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CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.

Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.

DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.

A new study reports biallelic mutations in RAD51C in a Fanconi anemia-like disorder, while a second study reports monoallelic mutations in the same gene associated with increased breast cancer risk. These findings strengthen the link between Fanconi anemia and breast cancer-associated pathways.

Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

PALB2 was recently identified as a nuclear binding partner of BRCA2. Biallelic BRCA2 mutations cause Fanconi anemia subtype FA-D1 and predispose to childhood malignancies. We identified pathogenic mutations in PALB2 (also known as FANCN) in seven families affected with Fanconi anemia and cancer in early childhood, demonstrating that biallelic PALB2 mutations cause a new subtype of Fanconi anemia, FA-N, and, similar to biallelic BRCA2 mutations, confer a high risk of childhood cancer.

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

BRIP1 (also called BACH1) is a DEAH helicase that interacts with the BRCT domain of BRCA1 (refs. 1-6) and has an important role in BRCA1-dependent DNA repair and checkpoint functions. We cloned the chicken ortholog of BRIP1 and established a homozygous knockout in the avian B-cell line DT40. The phenotype of these brip1 mutant cells in response to DNA damage differs from that of brca1 mutant cells and more closely resembles that of fancc mutant cells, with a profound sensitivity to the DNA-crosslinking agent cisplatin and acute cell-cycle arrest in late S-G2 phase. These defects are corrected by expression of human BRIP1 lacking the BRCT-interaction domain. Moreover, in human cells exposed to mitomycin C, short interfering RNA-mediated knock-down of BRIP1 leads to a substantial increase in chromosome aberrations, a characteristic phenotype of cells derived from individuals with Fanconi anemia. Because brip1 mutant cells are proficient for ubiquitination of FANCD2 protein, our data indicate that BRIP1 has a function in the Fanconi anemia pathway that is independent of BRCA1 and downstream of FANCD2 activation.

Seven Fanconi anemia-associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.

Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including nonsense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Fanconi anaemia (FA) is an autosomal-recessive disorder characterized by genomic instability, developmental defects, DNA crosslinking agent hypersensitivity and cancer susceptibility. Somatic-cell hybrid studies have revealed five FA complementation groups (A-E; refs 4-6) displaying similar phenotypes, suggesting that FA genes are functionally related. The two cloned FA genes, FAA and FAC, encode proteins that are unrelated to each other or to other proteins in GenBank. In the current study, we demonstrate the FAA and FAC bind each other and form a complex. Protein binding correlates with the functional activity of FAA and FAC, as patient-derived mutant FAC (L554P) fails to bind FAA. Although unbound FAA and FAC localize predominantly to the cytoplasm, the FAA-FAC complex is found in similar abundance in both cytoplasm and nucleus. Our results confirm the interrelatedness of the FA genes in a pathway, suggesting the cooperation of FAA and FAC in a nuclear function.

Diamond-Blackfan anaemia (DBA; MIM#205900) is a rare disorder manifested as a pure red-cell aplasia in the neonatal period or in infancy. The clinical hallmark of DBA is a selective decrease in erythroid precursors and anaemia. Other lineages are usually normal and the peripheral white blood cell count is normal. In approximately one-third of cases, the disease is associated with a wide variety of congenital anomalies and malformations. Most cases are sporadic, but 10-20% of them follow a recessive or a dominant inheritance pattern. A female with DBA and a chromosomal translocation involving chromosome 19q was recently identified. We undertook a linkage analysis with chromosome 19 markers in multiplex DBA families of Swedish, French, Dutch, Arabic and Italian origin. Significant linkage to chromosome 19q13 was established for dominant and recessive inherited DBA with a peak lod score at D19S197 (Zmax = 7.08, theta = 0.00). Within this region, a submicroscopic de novo deletion of 3.3 Mb was identified in a patient with DBA. The deletion coincides with the translocation break-point and, together with key recombinations, restricts the DBA gene to a 1.8-Mb region. The results suggest that, despite its clinical heterogeneity, DBA is genetically homogeneous for a gene in 19q13.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Fanconi anaemia (FA) is an autosomal recessive disorder associated with progressive bone-marrow failure, a variety of congenital abnormalities, and predisposition to acute myeloid leukaemia. Cells from FA patients show increased sensitivity to bifunctional DNA crosslinking agents such as diepoxybutane and mitomycin C, with characteristic chromosome breakage. FA is genetically heterogeneous, at least five different complementation groups (FA-A to FA-E) having been described. The gene for group C (FAC) was cloned by functional complementation and mapped to chromosome 9q22.3 (refs 3, 5), but the genes for the other complementation groups have not yet been identified. The group A gene (FAA) has recently been mapped to chromosome 16q24.3 by linkage analysis, and accounts for 60-65% of FA cases. We narrowed the candidate region by linkage and allelic association analysis, and have isolated a gene that is mutated in FA-A patients. The gene encodes a protein of 1,455 amino acids that has no significant homology to any other known proteins, and may therefore represent a new class of genes associated with the prevention or repair of DNA damage.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

