Honors & Awards

  • Research Scholar, American Cancer Society - Lisa Dean Moseley Foundation Stem Cell Consortium
  • V Scholar, V Foundation for Cancer Research
  • Career Development Award, Radiation Research Foundation

Professional Education

  • Research Fellow, Boston Children's Hospital, Program in Cellular and Molecular Medicine
  • PhD, University of Washington, Biochemistry (2009)
  • BA, Vassar College, Biochemistry (2001)


  • Frederick W Alt, Richard L Frock, Jiazhi Hu, Robin M Meyers. " Patent WO2016081798 Methods relating to the detection of recurrent and non-specific double strand breaks in the genome", Nov 20, 2015

Current Research and Scholarly Interests

We are a functional genomics laboratory interested in elucidating mechanisms of DNA repair pathway choice and genome instability. We employ a powerful discovery platform, High-Throughput Genome-wide Translocation Sequencing (HTGTS), which maps DNA junctions at single nucleotide resolution. Our expertise overlaps many different fields including: genome editing, ionizing radiation and cancer therapeutics, V(D)J and IgH class switch recombination, and meiosis.

2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • DNA-PKcs suppresses illegitimate chromosome rearrangements. Nucleic acids research Wang, J., Sadeghi, C. A., Frock, R. L. 2024


    Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.

    View details for DOI 10.1093/nar/gkae140

    View details for PubMedID 38412274

  • Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation. Nature structural & molecular biology Wang, J., Le Gall, J., Frock, R. L., Strick, T. R. 2023


    Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.

    View details for DOI 10.1038/s41594-023-01104-6

    View details for PubMedID 37828409

    View details for PubMedCentralID 5898235

  • FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Barghouth, P. G., Melemenidis, S., Montay-Gruel, P., Ollivier, J., Viswanathan, V., Jorge, P. G., Soto, L. A., Lau, B. C., Sadeghi, C., Edlabadkar, A., Zhang, R., Ru, N., Baulch, J. E., Manjappa, R., Wang, J., Le Bouteiller, M., Surucu, M., Yu, A., Bush, K., Skinner, L., Maxim, P. G., Loo, B. W., Limoli, C. L., Vozenin, M. C., Frock, R. L. 2023: 109906


    The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT.We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13Gy/s) and FLASH (1x102-5x106 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects.Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival.Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

    View details for DOI 10.1016/j.radonc.2023.109906

    View details for PubMedID 37690668

  • Development of beta-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Science translational medicine Lattanzi, A., Camarena, J., Lahiri, P., Segal, H., Srifa, W., Vakulskas, C. A., Frock, R. L., Kenrick, J., Lee, C., Talbott, N., Skowronski, J., Cromer, M. K., Charlesworth, C. T., Bak, R. O., Mantri, S., Bao, G., DiGiusto, D., Tisdale, J., Wright, J. F., Bhatia, N., Roncarolo, M. G., Dever, D. P., Porteus, M. H. 2021; 13 (598)


    Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the beta-globin gene (HBB). Ex vivo beta-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

    View details for DOI 10.1126/scitranslmed.abf2444

    View details for PubMedID 34135108

  • Ku70 suppresses alternative end joining in G1-arrested B cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liang, Z., Kumar, V., Bouteiller, M., Zurita, J., Kenrick, J., Lin, S. G., Lou, J., Hu, J., Ye, A., Boboila, C., Alt, F. W., Frock, R. L. 2021; 118 (21)
  • Mechanism of tandem duplication formation in BRCA1-mutant cells. Nature Willis, N. A., Frock, R. L., Menghi, F. n., Duffey, E. E., Panday, A. n., Camacho, V. n., Hasty, E. P., Liu, E. T., Alt, F. W., Scully, R. n. 2017; 551 (7682): 590–95


    Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

    View details for PubMedID 29168504

  • PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kumar, V., Alt, F. W., Frock, R. L. 2016; 113 (38): 10619-10624


    Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.

