Biology subjects

Boeke, J. D.

Publications and source records attributed to Boeke, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

ZATELLITE: a toolkit to visualize and manipulate human centromeres in live cells using synthetic zinc fingers

Changes in the number of chromosomes or their spatial organization within the nucleus have critical consequences for cell fate. Yet capturing the karyotype or three-dimensional architecture of chromatin in living cells remains limited by the difficulty in labeling endogenous loci non-invasively. The most widely used tools rely on dCas9, a bulky protein whose persistent DNA binding interferes with DNA and RNA metabolism, causes DNA damage, and is hard to multiplex. We developed ZATELLITE, an AI-enabled tool to target endogenous repetitive sequences with fluorescently-tagged synthetic zinc fingers. ZATELLITE probes have key advantages over dCas9: they are smaller, easier to multiplex and, critically, they do not cause DNA damage or chromosomal abnormalities, even after long-term labeling. We generated a collection of ZATELLITE probes to label the centromeres of nearly all human chromosomes, enabling the capture of genome organization and karyotype alterations in real time in living cells. Finally, we show that ZATELLITE can be used for simultaneous labeling and epigenetic editing of centromeres. Thus, ZATELLITE is a non-toxic, versatile tool for genome visualization and manipulation.

cell biology↗

bioRxiv2026

BxbI-mediated insertion of a 77kb human RET sensitive haplotype into the mouse genome to generate a humanized model of Hirschsprung disease

Hirschsprung disease (HSCR) is a complex developmental disorder of the enteric nervous system, primarily driven by regulatory variants within enhancer elements of the RET gene. To investigate how these variants lead to aganglionosis, we developed a humanized mouse model by inserting an intact 77kb human RET genomic locus into the Rosa26 safe-harbor locus. Utilizing "big DNA" synthetic biology and Bxb1-mediated recombination, we integrated the complete human locus including all exons, introns, and upstream regulatory elements which we validated via nanopore and short-read sequencing. Functional analysis confirmed in vivo human RET expression; however, our initial HSCR-associated "sensitive" haplotype expressed at only 21% of wild-type levels. This significant reduction proved insufficient to rescue the viability when endogenous mouse Ret was deleted. We identified that this deficiency is partially driven by five risk SNPs within established enhancers. Specifically, using CRISPR/Cas9 to restore a conserved Sox10 binding site (converting a sensitive SNP to a protective one) increased RET expression by 1.9-fold and restored transcription factor binding. This study provides a robust framework for modeling human-specific regulatory disorders and demonstrates the critical impact of non-coding variation on disease pathogenesis.

synthetic biology↗

bioRxiv2026

Disruption of a CCR5-like immunoglobulin gene is linked to plague susceptibility in black-footed ferrets

Black-footed ferrets (Mustela nigripes) are among the worlds most endangered mammals and remain highly vulnerable to sylvatic plague caused by Yersinia pestis, yet the genetic basis of this susceptibility has remained unknown. Current conservation strategies rely on vaccination of captive-bred animals and large-scale flea control with insecticides, approaches that are costly, labor-intensive, and difficult to implement across the species natural range. Several closely related mustelid species, including the domestic ferret, are substantially more resistant to plague, providing an opportunity to identify naturally evolved immune mechanisms through comparative immunogenomics. Here we identify a conserved class of immunoglobulin lambda variable genes encoding unusually long antigen-binding loops with CCR5-mimicking sequence features that are widespread among Caniformia species. Because CCR5 has been implicated in host interactions with Yersinia species, we hypothesized that antibodies encoded by these germline genes contribute to plague resistance through receptor-like molecular mimicry. Consistent with this hypothesis, we show that these genes are under strong purifying selection in mustelids, are actively expressed in antibody repertoires, and monoclonal antibodies encoded by them reduced intracellular Y. pestis survival in macrophages. In contrast, all analyzed black-footed ferrets carried a frameshifting deletion resulting in loss of gene expression. These findings identify a naturally disrupted germline antibody gene as a candidate determinant of plague susceptibility in black-footed ferrets, demonstrating that variation in germline immunoglobulin genes can influence susceptibility to a lethal infectious disease. Ultimately, these findings lay the groundwork for genetically informed conservation management and the development of new antibody-based anti-plague strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/734856v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@19d8ac1org.highwire.dtl.DTLVardef@a37aorg.highwire.dtl.DTLVardef@1ecd1e0org.highwire.dtl.DTLVardef@c6fd67_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Improved vector toolkit for genome writing in mammalian cells