Fanconi anaemia (FA) is an autosomal recessive disorder associated with diverse developmental abnormalities, bone-marrow failure and predisposition to cancer. FA cells show increased chromosome breakage and hypersensitivity to DNA cross-linking agents such as diepoxybutane and mitomycin C. Somatic-cell hybridisation analysis of FA cell lines has demonstrated the existence of at least five complementation groups (FA-A to FA-E), the most common of which is FA-A. This genetic heterogeneity has been a major obstacle to the positional cloning of FA genes by classical linkage analysis. The FAC gene was cloned by functional complementation, and localised to chromosome 9q22.3 (ref. 2), but this approach has thus far failed to yield the genes for the other complementation groups. We have established a panel of families classified as FA-A by complementation analysis, and used them to search for the FAA gene by linkage analysis. We excluded the previous assignment by linkage of an FA gene to chromosome 20q, and obtained conclusive evidence for linkage of FAA to microsatellite markers on chromosome 16q24.3. Strong evidence of allelic association with the disease was detected with the marker D16S303 in the Afrikaner population of South Africa, indicating the presence of a founder effect.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopenia, short stature, radial ray defects, skin hyperpigmentation and a predisposition to cancer. Cells from FA patients are hypersensitive to cell killing and chromosome breakage induced by DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Consequently, the defect in FA is thought to be in DNA crosslink repair. Additional cellular phenotypes of FA include oxygen sensitivity, poor cell growth and a G2 cell cycle delay. At least 5 complementation groups for Fanconi anaemia exist, termed A through E. One of the five FA genes, FA(C), has been identified by cDNA complementation, but no other FA genes have been mapped or cloned until now. The strategy of cDNA complementation, which was successful for identifying the FA(C) gene has not yet been successful for cloning additional FA genes. The alternative approach of linkage analysis, followed by positional cloning, is hindered in FA by genetic heterogeneity and the lack of a simple assay for determining complementation groups. In contrast to genetic linkage studies, microcell mediated chromosome transfer utilizes functional complementation to identify the disease bearing chromosome. Here we report the successful use of this technique to map the gene for the rare FA complementation group D (FA(D)).

Fanconi anaemia is an autosomal recessive disease for which four known complementation groups exist. Recently, the gene defective in complementation group C (FACC) has been cloned. In order to determine the fraction of Fanconi anaemia caused by FACC mutations, we used reverse transcription PCR and chemical mismatch cleavage (CMC) to examine the FACC cDNA in 17 FA cell lines. 4/17 patients (23.5%) had mutations in this gene. Two Ashkenazi-Jewish individuals were homozygous for an identical splice mutation. Three additional Jewish patients bearing this allele were found upon screening 21 other families. We conclude that a common mutation in FACC accounts for the majority of Fanconi anaemia in Ashkenazi-Jewish families.

Fanconi anaemia (FA) is a DNA repair disorder characterized by cellular hypersensitivity to DNA cross-linking agents and extensive phenotypic heterogeneity. To determine the extent of genetic heterogeneity present in FA, a panel of somatic cell hybrids was constructed using polyethylene glycol-mediated cell fusion. Three new complementation groups were identified, designated FA(B), FA(C) and FA(D), and the gene defective in FA(C) which we have recently cloned was localized to chromosome 9q22.3 through in situ hybridization. These results suggest that mutations in at least four different genes lead to FA, a degree of genetic heterogeneity comparable to that of other DNA repair disorders.