CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

The efficiency of conventional CRISPR knockout strategy relies on error-prone repair of DNA double strand breaks at target sites. However, this may result in excessive DNA damage response and deleterious effects particularly at high copy genomic regions, thus confounding experimental results. Here, we repurposed a CRISPR base editor as an efficient and less toxic gene silencing method. We demonstrate that Cas9n-fused to APOBEC1 cytosine deaminase can efficiently edit Arg, Gln and Trp coding sequences into STOP codons, leading to loss of function gene mutations (CRISPR-STOP). Importantly, CRISPR-STOP has higher gene KO efficiency at high copy regions and results in significantly less DNA damage response and cytotoxicity compared to WT-Cas9. Genome-wide computational analyses show that ~17,000 genes can be targeted to introduce early stop codons. Our targeted screening indicates the suitability of genetic code editing and CRISPR-STOP approach as an efficient functional screening tool. This protocol accompanies Kuscu et al, Nature Methods, published

Genetic and functional studies have revealed that both common and rare variants of several nicotinic acetylcholine receptor (nAChR) subunits are associated with nicotine dependence (ND). In this study, we identified variants in 30 candidate genes including nicotinic receptors in 200 sib pairs selected from the Mid-South Tobacco Family (MSTF) population with equal numbers of African Americans (AAs) and European Americans (EAs). We selected 135 of the rare and common variants and genotyped them in the Mid-South Tobacco Case-Control (MSTCC) population, which consists of 3088 AAs and 1430 EAs. None of the genotyped common variants showed significant association with smoking status (smokers vs. non-smokers), Fagerström Test for Nicotine Dependence (FTND) scores, or indexed cigarettes per day (CPD) after Bonferroni correction. Rare variants in NRXN1, CHRNA9, CHRNA2, NTRK2, GABBR2, GRIN3A, DNM1, NRXN2, NRXN3, and ARRB2 were significantly associated with smoking status in the MSTCC AA sample, with Weighted Sum Statistic (WSS) P values ranging from 2.42 × 10−3 to 1.31 × 10−4 after 106 phenotype rearrangements. We also observed a significant excess of rare nonsynonymous variants exclusive to EA smokers in NRXN1, CHRNA9, TAS2R38, GRIN3A, DBH, ANKK1/DRD2, NRXN3, and CDH13 with WSS P values between 3.5 × 10−5 and 1 × 10−6. Variants rs142807401 (A432T) and rs139982841 (A452V) in CHRNA9 and variants V132L, V389L, rs34755188 (R480H), and rs75981117 (N549S) in GRIN3A are of particular interest because they are found in both the AA and EA samples. A significant aggregate contribution of rare and common coding variants in CHRNA9 to the risk for ND (SKAT-C: P= 0.0012) was detected by applying the combined sum test in MSTCC EAs. Together, our results indicate that rare variants alone or combined with common variants in a subset of 30 biological candidate genes contribute substantially to the risk of ND.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through smallmolecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresis-tance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers.Significance: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.