Efficient genome writing in mammalian cells requires robust methods for integrating large DNA payloads. The previously described method mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) enables iterative, biallelic genome rewriting in mammalian stem cells with DNA payloads exceeding 100 kb. However, the lack of standardized vectors and certain technical constraints have limited its broader adoption. Here we present an improved plasmid toolkit designed to streamline the implementation of mSwAP-In. The toolkit includes two core vectors. pLP-TK (pCTC174) is a landing-pad plasmid compatible with Golden Gate assembly of genomic homology arms and supports both mSwAP-In and the recombinase-mediated cassette exchange method Big-IN. mSwAP-In MC2v2 (pKBA135) is a versatile Big DNA assembly and delivery vector that supports Gibson-based assembly and incorporates positive, negative, and fluorescent selection markers, as well as a backbone counterselection cassette to minimize unwanted plasmid integration. The vector architecture also enables propagation in yeast and bacterial hosts, inducible plasmid copy-number amplification in standard E. coli strains, and CRISPR/Cas9-mediated payload release through preinstalled guide RNA target sites. We further characterize the FCU1/5-FC counterselection system in mouse embryonic stem cells and define conditions that minimize its bystander toxicity. Finally, we provide a set of Cas9-gRNA expression plasmids optimized for common mSwAP-In applications. Together, these reagents constitute a standardized and experimentally validated toolkit that simplifies large-scale genome writing using mSwAP-In.

synthetic biology↗

bioRxiv2026

Intrahepatic reporter assay reveals leaky somatic blockade of L1 retrotransposition in mice

Long interspersed element-1 (LINE-1, L1) retrotransposition has long been proposed to occur in somatic tissues, yet direct experimental evidence distinguishing adult somatic events from early embryonic insertions has remained limited. Here we establish an intrahepatic L1 reporter assay that enables immunohistochemical detection and quantitative analysis of L1 retrotransposition in vivo. Using autonomous and non-autonomous L1 reporter variants, we demonstrate clearly detectable somatic L1 activity in the mouse liver. Comparative analysis of L1 activity in liver tissue and tumor-derived cell culture reveals that tumor cells preferentially restrict L1 at early regulatory stages, consistent with epigenetic control, whereas downstream defence mechanisms are comparatively permissive. In contrast, normal liver tissue shows stronger restriction at later stages of the L1 life cycle. Together, our results provide direct experimental evidence for somatic L1 retrotransposition in vivo in adult liver and reveal distinct regulatory strategies that shape L1 activity in tumor versus normal somatic cells. TeaserGenome destabilizing L1 retrotransposon activity is present in somatic tissues, where it likely contributes to cancer development.

genetics↗

bioRxiv2026

Genome writing and Targeted Delivery of the NKX6-3/ANK1 gene cluster and its Type 2 Diabetes GWAS Variants to Human iPSCs

