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1.
Talanta ; 233: 122554, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215057

RESUMO

Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and pathological studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA. PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3'-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H2O2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%-109.61% for ssRNA and 93.25%-111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.


Assuntos
Sistemas CRISPR-Cas , Peróxido de Hidrogênio , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Humanos , RNA
2.
Talanta ; 233: 122591, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215080

RESUMO

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.


Assuntos
COVID-19 , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
3.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203807

RESUMO

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Reparo do DNA por Junção de Extremidades/genética , Sequência de Bases , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oligonucleotídeos/metabolismo , RNA Guia/genética , Deleção de Sequência/genética
4.
Talanta ; 233: 122591, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1267931

RESUMO

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.


Assuntos
COVID-19 , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
5.
Anal Chim Acta ; 1174: 338747, 2021 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-34247734

RESUMO

In this work, hydrazone ligation assisted DNAzyme walking nanomachine is explored to couple with CRISPR-Cas12a trans-cleavage. Hydrazone ligation with high efficiency can mediate signal input which can be induced by target binding, thereby regulating the performance of DNAzyme walking nanomachine. The product strand from DNAzyme walking nanomachine can further activate the trans-cleavage of Cas12a. So, cascade signal amplification can be achieved to enhance the sensitivity for target detection. Subsequently, hydrazone ligation assisted DNAzyme walking nanomachine coupled with CRISPR-Cas12a has been further developed as a biosensor to analyze lipopolysaccharides. The developed biosensor exhibits a linear range from 0.05 ng/mL to 106 ng/mL and a lowest limit of detection of 7.31 fg/mL. This research provides a new mode for the signal output of DNAzyme walking nanomachine, so as to sensitively analyze different biomolecules.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Hidrazonas , Lipopolissacarídeos , Caminhada
6.
Nat Commun ; 12(1): 4270, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257311

RESUMO

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.


Assuntos
Mutação , SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Animais , Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Sistemas CRISPR-Cas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenvolvimento de Medicamentos , Genoma Viral , Células HEK293 , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genética
7.
J Agric Food Chem ; 69(23): 6379-6395, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34097395

RESUMO

The natural increase of the world's population implies boosting agricultural demand. In the current non-optimistic global scenario, where adverse climate changes come associated with substantial population growth, the main challenge in agribusiness is food security. Recently, the CRISPR/Cas system has emerged as a friendly gene editing biotechnological tool, enabling a precise manipulation of genomes and enhancement of desirable traits in several organisms. This review highlights the CRISPR/Cas system as a paramount tool for the improvement of agribusiness products and brings up-to-date findings showing its potential applications in improving agricultural-related traits in major plant crops and farm animals, all representing economic-relevant commodities responsible for feeding the world. Several applied pieces of research have successfully demonstrated the CRISPR/Cas ability in boosting interesting traits in agribusiness products, including animal productivity and welfare, crop yield growth, and seed quality, reflecting positive impacts in both socioeconomics and human health aspects. Hence, the CRISPR/Cas system has revolutionized bioscience and biotechnology, and its concrete application in agribusiness goods is on the horizon.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Animais , Sistemas CRISPR-Cas , Genoma de Planta , Humanos , Plantas Geneticamente Modificadas/genética
8.
Stem Cell Res ; 53: 102363, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087992

RESUMO

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.


Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Proteínas Luminescentes
9.
Biomed Pharmacother ; 140: 111772, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062417

RESUMO

The recent pandemic of novel coronavirus disease (COVID-19) has spread globally and infected millions of people. The quick and specific detection of the nucleic acid of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) remains a challenge within healthcare providers. Currently, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is the widely used method to detect the SARS-CoV-2 from the human clinical samples. RT-qPCR is expensive equipment and needs skilled personnel as well as lengthy detection time. RT-qPCR limitation needed an alternative healthcare technique to overcome with a fast and cheaper detection method. By applying the principles of CRISPR technology, several promising detection methods giving hope to the healthcare community. CRISPR-based detection methods include SHERLOCK-Covid, STOP-Covid, AIOD-CRISPR, and DETECTR platform. These methods have comparative advantages and drawbacks. Among these methods, AIOD-CRISPR and DETECTR are reasonably better diagnostic methods than the others if we compare the time taken for the test, the cost associated with each test, and their capability of detecting SARS-CoV-2 in the clinical samples. It may expect that the promising CRISPR-based methods would facilitate point-of-care (POC) applications in the CRISPR-built next-generation novel coronavirus diagnostics.