    View details for DOI 10.1073/pnas.1611882113

    View details for Web of Science ID 000383622600051

    View details for PubMedID 27601633

    View details for PubMedCentralID PMC5035843

  • Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing NATURE PROTOCOLS Hu, J., Meyers, R. M., Dong, J., Panchakshari, R. A., Alt, F. W., Frock, R. L. 2016; 11 (5): 853-871


    Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

    View details for DOI 10.1038/nprot.2016.043

    View details for Web of Science ID 000374445900002

    View details for PubMedID 27031497

    View details for PubMedCentralID PMC4895203

  • Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases NATURE BIOTECHNOLOGY Frock, R. L., Hu, J., Meyers, R. M., Ho, Y., Kii, E., Alt, F. W. 2015; 33 (2): 179-186


    Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

    View details for DOI 10.1038/nbt.3101

    View details for Web of Science ID 000349198800024

    View details for PubMedID 25503383

    View details for PubMedCentralID PMC4320661

  • Genome-wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells CELL Chiarle, R., Zhang, Y., Frock, R. L., Lewis, S. M., Molinie, B., Ho, Y., Myers, D. R., Choi, V. W., Compagno, M., Malkin, D. J., Neuberg, D., Monti, S., Giallourakis, C. C., Gostissa, M., Alt, F. W. 2011; 147 (1): 107-119


    Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

    View details for DOI 10.1016/j.cell.2011.07.049

    View details for Web of Science ID 000295396700019

    View details for PubMedID 21962511

    View details for PubMedCentralID PMC3186939

  • Increased AID Results in Mutations at the CRLF2 Locus Implicated in Latin American ALL Health Disparities. Research square Pannunzio, N., Rangel, V., Sterrenberg, J., Garawi, A., Mezcord, V., Folkerts, M., Caulderon, S., Wang, J., Soyfer, E., Eng, O., Valerin, J., Tanjasiri, S., Quintero-Rivera, F., Masri, S., Seldin, M., Frock, R., Fleischman, A. 2023


    Activation-induced cytidine deaminase (AID) is a B cell-specific base editor required during class switch recombination and somatic hypermutation for B cell maturation and antibody diversification. However, it has also been implicated as a factor in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain types of blood cancers is critical in assessing disease severity and treatment options. Here, we have developed a digital PCR (dPCR) assay that allows us to track the mutational landscape resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this new assay showed that increased AID levels in immature B cells increases genome instability at loci linked to translocation formation. This included the CRLF2 locus that is often involved in chromosomal translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Latin Americans (LAs). To support this LA-specific identification of AID mutation signatures, we characterized DNA from immature B cells isolated from the bone marrow of ALL patients. Our ability to detect and quantify these mutation signatures will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

    View details for DOI 10.21203/rs.3.rs-3332673/v1

    View details for PubMedID 37790327

    View details for PubMedCentralID PMC10543404

  • DNA End Joining: G0-ing to the Core. Biomolecules Frock, R. L., Sadeghi, C., Meng, J., Wang, J. L. 2021; 11 (10)


    Humans have evolved a series of DNA double-strand break (DSB) repair pathways to efficiently and accurately rejoin nascently formed pairs of double-stranded DNA ends (DSEs). In G0/G1-phase cells, non-homologous end joining (NHEJ) and alternative end joining (A-EJ) operate to support covalent rejoining of DSEs. While NHEJ is predominantly utilized and collaborates extensively with the DNA damage response (DDR) to support pairing of DSEs, much less is known about A-EJ collaboration with DDR factors when NHEJ is absent. Non-cycling lymphocyte progenitor cells use NHEJ to complete V(D)J recombination of antigen receptor genes, initiated by the RAG1/2 endonuclease which holds its pair of targeted DSBs in a synapse until each specified pair of DSEs is handed off to the NHEJ DSB sensor complex, Ku. Similar to designer endonuclease DSBs, the absence of Ku allows for A-EJ to access RAG1/2 DSEs but with random pairing to complete their repair. Here, we describe recent insights into the major phases of DSB end joining, with an emphasis on synapsis and tethering mechanisms, and bring together new and old concepts of NHEJ vs. A-EJ and on RAG2-mediated repair pathway choice.

    View details for DOI 10.3390/biom11101487

    View details for PubMedID 34680120

  • Ultra-high dose rate (FLASH) irradiation does not alter microhomology mediated recombination under varying oxygen tension when compared to standard clinical dose rates. Barghouth, P., Ollivier, J., Montay-Gruel, P., Loo, B. W., Vozenin, M., Limoli, C., Frock, R. AMER ASSOC CANCER RESEARCH. 2021
  • Precise and broad scope genome editing based on high-specificity Cas9 nickases. Nucleic acids research Wang, Q., Liu, J., Janssen, J. M., Le Bouteiller, M., Frock, R. L., Goncalves, M. A. 2021


    RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

    View details for DOI 10.1093/nar/gkaa1236

    View details for PubMedID 33398349

  • Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells Frock, R. L., Kumar, V., Liang, Z., Zurita, J., Du, Z., Lin, S. G., Boboila, C., Alt, F. W. AMER ASSOC CANCER RESEARCH. 2019
  • Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking. Nucleic acids research Chen, X. n., Tasca, F. n., Wang, Q. n., Liu, J. n., Janssen, J. M., Brescia, M. D., Bellin, M. n., Szuhai, K. n., Kenrick, J. n., Frock, R. L., Gonçalves, M. A. 2019


    Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.