Genome-wide association studies (GWAS) identified over 600 loci containing single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes (T2D), most of which reside in non-coding regions. Among the set of T2D SNPs, linking causal genome variants to disease risk experimentally has remained a challenge; however, advances in synthetic mammalian genome writing techniques now enable the delivery of multiple haplotypes to human induced pluripotent stem cells (hiPSCs) to create a series of isogenic cell lines that can be differentiated and phenotyped in vitro. Here, to begin efforts in dissecting a T2D GWAS locus, we engineered an NKX6-3/ANK1 gene cluster knockout hiPSC line and introduced a landing pad facilitating the delivery of synthetic haplotype payloads. We built four haplotypes, including several that are not observed in nature, containing risk SNPs spanning the NKX6-3/ANK1 gene cluster using a method called "variant Switching Auxotrophic markers for Integration" (vSwAP-In), and integrated them precisely into hiPSCs. NKX6-3/ANK1 deletion blocked pancreatic progenitor and skeletal muscle differentiation, suggesting that NKX6-3 and ANK1 are required for early pancreatic and skeletal muscle development, and perhaps related to the existence of two nonoverlapping sets of SNPs in linkage disequilibrium that associate with the expression of the two adjacent genes. When NKX6-3/ANK1 T2D "Risk" haplotypes were reintroduced, skeletal muscle and pancreatic progenitor differentiation capabilities were restored. ANK1 expression was elevated in the ANK1 Risk and All-Risk haplotypes compared to the NKX6-3 Risk and Non-Risk haplotypes, establishing a functional experimental platform to examine risk SNP clusters in their native contexts. Overall, this work establishes a platform for the dissection of GWAS loci using synthetic haplotype genomics in hiPSCs. Significance StatementGenome-wide association studies have been used to identify disease-associated SNPs; however, most SNPs lie in non-coding regions, making functional experimentation difficult to perform. Using vSwAP-In, a yeast-based DNA variant-building method, and mSwAP-In, a mammalian genome engineering approach, we establish a platform for functional GWAS dissection in hiPSCs. This platform allows us to build DNA harboring virtually any combination of disease-risk SNPs, allowing for functional characterization of SNPs without the limitations of linkage disequilibrium. We demonstrate this approach using a Type 2 diabetes GWAS gene cluster, NKX6-3/ANK1.

genetics↗

bioRxiv2025

Retrotransposon Activation in the Aged and Alzheimer's Disease Brain Examined by Nanopore Long-read DNA Sequencing

BackgroundCellular defenses against retrotransposable elements (RTEs) weaken with age and RTEs have been reported to contribute to Alzheimers disease (AD) pathogenesis by promoting neuroinflammation. The mechanisms implicated include DNA damage promoted by retrotransposition and interferon system activation by RTE-derived cDNA intermediates. LINE-1 (L1) retrotransposons are of particular interest because they are the only autonomously active RTEs in the human genome. ResultsTo investigate L1 activation and retrotransposition in AD, we performed Nanopore long-read DNA sequencing on six late-onset AD (LOAD) and six age-matched control human prefrontal cortex (PFC) samples. We developed and validated a stringent RTE insertion calling pipeline and identified two high-confidence somatic insertions, one AluY and one L1HS. We estimate that [~]1% of cells in the aged PFC have a somatic RTE insertion. AD samples were hypomethylated, and genome-wide analysis of differentially methylated regions (DMRs) supports a process of epigenetic drift in AD. DMR-associated gene sets primarily related to brain function and inflammation. To investigate L1 activation we used CpG methylation as a proxy for L1 expression. We observed decreased methylation at young L1 elements. While most reads overlapping the L1HS promoter were highly methylated (>80% methylated), 7% were <50% methylated, 1% were <25%, and the highly demethylated read fraction increased in AD. L1HS 5 UTR methylation was strongly correlated with RNA expression. ConclusionsCpG methylation-mediated repression of young RTEs is compromised in old age - our findings indicate that this is further exacerbated in AD. Amid these failing defenses, we report somatic retrotransposition events in the aging and demented brain.

genomics↗

bioRxiv2025

Genome writing to dissect consequences of SVA retrotransposon disease X-Linked Dystonia Parkinsonism

