stranded break (DSB) to capture genome-wide "prey" DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.

Three classes of recurrent DNA break clusters in brain progenitors identified by 3D proximity-based break joining assay

We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.

Abstract A033: Mechanisms that mediate intralocus and interlocus regulation of V(D)J recombination at immunoglobulin light chain loci

Abstract The N-terminal variable regions of immunoglobulin (Ig) heavy (IgH) and light (IgL) chains are involved in specific antigen binding and assembled from germline variable (V), diversity (D), and joining (J) gene segments through a somatic recombination reaction known as V(D)J recombination. V(D)J recombination is initiated by RAG endonuclease and completed by non-homologous DNA end-joining. V(D)J recombination is tightly regulated in the contexts of order, lineage, and allelic exclusion. For IgL this regulation additionally involves isotypic exclusion, in which most B cells express Ig molecules containing only one of the two IgL isotypes, κ and λ. Controlled V(D)J recombination is important beyond its role in generating diverse antibody repertoires against diverse antigens, since dysregulation of V(D)J recombination can not only underlie various immune disorders, but lead to oncogenic translocations that are frequently found in human B and T cell leukemias and lymphomas. Many of such translocations involve IgL chain loci. To provide new insights into mechanisms of chromosomal translocations involving IgL chain loci, we sought to investigate mechanisms of normal V(D)J recombination at IgL loci by developing and applying multiple new approaches. In this study, we employed high-throughput genome-wide translocation sequencing (HTGTS) to identify RAG activities at Igκ and Igλ loci in v-Abl transformed progenitor B (pro-B) line. Remarkably, we found such RAG activities were largely confined to unanticipated distinct chromatin topologically associated loop domains within Igκ and Igλ loci, adding a new layer of regulation of gene rearrangements at IgL loci. Furthermore, we developed a highly sensitive and unbiased assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify Igκ and Igλ repertoires in mouse bone marrow precursor B (pre-B) and splenic B cell populations. HTGTS-Rep-seq detected diverse Igκ and Igλ VJ rearrangements involving individual functional Jκ and Jλ segments, respectively. Currently we are integrating such enormous Igκ and Igλ repertoire data with long-range chromatin interaction map data revealed by Hi-C and our recently developed high-resolution HTGTS chromosome conformation capture sequencing (HTGTS-3C-seq) methods to further address the mechanisms of gene rearrangement regulation by chromatin loop domains at these loci. In addition, to address the mechanisms of isotypic exclusion that has been debated, we generated germline mouse models harboring endogenously-generated Igκ deletions and a productively assembled IgH variable region exon by generating induced-pluripotent stems cells (iPSCs) from peripheral B cells. We then applied global nuclear run-on sequencing (GRO-seq) to bone marrow pre-B cells purified from iPSC-generated mice in a RAG-deficient background (which abrogates V(D)J recombination) to elucidate transcriptional features at Igκ and Igλ loci and specifically to reveal potential transcriptional alterations at Igλ in the context of Igκ deletion. Compared with Igκ-intact RAG-deficient pre-B cells, Igκ-deleted RAG-deficient pre-B cells showed remarkably increased transcription across the whole Igλ locus, indicating specific Igκ-deletion-triggered transcriptional activation at Igλ. Citation Format: Zhaoqing Ba, Jiazhi Hu, Zhou Du, Sherry G. Lin, Duane R. Wesemann, Frederick W. Alt. Mechanisms that mediate intralocus and interlocus regulation of V(D)J recombination at immunoglobulin light chain loci [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A033.

Abstract B087: Revealing antibody repertoires and RAG tracking by linear amplification mediated-high-throughput genome-wide translocation sequencing

Abstract B lymphocytes generate antibodies to neutralize numerous antigens via RAG endonuclease-initiated V(D)J recombination which joins diverse V and D segments into several J segments. High-throughput genome-wide translocation sequencing (HTGTS) was developed to identify genomic fragments that translocate to a distinct “bait” fragment associated with DNA double-stranded breaks (DSBs). With a single bait primer to linearly amplify across bait-prey junctions for “prey” sequence identification, the linear amplification-mediated HTGTS (LAM-HTGTS) could sensitively detect genome-wide DSBs or translocation in an unbiased way independent of prey sequences. Employing the J-segment-associated coding ends cleaved by RAG as bait fragments, we can capture all the Vs and Ds joined to the bait J segments and thus reveal antibody repertoires in B lymphocyte populations. Such LAM-HTGTS adapted repertoire sequencing (HTGTS-Rep-seq) can easily uncover the productive and non-productive V(D)J joins and complementary determining region 3 (CDR3) information that is important for antigen contact, as well as D usage and RAG “off-targets” that are inviable to previous assays. HTGTS-Rep-seq analysis on the Igk repertoires showed that Vks were widely utilized in spite of their transcription orientations convergent or tandem to those of Jks. However, deletion of the four CTCF-binding elements (CBEs) in the Vk-Jk intervening region greatly increased the usage of Vks proximal to Jks. Strikingly, the usage of Vks transcriptionally convergent to Jks was far more elevated than those in the same transcriptional orientation to Jks, which strongly suggested a role of RAG tracking in mediating increased proximal Vk usage in the absence of Vk-Jk intervening CBEs. Citation Format: Jiazhi Hu, Sherry G. Lin, Zhaoqing Ba, Zhou Du, Yu Zhang, Frederick W. Alt. Revealing antibody repertoires and RAG tracking by linear amplification mediated-high-throughput genome-wide translocation sequencing [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B087.

Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.

T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ thymocytes. Here, we use linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4(-)CD8(-) double-negative thymocyte progenitors differentiated in vitro from bone marrow-derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement. We also find frequent, previously unappreciated, Trdd1 and Trdd2 rearrangements that inactivate Tcrd, including sequential rearrangements from V(D)J recombination signal sequence fusions. Moreover, we find dozens of RAG off-target sequences that are generated via RAG tracking both upstream and downstream from the Trdd2 recombination center across the Tcrd loop domain that is bounded by the upstream INT1-2 and downstream TEA elements. Disruption of the upstream INT1-2 boundary of this loop domain allows spreading of RAG on- and off-target activity to the proximal Trdv domain and, correspondingly, shifts the Tcrd V(D)J recombination landscape by leading to predominant V(D)J joining to a proximal Trdv3 pseudogene that lies just upstream of the normal boundary.

568. Transient Manipulation of DNA Damage Repair Pathway Choice Improves Homology-Directed Repair During CRISPR/Cas9-Mediated Genome Editing

CRISPR/Cas9 system allows efficient gene ablation through error-prone non-homologous end joining DNA repair. However, very low efficiency of homology-directed DNA repair (HDR) is the bottleneck in correcting genetic mutations of clinical relevance. Here we report that transient manipulation of DNA damage repair pathways increases the HDR frequency by 3-5 fold. Furthermore, we show that this approach is applicable to introduce precise genetic modifications at many genetic loci in multiple cell-types including human induced pluripotent stem (hiPS) cells. Furthermore, unbiased off-target mutational analysis using High Throughput Genome-wide Translocation Sequencing (HTGTS) suggests that transient manipulation of DNA damage repair pathways have no adverse effect on either CRISPR/Cas9 specificity or genomic integrity. Our data suggests that this approach could be used for correcting various genetic disorders in relevant cell types.