CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.

Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs1) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector "handles". We asked whether coupling de novo protein design to a multiplex screening framework could address this gap by accelerating the discovery of effector handles for intracellular protein degradation, stabilization, or relocalization. Using LABEL-seq2, a sequencing-based assay that enables multiplex, quantitative measurement of protein abundance, we screened 9,715 de novo designed candidate effector handles for their ability to recruit a target protein to components of the ubiquitin-proteasome system3 (UPS) (FBXL12, TRAF2, UCHL1, USP38) or the autophagy pathway4 (GABARAP, GABARAPL2, MAP1LC3A). In a single experiment, we discovered hundreds of de novo designed effector handles that reproducibly drove either intracellular degradation (n = 277) or stabilization (n = 204) of a reporter protein. Validation of a subset of these hits in an orthogonal assay confirmed that sequencing-based measurements from the primary screen reliably reflected changes in intracellular abundance of the target protein. Successful effector handles were discovered for both the UPS (n = 194) and autophagy (n = 287) pathways, which provide complementary routes for programmable proteome editing. Autophagy-recruiting effector handles generalized to endogenous targets, as substituting the reporter-specific target handle with a high-affinity MCL1 binder5 reduced endogenous levels of this intracellular oncoprotein6. Moreover, directing autophagy-recruiting effector handles to the outer mitochondrial membrane dramatically perturbed mitochondrial networks in a manner consistent with synthetic tethering and sequestration7,8. Beyond generating a diverse repertoire of protein abundance or localization effector handles, our results establish a scalable, low-cost platform that links deep learning-guided protein design to functional cellular readouts, and chart a course toward a general framework for programmable proteome editing.

E3 ubiquitin ligases (E3s) confer specificity of protein degradation through ubiquitination of substrate proteins. Yet, the vast majority of the >600 human E3s have no known substrates. To identify proteolytic E3-substrate pairs at scale, we developed combinatorial mapping of E3 targets (COMET), a framework for testing the role of many E3s in degrading many candidate substrates within a single experiment. We applied COMET to SCF ubiquitin ligase subunits that mediate degradation of target substrates (6,716 F-box-ORF [open reading frame] combinations) and E3s that degrade short-lived transcription factors (TFs) (26,028 E3-TF combinations). Our data suggest that many E3-substrate relationships are complex rather than 1:1 associations. Finally, we leverage deep learning to predict the structural basis of E3-substrate interactions and probe the strengths and limits of such models. Looking forward, we consider the practicality of transposing this framework, i.e., computational structural prediction of all possible E3-substrate interactions, followed by multiplex experimental validation.

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct ‘symbols’ that can be concurrently recorded and/or by a failure to capture the order in which events occur 1 . Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium (‘DNA Tape’) consists of a tandem array of partial CRISPR–Cas9 target sites, with all but the first site truncated at their 5′ ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA 2 mediating the edit while also shifting the position of the ‘type guide’ by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct ‘long tape’ potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion.

DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events 1 . Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5’ ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs 2 . Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the “write head” by one unit along the tandem array, i.e. sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct “long tape” potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.

Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR–Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1–30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements. Paired prime editing is used to precisely delete genomic sequences up to 10 kb.

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15 -informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants’ effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino-acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.

Metabolic syndrome induces an increased cardiovascular morbidity and mortality. Most importantly, the prevalence of metabolic syndrome in adult population is expanding. Both clinical and preclinical studies indicate that increased Free Fatty Acids (FFAs) are involved in the pathogenesis of insulin resistance and subsequent development of metabolic syndrome. The relevance of FFAs in protecting and restoring tissue function is quite vast. The search to correlate the functional deterioration of the tissues within the cardiovascular system and increased plasma concentrations of FFAs has been reported. The importance of reduction in the consumption of dietary fatty acids along with the identification of dysregulated genes responsible for persistent increased FFAs uptake and mitochondrial β-oxidation has been increasingly recognized. This review discusses the current empirical understanding of the different types of fatty acids and their metabolism and functions both in physiological and pathophysiological conditions. We also discuss in detail about the molecular and pathophysiological basis of increased FFAs, which augments Cardiovascular Disease (CVD).

s of 2018 Annual Meeting* Hilton Orlando, Orlando, FL, March 21–24, 2018 Presidential Trainee Showcase Wednesday, March 21, 5:00 PM–6:30 PM

Prolonged exposure to thiopurines (e.g., 6-mercaptopurine [MP]) is an essential component of curative therapy of acute lymphoblastic leukemia (ALL) in children and adults. However, this class of drugs are also associated with frequent dose-limiting hematopoietic and liver toxicities, at least in part explained by genetic polymorphisms in metabolizing enzymes such as TPMT . In fact, pre-emptive TPMT- genotype guided thiopurine dose reduction has been implemented in clinical practice and shown to effectively mitigate toxicity without compromising therapeutic efficacy. More recently, we and others identified NUDT15 as a novel genetic determinant of thiopurine toxicity (Nature Genetics , 2014 and 2016), particularly in Asian and Hispanic populations. While results from correlative studies in patients point to potential effectiveness of NUDT15 -genotype based thiopurine individualization, the exact dosing algorithm remains unknown and there have been no laboratory models to test such strategies. To this end, we established a Nudt15 knockout mouse model on FVB/N background by CRISPR-Cas9 genome editing, with which we systematically evaluated NUDT15 -genotype directed thiopurine dose reduction and its effects on hematopoietic toxicity in vivo . Nudt15 +/+ and Nudt15 -/- mice received daily intra-peritoneal MP injection at the dosage of 1, 5 or 20 mg/kg. Consistently across dosage levels, Nudt15-/- mice experienced more severe and precipitous myelosuppression (especially reduction in neutrophil count), more rapid weight loss, and earlier toxic death, compared to wildtype mice (P To assess the impacts of Nudt15 genotypes on thiopurine activation, we also characterized the acute accumulation of MP metabolite (i.e., DNA incorporated thioguanine, DNA-TG) following daily MP injection at increasing dosages (1, 5, 20 and 40 mg/kg/day). Although the level of DNA-TG in hematopoietic cells was positively correlated with MP dosage in both genotype groups, this active metabolite accumulated much more rapidly in Nudt15-/- mice with increasing MP dosages than in wildtype mice (p In conclusion, we established a Nudt15 knockout mouse model, with which we comprehensively evaluated NUDT15 -genotype guided thiopurine dose individualization in vivo . Disclosures No relevant conflicts of interest to declare.