High-throughput Identification Of Pathogenic Mutations In Tumor Genes Using Single-base Editing Tools

With the development of next generation sequencing,millions of single nucleotide variants(SNVs),including germline and somatic variants,have been identified through genome sequencing of clinical cancer samples.Some mutations play an important role in the initiation and progression of cancer,so the ability for us to interpret the functional impacts of these variants on cancer development is required for cancer risk assessment and therapy.There are not enough high-throughput screening methods on assessment of single nucleotide variants.Even for variants in the well-known cancer genes,it is unclear whether they are pathogenic,which limited the usage of the variant information for cancer risk management.For example,clinical genetic testing has identified many variants including variants of unknown significance(VUS)in the BRCA1/2,which are known to confer the greatest risks for developing breast and ovarian cancer.Assessment of functional impacts of SNVs in a high-throughput manner is a still challenge.Here we used base editors developed from CRISPR-mediated gene editing technology to detect impact for single nucleotide variants(SNVs)of BRCA1/2 exons in a full-length tiling manner.We calculated the impacts of variants on the growth of eHAP cells.We used two editors recognizing NGG PAM sequences which induce C to T and A to G editing,respectively.In order to expand the scope of target region,we developed two base editors with PAM requirement relaxed to "NG",making it possible for large-scale base-editing screens to assess a group of densely clustered SNVs.Totally we designed 4032 and 5298 sgRNAs to target all exons in BRCA1 and BRCA2,using cytosine and adenine base editors according to their PAM requirement and activity window.In order to avoid the impact of the variability in editing activities,we developed an RNA target pair reporter system to verify editing activities of sgRNAs,and then developed a framework to incorporate editing efficiency correction in base editing screens.Applying base editing screens with efficiency corrections,we assessed functional impacts of base conversions for about 9000 sites in BRCA1/2 genes.We identified 919 mutations that have deleterious effects on BRCA1/2 function,including a group of mutations in known functional domains and 154 mutations at UTR(Untranslated Regions),some of which have been identified as "pathogenic"mutations in clinical testing.Our cell viability functional assays results suggested that the variants in UTR,splicing regions and nonsynonymous mutations have significantly delayed the growth of cells.Our results suggested that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner,and it was the first screen using NG base editors.Applying this strategy to assess variants in both coding and noncoding regions of the genome could help us to understand functional impacts of cancer variants.Here we demonstrated the possibility to identify pathogenic variants using base editors and our results also indicated noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.Our system could greatly facilitate the high-throughput analysis of variants.The results will also helped us to illustrate the "genotype-phenotype" correlation,which could benefit interpretation of variants in cancer samples for better risk assessment and therapeutic applications.