    View details for DOI 10.1093/nar/gkz1121

    View details for PubMedID 31799604

  • Parp3 promotes long-range end joining in murine cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Layer, J., Cleary, J., Brown, A. J., Stevenson, K. E., Morrow, S. N., Van Scoyk, A., Blasco, R. B., Karaca, E., Meng, F., Frock, R. L., Tivey, T., Kim, S., Fuchs, H., Chiarle, R., Alt, F. W., Roberts, S. A., Weinstock, D. M., Day, T. A. 2018; 115 (40): 10076–81


    Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

    View details for DOI 10.1073/pnas.1801591115

    View details for Web of Science ID 000446078700075

    View details for PubMedID 30213852

    View details for PubMedCentralID PMC6176633

  • Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing. Nature biomedical engineering Paulsen, B. S., Mandal, P. K., Frock, R. L., Boyraz, B., Yadav, R., Upadhyayula, S., Gutierrez-Martinez, P., Ebina, W., Fasth, A., Kirchhausen, T., Talkowski, M. E., Agarwal, S., Alt, F. W., Rossi, D. J. 2017; 1 (11): 878-888


    Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

    View details for DOI 10.1038/s41551-017-0145-2

    View details for PubMedID 31015609

  • Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development. journal of experimental medicine Zhao, L., Frock, R. L., Du, Z., Hu, J., Chen, L., Krangel, M. S., Alt, F. W. 2016; 213 (9): 1921-1936


    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.

    View details for DOI 10.1084/jem.20160670

    View details for PubMedID 27526713

    View details for PubMedCentralID PMC4995090

  • Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes CELL Hu, J., Zhang, Y., Zhao, L., Frock, R. L., Du, Z., Meyers, R. M., Meng, F., Schatz, D. G., Alt, F. W. 2015; 163 (4): 947-959


    RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

    View details for DOI 10.1016/j.cell.2015.10.016

    View details for Web of Science ID 000364829700019

    View details for PubMedID 26593423

    View details for PubMedCentralID PMC4660266

  • Mechanisms of Recurrent Chromosomal Translocations Chromosomal Translocations and Genome Rearrangements in Cancer Frock, R. L., Hu, J., Alt, F. W. Springer International Publishing. 2015; 1: 27–51
  • Cardiomyocyte-Specific Expression of Lamin A Improves Cardiac Function in Lmna(-/-) Mice PLOS ONE Frock, R. L., Chen, S. C., Da, D., Frett, E., Lau, C., Brown, C., Pak, D. N., Wang, Y., Muchir, A., Worman, H. J., Santana, L. F., Ladiges, W. C., Rabinovitch, P. S., Kennedy, B. K. 2012; 7 (8)


    Lmna(-/-) mice display multiple tissue defects and die by 6-8 weeks of age reportedly from dilated cardiomyopathy with associated conduction defects. We sought to determine whether restoration of lamin A in cardiomyocytes improves cardiac function and extends the survival of Lmna(-/-) mice. We observed increased total desmin protein levels and disorganization of the cytoplasmic desmin network in ~20% of Lmna(-/-) ventricular myocytes, rescued in a cell-autonomous manner in Lmna(-/-) mice expressing a cardiac-specific lamin A transgene (Lmna(-/-); Tg). Lmna(-/-); Tg mice displayed significantly increased contractility and preservation of myocardial performance compared to Lmna(-/-) mice. Lmna(-/-); Tg mice attenuated ERK1/2 phosphorylation relative to Lmna(-/-) mice, potentially underlying the improved localization of connexin43 to the intercalated disc. Electrocardiographic recordings from Lmna(-/-) mice revealed arrhythmic events and increased frequency of PR interval prolongation, which is partially rescued in Lmna(-/-); Tg mice. These findings support our observation that Lmna(-/-); Tg mice have a 12% median extension in lifespan compared to Lmna(-/-) mice. While significant, Lmna(-/-); Tg mice only have modest improvement in cardiac function and survival likely stemming from the observation that only 40% of Lmna(-/-); Tg cardiomyocytes have detectable lamin A expression. Cardiomyocyte-specific restoration of lamin A in Lmna(-/-) mice improves heart-specific pathology and extends lifespan, demonstrating that the cardiac pathology of Lmna(-/-) mice limits survival. The expression of lamin A is sufficient to rescue certain cellular defects associated with loss of A-type lamins in cardiomyocytes in a cell-autonomous fashion.

