RESUMEN
Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely unknown. We investigated the relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNAPII and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific genes. By contrast, the short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly housekeeping. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate genes located in early initiation zones (EIZ) of DNA replication, and the EIZ signals were amplified drastically without RAD21. Our results revealed new mechanistic insights of cohesin's multifaceted roles in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes, and maintaining timely order of DNA replication.
RESUMEN
A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.
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Metilación de ADN , Células Madre Pluripotentes Inducidas , Metilación de ADN/genética , Reprogramación Celular/genética , Haploinsuficiencia , Fibroblastos/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismoRESUMEN
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN Guía de Kinetoplastida , Sistemas CRISPR-Cas/genética , Cromatina/genética , Cromosomas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genómica , ARN Guía de Kinetoplastida/genéticaRESUMEN
Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.See related commentary by Henssen, p. 293.This article is highlighted in the In This Issue feature, p. 275.
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ADN/genética , Herencia Extracromosómica , Amplificación de Genes , Neoplasias/genética , Microambiente Tumoral , HumanosRESUMEN
Here we describe TALE.Sense, a versatile platform for sensing DNA sequences in live mammalian cells enabling programmable generation of a customable response that discerns cells containing specified sequence targets. The platform is based on the programmable DNA binding of transcription activator-like effector (TALE) coupled to conditional intein-reconstitution producing a trans-spliced ON-switch for a response circuit. TALE.Sense shows higher efficiency and dynamic range when compared to the reported zinc-finger based DNA-sensor in detecting same DNA sequences. Swapping transcriptional activation modules and introducing SunTag-based amplification loops to TALE.Sense circuits augment detection efficiency of the DNA sensor. The TALE.Sense platform shows versatility when applied to a range of target sites, indicating its suitability for applications to identify live cell variants with anticipated DNA sequences. TALE.Sense could be integrated with other cellular or synthetic circuits by using specified DNA sequences as control-switches, thus expanding the scope in connecting inducible modules for synthetic biology.
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ADN , Efectores Tipo Activadores de la Transcripción , Animales , ADN/genética , ADN/metabolismo , Inteínas , Mamíferos/genética , Biología Sintética , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
FOCUSED CLINICAL QUESTION: How should clinicians manage dental extraction sockets when immediate implant placement is contraindicated, and alveolar ridge preservation is expected to result in inadequate bone volume for implant placement? SUMMARY: Three fundamental options for extraction socket management form a hierarchical continuum in sites where dental implant placement is planned: place an immediate implant, perform ridge preservation, or perform ridge augmentation. The available volume and quality of bone and keratinized mucosa are the primary considerations driving the decision, and each tier in the continuum encompasses a variety of techniques with attendant advantages and disadvantages. CONCLUSIONS: Some immediate implant protocols require no mucoperiosteal flap and possibly produce the most favorable clinical and patient-centered outcomes compared with other extraction socket management approaches. Conversely, guided bone regeneration at dental extraction sites can result in substantial gains in alveolar ridge dimensions, although this treatment may adversely influence mucosal architecture and carry increased risk of postoperative morbidity. When favorable bone and mucosa are present at a dental extraction site, immediate implant placement may be the treatment of choice, barring unusual circumstances. Ridge preservation, typically associated with minimal postoperative morbidity, is a rational second choice when acceptable ridge dimensions are anticipated after healing.
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Aumento de la Cresta Alveolar , Implantes Dentales , Alveolo Dental , Proceso Alveolar , Humanos , Extracción DentalRESUMEN
INTRODUCTION: Postoperative discomfort is a documented complication of the epithelialized palatal graft (EPG) procedure, and the expectation of an unpleasant patient experience may cause some practitioners to avoid EPG altogether. However, EPG affords distinct advantages in a variety of clinical situations, and the postoperative discomfort associated with the procedure can be minimized. CASE SERIES: Three generally and periodontally healthy patients with gingival recession defects and minimal zones of attached gingiva received mandibular anterior EPG procedures. In all cases, collagen membranes were trimmed to fit the palatal donor sites and sutured in place. Two patients reported minimal donor site discomfort at any time point. One patient with large bilateral donor sites reported moderate palatal discomfort limited to the first postoperative week. All patients reported overall positive treatment experiences. CONCLUSIONS: Placement of a resorbable collagen membrane at large EPG harvest sites appears to limit topical irritation of the wound and may substantially improve patient comfort postoperatively. Combining local and systemic measures to minimize patient discomfort may render EPG procedures very tolerable for patients. Controlled clinical trials comparing patient-centered outcomes following EPG harvest with and without collagen membrane placement appear warranted.
