RESUMO
There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.
Assuntos
Proteína 9 Associada à CRISPR/genética , DNA/genética , RNA/genética , Imagem Individual de Molécula/métodos , Pareamento de Bases , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , DNA/química , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Mutação , Hibridização de Ácido Nucleico , Conformação Proteica , Engenharia de Proteínas/métodos , RNA/química , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteína 9 Associada à CRISPR/genética , DNA/genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos TestesRESUMO
Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.
Assuntos
Proteína BRCA1/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Variação Genética , Sequência de Bases , Neoplasias da Mama/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliais/metabolismo , Edição de Genes , Mesoderma/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Imunofluorescência , Camundongos , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Cytosine base editors (CBEs) enable targeted Câ¢G-to-Tâ¢A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target Câ¢G-to-Tâ¢A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Citosina/metabolismo , Escherichia coli/genética , Edição de Genes/métodos , Edição de Genes/economia , Células HEK293 , Humanos , Mutação , Sequenciamento Completo do GenomaRESUMO
CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Microscopia/métodos , Plasmídeos/genética , Imagem Individual de Molécula/métodos , Proteína 9 Associada à CRISPR/genética , Dosagem de Genes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia/instrumentação , Modelos Genéticos , Método de Monte Carlo , Motivos de Nucleotídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula/instrumentação , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
Dynamic changes in DNA (hydroxy-)methylation are fundamental for stem cell differentiation. However, the signature of these epigenetic marks in specific cell types during corticogenesis is unknown. Moreover, site-specific manipulation of cytosine modifications is needed to reveal the significance and function of these changes. Here, we report the first assessment of (hydroxy-)methylation in neural stem cells, neurogenic progenitors, and newborn neurons during mammalian corticogenesis. We found that gain in hydroxymethylation and loss in methylation occur sequentially at specific cellular transitions during neurogenic commitment. We also found that these changes predominantly occur within enhancers of neurogenic genes up-regulated during neurogenesis and target of pioneer transcription factors. We further optimized the use of dCas9-Tet1 manipulation of (hydroxy-)methylation, locus-specifically, in vivo, showing the biological relevance of our observations for Dchs1, a regulator of corticogenesis involved in developmental malformations and cognitive impairment. Together, our data reveal the dynamics of cytosine modifications in lineage-related cell types, whereby methylation is reduced and hydroxymethylation gained during the neurogenic lineage concurrently with up-regulation of pioneer transcription factors and activation of enhancers for neurogenic genes.
Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , 5-Metilcitosina/fisiologia , Animais , Proteína 9 Associada à CRISPR/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Linhagem da Célula/fisiologia , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/metabolismo , Gravidez , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , TranscriptomaRESUMO
Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38â¯cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection.