CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knockin of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on longread sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.Recent developments in gene editing technologies have transformed our ability to manipulate genomes. Programmable site-specific nucleases -in particular CRISPR/Cas systems -introduce double-strand breaks (DSBs) at chosen genomic locations, prompting the activation of two separate DNA repair pathways which can be leveraged for genome engineering 1 . On the one hand, nonhomologous end-joining (NHEJ) can introduce insertions or deletions (in-dels) at the DSB site to inactivate gene products or regulatory elements. On the other hand, homology-directed repair (HDR)can use exogenous DNA sequences as templates to integrate (knock-in) new genetic information in the locus of interest 2 . Knock-in strategies have wide applications ranging from correcting diseasecausing mutations or inserting therapeutic payloads in a clinical context 3-5 , to introducing functional reporters for cell biology research 6,7 .Ultimately, the power of genetic engineering will rely on our ability to predictably edit genomes in order to precisely control cellular behaviors. Traditionally, NHEJ and HDR have been set apart by their degree of predictability: NHEJ is often thought to drive random repair outcomes, while HDR is considered to enable precise and templated editing. Recent data is challenging this simple distinction, however. High-throughput sequencing of NHEJ repair has uncovered complex but actionable sequence patterns that control in-del outcomes at DSBs 8-13 . In some cases, predictable NHEJ outcomes can be leveraged to precisely correct pathogenic human mutations, paving the way for template-free therapeutic genome editing 9 . By contrast, while accumulating evidence suggests that the integration of payload in knock-in experiments is not always precise 7,14,15 , a systematic and quantitative profiling of the full spectrum of HDR repair outcomes is still missing.Given its wide range of applications in both clinical and research settings, understanding the parameters that govern the efficiency and precision of knock...

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

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