    View details for DOI 10.1371/journal.pone.0042918

    View details for Web of Science ID 000307823600034

    View details for PubMedID 22905185

    View details for PubMedCentralID PMC3419749

  • Cell-Extrinsic Defective Lymphocyte Development in Lmna(-/-) Mice PLOS ONE Hale, J. S., Frock, R. L., Mamman, S. A., Fink, P. J., Kennedy, B. K. 2010; 5 (4)


    Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna(-/-) mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(-/-) mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development.Lmna(-/-) mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna(-/-) bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4(+) and CD8(+) T cells. Transplantation of Lmna(-/-) neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development.Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(-/-) mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(-/-) mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.

    View details for DOI 10.1371/journal.pone.0010127

    View details for Web of Science ID 000276705900011

    View details for PubMedID 20405040

    View details for PubMedCentralID PMC2853576

  • A-type lamins, nuclear structure and disease Kennedy, B., Frock, R., Kudlow, B., Lee, D., Nitta, R., Johnston, E., Hauschka, S. SPRINGER. 2007: 13
  • Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation GENES & DEVELOPMENT Frock, R. L., Kudlow, B. A., Evans, A. M., Jameson, S. A., Hauschka, S. D., Kennedy, B. K. 2006; 20 (4): 486-500


    Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and beta-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(-/-) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(-/-) myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.

    View details for DOI 10.1101/gad.1364906

    View details for Web of Science ID 000235428600011

    View details for PubMedID 16481476

    View details for PubMedCentralID PMC1369050

  • A-type nuclear lamins, progerias and other degenerative disorders MECHANISMS OF AGEING AND DEVELOPMENT Smith, E. D., Kudlow, B. A., Flock, R. L., Kennedy, B. K. 2005; 126 (4): 447-460


    Nuclear lamins were identified as core nuclear matrix constituents over 20 years ago. They have been ascribed structural roles such as maintaining nuclear integrity and assisting in nuclear envelope formation after mitosis, and have also been linked to nuclear activities including DNA replication and transcription. Recently, A-type lamin mutations have been linked to a variety of rare human diseases including muscular dystrophy, lipodystrophy, cardiomyopathy, neuropathy and progeroid syndromes (collectively termed laminopathies). Most diseases arise from dominant, missense mutations, leading to speculation as to how different mutations in the same gene can give rise to such a diverse set of diseases, some of which share little phenotypic overlap. Understanding the cellular dysfunctions that lead to laminopathies will almost certainly provide insight into specific roles of A-type lamins in nuclear organization. Here, we compare and contrast the LMNA mutations leading to laminopathies with emphasis on progerias, and discuss possible functional roles for A-type lamins in the maintenance of healthy tissues.

    View details for DOI 10.1016/j.mad.2004.10.006

    View details for Web of Science ID 000227605900001

    View details for PubMedID 15722103

  • A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnson, B. R., Nitta, R. T., Frock, R. L., Mounkes, L., Barbie, D. A., Stewart, C. L., Harlow, E., Kennedy, B. K. 2004; 101 (26): 9677-9682


    The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G(1) phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna(-/-) cells resemble Rb(-/-) cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.

    View details for DOI 10.1073/pnas.0403250101

    View details for Web of Science ID 000222405600030

    View details for PubMedID 15210943

    View details for PubMedCentralID PMC470734

  • Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit MOLECULAR AND CELLULAR BIOLOGY Barbie, D. A., Kudlow, B. A., Frock, R., Zhao, J. Y., Johnson, B. R., Dyson, N., Harlow, E., Kennedy, B. K. 2004; 24 (2): 595-607


    In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.

    View details for DOI 10.1128/MCB.24.2.595-607.2004

    View details for Web of Science ID 000188211200010

    View details for PubMedID 14701733

    View details for PubMedCentralID PMC343811

  • Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila DEVELOPMENT Deng, W. M., Schneider, M., Frock, R., Castillejo-Lopez, C., Baumgartner, S., Ruohola-Baker, H. 2003; 130 (1): 173-184


    The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.

    View details for DOI 10.1242/dev.00199

    View details for Web of Science ID 000180537900016

    View details for PubMedID 12441301