Assuntos
COVID-19/virologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , SARS-CoV-2/genética , Teste para COVID-19/métodos , Humanos , Pandemias/prevenção & controle
10.
Biomed Pharmacother ; 140: 111772, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: covidwho-1244709

RESUMO

The recent pandemic of novel coronavirus disease (COVID-19) has spread globally and infected millions of people. The quick and specific detection of the nucleic acid of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) remains a challenge within healthcare providers. Currently, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is the widely used method to detect the SARS-CoV-2 from the human clinical samples. RT-qPCR is expensive equipment and needs skilled personnel as well as lengthy detection time. RT-qPCR limitation needed an alternative healthcare technique to overcome with a fast and cheaper detection method. By applying the principles of CRISPR technology, several promising detection methods giving hope to the healthcare community. CRISPR-based detection methods include SHERLOCK-Covid, STOP-Covid, AIOD-CRISPR, and DETECTR platform. These methods have comparative advantages and drawbacks. Among these methods, AIOD-CRISPR and DETECTR are reasonably better diagnostic methods than the others if we compare the time taken for the test, the cost associated with each test, and their capability of detecting SARS-CoV-2 in the clinical samples. It may expect that the promising CRISPR-based methods would facilitate point-of-care (POC) applications in the CRISPR-built next-generation novel coronavirus diagnostics.


Assuntos
COVID-19/virologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , SARS-CoV-2/genética , Teste para COVID-19/métodos , Humanos , Pandemias/prevenção & controle
11.
Lab Chip ; 21(14): 2730-2737, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34100058

RESUMO

The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.


Assuntos
COVID-19 , Pandemias , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
13.
Talanta ; 232: 122415, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074403

RESUMO

Low abundance gene-PIK3CAH1047R mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CAH1047R mutation. The mutated gene-PIK3CAH1047R can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CAH1047R mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CAH1047R mutation detection.


Assuntos
Técnicas Biossensoriais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Mutação
17.
Methods Mol Biol ; 2298: 399-414, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085257

RESUMO

N6-methyladenosine (m6A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m6A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m6A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m6A modification enzymes, the programmable m6A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m6A and diversifying the toolkits for RNA manipulation.


Assuntos
Adenosina/análogos & derivados , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA/genética , Adenosina/genética , Sequência de Bases/genética , Linhagem Celular Tumoral , Edição de Genes/métodos , Células HeLa , Humanos , Metilação , RNA Mensageiro/genética
18.
Nat Commun ; 12(1): 3281, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078888

RESUMO

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Drosophila melanogaster/genética , Tecnologia de Impulso Genético/métodos , Genes Letais , Especiação Genética , Dinâmica Populacional , Animais , Proteína 9 Associada à CRISPR/deficiência , Proteína 9 Associada à CRISPR/genética , Drosophila melanogaster/metabolismo , Feminino , Fluxo Gênico , Mutação INDEL , Masculino , RNA Guia/genética , RNA Guia/metabolismo , Isolamento Reprodutivo
19.
BMC Bioinformatics ; 22(1): 344, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34167459

RESUMO

BACKGROUND: VISPR is an interactive visualization and analysis framework for CRISPR screening experiments. However, it only supports the output of MAGeCK, and requires installation and manual configuration. Furthermore, VISPR is designed to run on a single computer, and data sharing between collaborators is challenging. RESULTS: To make the tool easily accessible to the community, we present VISPR-online, a web-based general application allowing users to visualize, explore, and share CRISPR screening data online with a few simple steps. VISPR-online provides an exploration of screening results and visualization of read count changes. Apart from MAGeCK, VISPR-online supports two more popular CRISPR screening analysis tools: BAGEL and JACKS. It provides an interactive environment for exploring gene essentiality, viewing guide RNA (gRNA) locations, and allowing users to resume and share screening results. CONCLUSIONS: VISPR-online allows users to visualize, explore and share CRISPR screening data online. It is freely available at http://vispr-online.weililab.org , while the source code is available at https://github.com/lemoncyb/VISPR-online .


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Software , Internet , RNA Guia , Pesquisa
20.
BMC Genomics ; 22(1): 457, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34139989

RESUMO

BACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma , Mutagênese , Mutação
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