Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

Chemical screen for epigenetic barriers to single allele activation of Oct4.

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.