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1.
BMC Res Notes ; 14(1): 389, 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627381

RESUMO

OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection. RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.


Assuntos
Edição de Genes , Zigoto , Animais , Blastocisto , Sistemas CRISPR-Cas/genética , RNA Guia , Suínos
2.
BMC Plant Biol ; 21(1): 419, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517842

RESUMO

BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Interações Hospedeiro-Patógeno/genética , Vírus de Plantas/genética , Soja/genética , Soja/virologia , Viroses/genética , Produtos Agrícolas/genética , Produtos Agrícolas/virologia , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Genoma de Planta , Mutagênese , RNA Guia , RNA de Plantas , RNA Viral
3.
Methods Enzymol ; 658: 335-358, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517953

RESUMO

Site-directed RNA editing (SDRE) exploits the enzymatic activity of Adenosine Deaminases Acting on RNAs (ADAR) to program changes in genetic information as it passes through RNA. ADARs convert adenosine (A) to inosine (I) through a hydrolytic deamination and since I can be read as guanosine (G) during translation, this change can regulate gene function and correct G→A genetic mutations. In SDRE, ADARs are redirected to convert user-defined A's to I's. SDRE also has certain advantages over genome editing because the changes in RNA are reversible and thus safer. In addition, ADARs are endogenously expressed in humans and therefore unlikely to provoke immunological complications when administered. Recently, a variety of systems for SDRE have been developed. Some rely on harnessing endogenously expressed ADARs and other deliver engineered versions of ADAR's catalytic domain. All systems are currently under refinement, and there are still challenges associated with raising their efficiency and specificity to levels that are adequate for therapeutics. This chapter provides a detailed protocol for in vitro and in cellula editing assays using the λNDD-BoxB system, one of the first systems developed for SDRE. The λNDD-BoxB system relies on gRNAs that are linked to the catalytic domain of human ADAR2 through a small RNA binding protein-RNA stem/loop interaction. We provide step-by-step protocols for (a) the construction of guide RNAs and editing enzyme plasmids, and (b) their use in vitro and in cellula for editing assays using a fluorescent protein-based reporter system containing a premature termination codon that can be corrected by editing.


Assuntos
Adenosina Desaminase , Edição de RNA , Adenosina Desaminase/genética , Humanos , Inosina , RNA Guia/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
4.
Methods Enzymol ; 658: 435-452, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517958

RESUMO

Though over 100 distinct RNA modifications have been identified, the roles for many of these modifications in vivo remain unknown. Genome editing is one tool investigators are using to better understand the roles these modifications play and the consequences of their absence. In this chapter, we describe how CRISPR mediated genome editing can be used to interrogate the process of RNA modification in C. albicans. Furthermore, we discuss how the protocols described can be altered to meet experimental demands. The underlying theory on which these protocols are based are applicable to a variety of model systems. The protocols described utilize the widely used S. pyogenes Cas9, but the field of genome editing is quickly evolving. We discuss the recent developments of more flexible CRISPR systems that can target a greater number of sites in the genome. These and other advancements make CRISPR mediated genome editing a practical methodology to investigate RNA modification.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , RNA/genética , RNA Guia/genética
5.
Nat Commun ; 12(1): 5206, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471126

RESUMO

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.


Assuntos
Sistemas CRISPR-Cas , Escherichia coli/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano , Genoma Bacteriano , Plasmídeos , RNA Guia/genética
6.
Biosensors (Basel) ; 11(9)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34562891

RESUMO

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Vírus da SARS/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Viroses/diagnóstico , Sistemas CRISPR-Cas , Corantes Fluorescentes/química , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Nariz/virologia , Testes Imediatos , RNA Guia/química , RNA Guia/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da SARS/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Escarro/virologia
7.
Biochemistry (Mosc) ; 86(9): 1151-1161, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34565318