Human retrotransposon insertions are often associated with diseases. In the case of the neurodegenerative X-Linked Dystonia-Parkinsonism disease, a human-specific SINE-VNTR-Alu subfamily F retrotransposon was inserted in intron 32 of the TAF1 gene. Here, we genomically rewrote a portion of the mouse Taf1 allele with the corresponding 78-kb XDP patient derived TAF1 allele. In mESCs, the presence of the intronic SVAs--rather than the hybrid gene structure--reduces hyTAF1 levels. This leads to transcriptional downregulation of genes with TATA box enriched in their promoters and triggering apoptosis. Chromatin and transcriptome profiling revealed that intronic SVAs are actively transcribed, forming barriers that likely impede transcription elongation. In mice, neuronal lineage TAF1 humanization resulted lethality of male progeny within two months. XDP male mice had severe atrophy centered on the striatum--the same affected brain region in XDP patients. Lastly, CRISPRa-mediated activation of hyTAF1 restored mESC viability, suggesting boosting TAF1 transcription as a therapeutic approach.

genetics↗

bioRxiv2025

Myelin pathology is a key feature of X-linked Dystonia Parkinsonism

X-linked Dystonia-Parkinsonism (XDP) is a progressive, adult-onset neurodegenerative movement disorder that predominantly affects males of Filipino descent1-3. The disease is caused by the insertion of a SINE-VNTR-Alu subfamily F (SVA_F) retrotransposon within an intron of the TATA-box binding protein-associated factor 1 (TAF1) gene4. A major barrier to understanding the pathophysiology of XDP has been the lack of relevant animal models. Here, we introduce a novel conditional humanized XDP mouse model harboring a hybrid mouse-human Taf1/TAF1 gene (hyTAF1) containing the pathogenic SVA_F insertion. We activated the hyTAF1 in Nestin+ neural progenitor cells and found that the resulting XDP male mice recapitulate features of the human disease including severe motor impairment, striatal atrophy, and reactive gliosis. Transcriptomic, histological, and electron microscopy analysis revealed a dramatic reduction in oligodendrocyte lineage cells and widespread myelin disruption. Consistent with these findings, postmortem brain tissue from XDP patients revealed similar myelin pathology, including near-complete loss of myelin in parts of the medial prefrontal cortex. Together, these results identify oligodendrocyte dysfunction and myelin loss as previously unrecognized contributors to XDP pathogenesis, providing new mechanistic insight into this debilitating disorder.

neuroscience↗

bioRxiv2025

Pan-cancer analysis reveals context-dependent roles of LINE-1 ORF1p in immune regulation and copy number alterations

L1 comprises 17% of the human genome, with 50-150 full-length sequences capable of retrotransposition. Although largely inactive in normal somatic tissues, tumorigenesis leads to L1 derepression and overexpression of its RNA binding chaperone protein, ORF1p. A potential cancer biomarker, ORF1p expression is a hallmark of multiple cancers and an early event in precursor lesions. Our study provides a comprehensive pan-cancer analysis of ORF1p using CPTAC proteogenomic data, revealing a dichotomous role in modulating immune responses. Integrated analyses and supervised learning models divide ORF1p-high tumors into two groups: (1) high ORF1p associates with immunosuppression, reduced interferon signaling and diminished immune cell infiltration (HNSCC and LSCC), and (2) ORF1p-high tumors associate with immune activation (UCEC). Linear regression models reveal that cancer-specific aneuploidies may underlie this immune dichotomy, highlighting the prognostic significance of ORF1p and informing new strategies to leverage detection of plasma-circulating ORF1p to enhance immunotherapeutic efficacy in different tumor contexts.

genomics↗

bioRxiv2025

Multimodal learning decodes the global binding landscape of chromatin-associated proteins

