Double strand break (DSB) repair mainly occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). We present an assay system that enables simultaneous measurement of all three pathways using Cas9-generated DSBs and next generation sequencing to profile and quantify pathway choice. The assay system has provided several insights. First, absence of the key Alt-EJ factor Pol q only abrogates ~50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. These fundamental differences between BRCA1 and BRCA2 deficiency have implications for therapeutic targeting of HR-deficient cancers. This assay can be used in any system which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

10.2139/ssrn.3720009

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SSI 2013 Abstract Submission Innate Immunity

Scandinavian Journal of Immunology (2013)

Olivier Demeure Edin Hamzić Hervé Juin Murielle Naciri Helle R. Juul‐Madsen

Background: Collectins and ficolins are important in the clearance of endogenous and exogenous danger materials.A new human collectin-11 was recently identified in serum with low concentration in complex with mannose-binding lectin (MBL)/ficolin-associated serine proteases, and found to recognize carbohydrate residues present on various microorganisms.Objectives: We hypothesized that collectin-11 could be a novel initiation molecule in the lectin complement pathway in addition to the MBL and the ficolins.Methods: The association of MASP-1, -2, -3 and MAP-1 with collectin-11 was analyzed by the ELISA platform and by flow cytometry using Candida albicans as collectin-11 binding matrix.The potential involvement of collectin-11 in activation of the lectin complement pathway was evaluated by addition of exogenous recombinant MASP-2 and purified human C4 to collectin-11 anchored on Candida albicans using flow cytometry.Moreover, we investigated the depositions of C4, C3 and the terminal complement complex (TCC) in the presence of collectin-11 on Candida albicans by using MBL-defect serum as complement source.Results: We found that collectin-11 could associate with all the known MASPs (MASP-1, -2, -3) as well as the lectin pathway regulator MAP-1, and revealed that collectin-11:MASP-2 complex formation on Candida albicans could mediate C4 deposition.Furthermore, collectin-11 anchored on Candida albicans induced depositions of C4, C3 and formation of TCC in the presence of MBL-defect serum.Conclusions: These results define collectin-11 as the fifth recognition molecule in the lectin complement pathway in addition to MBL, ficolin-1, ficolin-2 and ficolin-3.

10.1111/sji.12036

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Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell

Nucleic Acids Research (2021)

Suleman S. Hussain Rahul Majumdar Grace Moore Himanshi Narang Erika S. Buechelmaier

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Non-homologous end joining

10.1093/nar/gkab262

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Estimates of autozygosity derived from runs of homozygosity: empirical evidence from selected cattle populations

Journal of Animal Breeding and Genetics (2012)

Maja Ferenčaković Edin Hamzić Birgit Gredler Trygve Solberg G. Klemetsdal

Summary Using genome‐wide SNP data, we calculated genomic inbreeding coefficients ( F ROH > 1 Mb , F ROH > 2 Mb , F ROH > 8 Mb and F ROH > 16 Mb ) derived from runs of homozygosity ( ROH ) of different lengths (>1, >2, >8 and > 16 Mb) as well as from levels of homozygosity ( F HOM ). We compared these values of inbreeding coefficients with those calculated from pedigrees ( F PED ) of 1422 bulls comprising Brown Swiss (304), Fleckvieh (502), Norwegian Red (499) and Tyrol Grey (117) cattle breeds. For all four breeds, population inbreeding levels estimated by the genomic inbreeding coefficients F ROH > 8 Mb and F ROH > 16 Mb were similar to the levels estimated from pedigrees. The lowest values were obtained for Fleckvieh ( F PED = 0.014, F ROH > 8 Mb = 0.019 and F ROH > 16 Mb = 0.008); the highest, for Brown Swiss ( F PED = 0.048, F ROH > 8 Mb = 0.074 and F ROH > 16 Mb = 0.037). In contrast, inbreeding estimates based on the genomic coefficients F ROH > 1 Mb and F ROH > 2 Mb were considerably higher than pedigree‐derived estimates. Standard deviations of genomic inbreeding coefficients were, on average, 1.3–1.7‐fold higher than those obtained from pedigrees. Pearson correlations between genomic and pedigree inbreeding coefficients ranged from 0.50 to 0.62 in Norwegian Red (lowest correlations) and from 0.64 to 0.72 in Tyrol Grey (highest correlations). We conclude that the proportion of the genome present in ROH provides a good indication of inbreeding levels and that analysis based on ROH length can indicate the relative amounts of autozygosity due to recent and remote ancestors.

Runs of Homozygosity

Pedigree chart

10.1111/jbg.12012

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Additional file 4: Table S2. of RNA sequencing-based analysis of the spleen transcriptome following infectious bronchitis virus infection of chickens selected for different mannose-binding lectin serum concentrations

Figshare (2016)

Edin Hamzić Rikke Kjærup Núria Mach Guilietta Minozzi Francesco Strozzi

Differentially expressed genes (FDR

10.6084/m9.figshare.c.3597131_d7

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