RESUMO

Despite its similarity to protein biosynthesis in bacteria, translation in the mitochondria of modern eukaryotes has several unique features, such as the necessity for coordination of translation of mitochondrial mRNAs encoding proteins of the electron transport chain complexes with translation of other protein components of these complexes in the cytosol. In the mitochondria of baker's yeast Saccharomyces cerevisiae, this coordination is carried out by a system of translational activators that predominantly interact with the 5'-untranslated regions of mitochondrial mRNAs. No such system has been found in human mitochondria, except a single identified translational activator, TACO1. Here, we studied the role of the ZMYND17 gene, an ortholog of the yeast gene for the translational activator Mss51p, on the mitochondrial translation in human cells. Deletion of the ZMYND17 gene did not affect translation in the mitochondria, but led to the decrease in the cytochrome c oxidase activity and increase in the amount of free F1 subunit of ATP synthase. We also investigated the evolutionary history of Mss51p and ZMYND17 and suggested a possible mechanism for the divergence of functions of these orthologous proteins.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Evolução Molecular , Edição de Genes , Células HeLa , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Filogenia , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , RNA Guia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
8.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576035

RESUMO

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Genoma/genética , Sequência de Aminoácidos/genética , Animais , DNA/genética , Edição de Genes/instrumentação , Humanos , Mutação/genética , RNA Guia/genética
10.
Curr Protoc ; 1(8): e232, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34432381

RESUMO

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Mutação com Perda de Função , RNA Guia/genética
11.
Medicina (Kaunas) ; 57(7)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34356991

RESUMO

Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.


Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/genética , Metaloproteinase 8 da Matriz , Invasividade Neoplásica , RNA Guia
12.
Sheng Li Xue Bao ; 73(4): 559-570, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34405212

RESUMO

Prostaglandins are a class of poly-unsaturated fatty acids-derived bioactive lipids with important physiological function by binding to specific receptors. Prostaglandin receptors lack specific antibodies, which greatly impedes the research on our understanding of the signaling of prostaglandins. The aim of this study was to identify nine mouse lines with amino terminal (-NH2, -N) HA-tagged prostaglandin receptors by using the combination of artificial sperm and CRISPR-Cas9 technology. The guide RNA expression plasmid and labeled targeting vector plasmids were transferred into "artificial sperm cells". The "artificial sperm cells" containing labeled proteins were selected and injected into mouse oocytes, and implanted into pseudopregnant mice to obtain labeled mice. The genomic DNA of the prostaglandin receptor tagged mice was extracted, and the genotypes of mice were detected by PCR method. We also isolated mouse peritoneal macrophages to verify the protein expression of HA-labeled prostaglandin receptor by Western blot. Specific DNA bands were amplified in prostaglandin receptor labeled mice, and specific HA protein bands were detected in macrophage proteins, which was not detected in wild type mice. In summary, we successfully constructed 9 mouse lines with HA-tagged prostaglandin receptors, providing a powerful tool for further study of the pathophysiological functions of prostaglandin signaling both in vivo and in vitro.


Assuntos
RNA Guia , Receptores de Prostaglandina , Animais , Camundongos , Oócitos , Plasmídeos
13.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
14.
Methods Mol Biol ; 2351: 321-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382198

RESUMO

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Clonagem Molecular , Ordem dos Genes , Marcação de Genes/métodos , Humanos , Células Secretoras de Insulina/metabolismo , RNA Guia/química , RNA Guia/genética
15.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445123

RESUMO

Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.


Assuntos
Aterosclerose/genética , Sistemas CRISPR-Cas/genética , Animais , DNA/genética , Epigênese Genética/genética , Edição de Genes/métodos , Expressão Gênica/genética , Genoma/genética , Humanos , RNA/genética , RNA Guia/genética
16.
BMC Plant Biol ; 21(1): 363, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376154

RESUMO

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Pinus/genética , DNA de Plantas , Mutação INDEL , RNA Guia , Ribonucleoproteínas/genética
17.
Nat Commun ; 12(1): 5034, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413309

RESUMO

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Edição de Genes/métodos , RNA Guia , Animais , Humanos , Estudos Retrospectivos , Especificidade da Espécie
18.
Mol Cell ; 81(17): 3650-3658.e5, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34390675

RESUMO

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.


Assuntos
Epitopos/genética , Edição de Genes/métodos , Biblioteca de Peptídeos , RNA Guia/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Epitopos/imunologia , Humanos , Mutagênese/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Guia/imunologia
19.
Biomolecules ; 11(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34439828

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.


Assuntos
Doenças Autoimunes/terapia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Metilação de DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Engenharia Genética/métodos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Guia/genética , RNA Guia/metabolismo
20.
Nat Struct Mol Biol ; 28(8): 652-661, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34381246

RESUMO

CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Conformação Molecular , Bacteriófagos/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Técnicas Genéticas , RNA Guia/metabolismo
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