The identity of a cell is governed by regulatory proteins binding to the genome to control gene expression. Mapping these genome-wide binding events across thousands of proteins and cell types is essential for understanding development and disease at scale, yet has remained a major experimental and computational barrier. Here we present Chromnitron, a multimodal foundation model that learns the rules of protein-DNA binding from protein sequence, DNA sequence, and context-specific chromatin states. Unlike prior single-task and multi-task learning approaches, Chromnitron implements a multimodal learning framework that accurately predicts the binding landscape for proteins and cell types not seen during training. Using Chromnitron, we discovered and experimentally validated new protein regulators of T cell exhaustion. Chromnitron also uncovered previously uncharacterized dynamic shifts in the binding landscape of regulatory proteins during neurogenesis. This marks a critical step toward a predictive model of interpretable gene regulatory programs across cell types, enabling rapid discovery of regulatory circuits and identification of new therapeutic targets.

systems biology↗

bioRxiv2025

Synthetic genomic dissection of enhancer context sensitivity and synergy

Noncoding disease and trait-associated genetic variation is frequently interpreted in the context of genomic regulatory elements such as DNase I hypersensitive sites (DHSs). But while most DHSs lie within a few kilobases of another DHS, regulatory elements are typically analyzed individually without accounting for their neighbors. We characterize multiple heterotypic DHS combinations from different critical mESC regulator loci, all delivered in a constant chromosomal context replacing the Sox2 Locus Control Region (LCR). We employ an optimized high-throughput multiplexed delivery pipeline enabling analysis of 213 distinct payloads in 641 mouse embryonic stem cell (mESC) clones. We identify widespread examples of context-dependent enhancers which have no activity on their own but can more than double the activity of a neighboring DHS. Enhancers exhibit synergy only with certain partners, and deliveries to the Igf2/H19 locus show that synergy is not constrained to a single genomic context. We further show that synergy between neighboring DHSs decays as a characteristic function of distance, with its influence extending up to 4 kilobases. We fine map this context dependency to the contribution of individual transcription factor recognition sequences. Our approach implicates the specific sequence and architectural features underpinning pervasive genomic context effects, and outlines a direction for modeling the functional impact of noncoding regulatory variation on common human traits and diseases.

genomics↗

bioRxiv2025

A shotgun approach for highly multiplexed mammalian metabolic engineering

Mammalian metabolic engineering is critical to advancing basic biology, bioproduction, and cell therapy. However, as pathway complexity increases, so does the size of both the combinatorial design space and the DNA constructs required, rendering unbiased screens intractable. To address this, we developed Shotgun Genetic Engineering (SGE), a scalable approach that exploits the ease of delivering many barcoded small constructs--rather than a single large one--into mammalian cells. This allows each cell to serve as an independent experiment, carrying a unique synthetic metabolic pathway that explores combinations of gene content, stoichiometry, and organellar localization. Functional pathways are identified by sequencing barcodes from cells exhibiting the desired phenotype. Using SGE, we screened millions of pathway combinations to engineer essential amino acid biosynthesis in two mammalian cell lines (CHO and Jurkat), achieving near-wild-type growth in valine-free medium and, for the first time, enabling isoleucine prototrophy in CHO cells. Successful solutions favored mitochondrial localization and required integration of 23-52 kb of synthetic DNA--lengths that are impractical to screen by conventional methods. The resulting datasets are compatible with machine learning frameworks, positioning SGE as a powerful platform for decoding and engineering complex biosynthetic traits in mammalian systems.

synthetic biology↗

bioRxiv2025

Assemblatron: An Automated Workflow for High-Throughput Assembly of Big-DNA Libraries

