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
A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Oryza/genética , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/genética , Genoma de Planta/genética , Mutação/genéticaRESUMO
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 FluorescenteAssuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Pesquisas com Embriões/ética , Edição de Genes/ética , China , Pesquisas com Embriões/legislação & jurisprudência , Edição de Genes/legislação & jurisprudência , Política de Saúde/legislação & jurisprudência , Humanos , Internacionalidade/legislação & jurisprudência , National Academies of Science, Engineering, and Medicine, U.S., Health and Medicine Division , Estados UnidosRESUMO
A typical molecular cloning procedure requires Sanger sequencing for sequence validation, which is cost-prohibitive and labor-intensive for large-scale clone analysis in genotype-phenotype studies. Here we present the cost-effective clone analysis platform TnClone, which uses next-generation sequencing based on Tn5 tagmentation to rapidly analyze a large number of clones from cell lysates. This method bypasses the extensive plasmid purification step. We also developed a user-friendly graphical user interface and provided general guidelines for conducting validation experiments. We tested our program with 1023 plasmids (222 from cell lysates and 801 from purified clones) and achieved 92% and 99.3% sensitivity with cell lysates and purified DNA, respectively. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation, as next-generation sequencing technology continues to evolve.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Software , Transposases/genética , Proteína 9 Associada à CRISPR/genética , Clonagem Molecular/métodos , Confiabilidade dos Dados , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala/economia , Inteínas/genética , Plasmídeos/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/economia , Anticorpos de Cadeia Única/genéticaRESUMO
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.
Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Vetores Genéticos/genética , RNA Guia de Cinetoplastídeos/genética , Ribonucleoproteínas/genética , Animais , Linhagem Celular Tumoral , Reparo do DNA/genética , Embrião de Mamíferos , Fibroblastos , Edição de Genes/economia , Genoma/genética , Células HEK293 , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas , Vírus da Leucemia Murina/genética , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Ativação Transcricional/genéticaRESUMO
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
Assuntos
Hemoglobina Fetal/genética , Filtração/métodos , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Ribonucleoproteínas/genética , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Eletroporação/métodos , Feminino , Hemoglobina Fetal/metabolismo , Filtração/economia , Filtração/instrumentação , Terapia Genética/economia , Terapia Genética/instrumentação , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Membranas Artificiais , Camundongos , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Transplante AutólogoRESUMO
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.