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Encía/trasplante , Recesión Gingival , Hueso Paladar , Comodidad del Paciente , Colágeno , Humanos , Hueso Paladar/cirugía , Donantes de TejidosRESUMEN
Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Desmetilación del ADN , Reparación del ADN , ARN Guía de Kinetoplastida/metabolismo , 5-Metilcitosina/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN Glicosilasas/metabolismo , Metilación de ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Edición Génica , Células HEK293 , Humanos , Oxigenasas de Función Mixta/genética , Oxidación-Reducción , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Análisis de Secuencia de ARNAsunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas/genética , Redes Reguladoras de Genes/genética , Aptámeros de Nucleótidos/genética , Sitios de Unión/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/genética , Genómica/métodos , ARN/genética , Proteínas de Unión al ARN/genéticaRESUMEN
CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR-Cas9 system holds promise as a "system of choice" for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRISPR-Cas9 system and its application to genome editing, with a focus on the basic considerations in designing the targeting guide RNA sequence.
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Sistemas CRISPR-Cas/genética , Edición de ARN/genética , ARN Guía de Kinetoplastida/genética , Animales , Secuencia de Bases/genética , Ingeniería Genética , GenomaRESUMEN
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.
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Sistemas CRISPR-Cas , Endonucleasas/genética , Genoma , Genómica , Cigoto/metabolismo , Animales , Secuencia de Bases , Electroporación , Femenino , Marcación de Gen , Sitios Genéticos , Mutación INDEL , Ratones , Datos de Secuencia Molecular , Edición de ARN , ARN Guía de Kinetoplastida/genética , ARN Mensajero/genética , Alineación de SecuenciaRESUMEN
OBJECTIVE: To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. APPROACH AND RESULTS: The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. CONCLUSIONS: CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.
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Sistemas CRISPR-Cas/genética , Proteínas de Unión al Calcio/genética , Proteínas de Microfilamentos/genética , Mutación Puntual , Elementos Reguladores de la Transcripción/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular , Regulación hacia Abajo , Homocigoto , Intrones , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fenotipo , ARN Mensajero/metabolismo , CalponinasRESUMEN
The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.
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Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Transcripción Genética , Empalme Alternativo/genética , Animales , Secuencia de Bases , Mama/crecimiento & desarrollo , Mama/patología , Neoplasias de la Mama/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/genética , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Modelos Biológicos , Datos de Secuencia Molecular , Morfogénesis , Células-Madre Neurales/metabolismo , Motivos de Nucleótidos/genética , Unión Proteica , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Receptores Notch/metabolismo , Proteínas Serrate-JaggedRESUMEN
Niemann-Pick type C (NPC) disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs) using transcription activator-like effector nucleases (TALENs). We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.
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Autofagia/fisiología , Colesterol/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neuronas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Adulto , Células Cultivadas , Niño , Preescolar , Hepatocitos/citología , Humanos , Neuronas/citologíaRESUMEN
The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation.
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Empalme Alternativo , Proteínas Portadoras/genética , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Eritropoyesis/fisiología , Regulación de la Expresión Génica , Precursores del ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Exones/genética , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Isoformas de Proteínas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
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Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/genética , Células Madre Embrionarias/fisiología , Genoma/genética , Modelos Genéticos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Ratones , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.
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Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Cuerpos Embrioides/citología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Metilación de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Cuerpos Embrioides/enzimología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.
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Células Madre Embrionarias/patología , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/patología , Biosíntesis de Proteínas/genética , Síndrome de Rett/genética , Síndrome de Rett/patología , Transcripción Genética/genética , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Mutación , Neuronas/metabolismoRESUMEN
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.