The ability to synthesize 50-kb+ DNA molecules has tremendous potential in the fields of genome engineering, metabolic engineering, and synthetic regulatory genomics. Despite tremendous achievements in these fields, such as the completion of the first synthetic eukaryotic genome, assembling custom big DNAs remains slow, expensive, and laborious. In this work, we present a set of improvements to yeast-based DNA assembly methods that enable medium-to high-throughput big DNA experiments. In particular, we i) developed two easy-to-use vector systems: Jack In the Box (JIB), which deploys a split marker and split centromere strategy to reduce background, and Selection of URS Recombinational Excision (SURE) which exploits URS silencer removal from the LEU2 marker. These strategies sharply decrease the number of colonies containing an empty vector, greatly reducing the amount of screening required to find correct clones. ii) We improved yeast transformation efficiency, increasing the likelihood that all required DNA segments are co-transformed into the same cell. iii) We developed an automation pipeline for segment mixing through single-colony isolation. We also established three phenotype-driven assays that directly and rapidly report the frequency of correctly assembled large DNA molecules. Harnessing these improvements, we show a high success rate in assembling diverse big DNA libraries by combinatorial assembly and oligo-guided architecture in yeast (OLIGARCHY), a novel method that allows cost-effective reuse of segments for distinct assemblies with reduced effort and cost. Using OLIGARCHY, we designed 96 structural variants of a [~]73kb construct, and a single person was able to correctly assemble 90 of these in 7 days, for a total of 7 Mb of assembled DNA. Finally, we used the SURE vector to greatly improve existing methods to turn oligonucleotides into gene-sized double-stranded DNA segments, efficiently turning 80 overlapping 60-mers into [~]3-kb DNAs directly, without PCR amplification.

genetics↗

bioRxiv2025

Ancient co-option of LTR retrotransposons as yeast centromeres

The evolutionary origins of the genetic point centromere in the brewers yeast Saccharomyces cerevisiae, a member of the order Saccharomycetales, are still unknown. Competing hypotheses suggest that the point centromere tripartite genetic centromere DNA elements (CDEs) either evolved from ancestral epigenetic centromeres by descent with modification or were gained through horizontal transfer from selfish DNA plasmids.1,2 Here, we identified centromeres in the sister order Saccharomycodales and termed them "proto-point centromeres" due to sequence features that bridge the evolutionary gap between point centromeres and ancestral centromeres types. Comparative genomic analyses across multiple yeast orders showed an unexpected evolutionary link between point and proto-point centromeres to the long terminal repeats (LTRs) of Ty5 retrotransposons. Strikingly, one Saccharomycodales species, Saccharomycodes ludwigii, harbors compact Ty5-based centromeres, where its CDEII elements are divergent AT-rich Ty5 LTRs. These living fossil centromeres show how retrotransposon cis-regulation was likely co-opted for genetic centromere specification. These insights show that point centromeres are direct descendants of retrotransposons and have evolved by descent with modification. Ultimately, the many diverse centromere types across the yeast subphylum may share a common ancestry rooted in retrotransposon activity.

evolutionary biology↗

bioRxiv2025

Selective depletion of cancer cells with extrachromosomal DNA via lentiviral infection

Dear Editor Extrachromosomal DNA (ecDNA), a major focal oncogene amplification mode found across cancer, has recently regained attention as an emerging cancer hallmark1,2, with a pervasive presence across cancers3,4. With technical advancements such as high-coverage sequencing and live-cell genome imaging, we can now investigate ecDNAs behaviors and functions 5-7. However, we still lack an understanding of how to eliminate ecDNA. We observed depletion of cells containing ecDNA during lentiviral but not transposon-based transduction while we sought to investigate the mechanism of ecDNA behavior. This discovery may provide critical information on utilizing a lentiviral system in emerging ecDNA research. Additionally, this observation suggests specific sensitivities for cells with ecDNA.

cancer biology↗

bioRxiv2024

Enhancer activation from transposable elements in extrachromosomal DNA

Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoral heterogeneity in aggressive cancers. While transposable element (TE) reactivation is common in cancer, its role on ecDNA remains unexplored. Here, we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements (EIEs)--genomic loci enriched for TEs that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA amplified context. Using CRISPR-CATCH, CRISPR interference, and reporter assays, we confirm its presence on ecDNA, enhancer activity, and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumor evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.

genetics↗

bioRxiv2023

Engineered transcription-associated Cas9 targeting in eukaryotic cells

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9s targeting activity at specific DNA sites may be engineered without modifying Cas9s core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.

bioengineering↗