The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.
53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini. This function of 53BP1 requires interactions with PTIP and RIF1, the latter of which recruits REV7 (also known as MAD2L2) to break sites. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.
The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genomewide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1, limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage, and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.53BP1 can influence the type of DNA repair at DSBs (1) as seen in immunoglobulin gene rearrangements (2-4) and in the fusion of telomeres rendered dysfunctional through the removal of the shelterin protein TRF2 (5), where 53BP1 enhances the mobility of damaged telomeres, thus potentially promoting the chance of telomeretelomere encounters. In Brca1-deficient cells, 53BP1 enhances aberrant NHEJ events that create lethal radial chromosomes in response to poly(ADP-ribose) polymerase PARP1 inhibitors (PARPi) (6). In this setting, 53BP1 may favor NHEJ-mediated mis-rejoining by blocking the DSB resection needed for HDR (6, 7). 53BP1 was shown to impede 5' end resection at dysfunctional telomeres lacking all shelterin proteins and similarly, telomeres lacking only TRF2 show evidence of 53BP1-dependent protection from resection (5,8). Based on the finding that an allele of 53BP1 (53BP1 28A ) lacking all potential ATM/ATR kinase S/TQ target sites did not support immunoglobulin Class Switch Recombination (CSR) and failed to generate radial chromosomes in Brca1-deficient cells (7), it appears that these functions of 53BP1 involve interacting partner(s) modulated by the S/TQ sites. One candidate 53BP1-interacting factor is Rif1, which localizes to DSBs and dysfunctional telomeres, in a manner that is dependent on ATM signaling (9-11). Rif1 was originally identified as part of the telomeric complex in budding yeast (12) and was recently shown to inhibit resection at yeast telomeres (13,14). In contrast, mammalian Rif1 has no known function at functional telomeres but contributes to the intra-S phase checkpoint, facilitates recovery from replication stress, and affects replication timing (10,(15)(16)(17) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe introduced 53BP1 28A and other 53BP1 mutant alleles (7) into immortalized TRF2 F/-53BP1 -/-mouse embryo fibroblasts (MEFs) and induced telomere dysfunction by deletion of TRF2 (Fig. 1A,B). The results showed that the S/TQ sites were required for the accumulation of Rif1 at deprotected telomeres, whereas the GAR, BRCT, and oligomerization domains of 53BP1 were not ( Fig. 1A-C; fig. S1). The functional significance of the Rif1-53BP1 interaction was addressed using a tel...
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