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1.
Nat Commun ; 15(1): 5471, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38942756

RESUMO

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Francisella , Edição de Genes , Humanos , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Amaurose Congênita de Leber/genética , Streptococcus pyogenes/genética , Células HEK293 , Mutação , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Engenharia de Proteínas/métodos , Genoma Humano
2.
Biosens Bioelectron ; 183: 113207, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33866136

RESUMO

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.


Assuntos
Técnicas Biossensoriais , COVID-19 , Teste para COVID-19 , Humanos , RNA Viral , SARS-CoV-2 , Sensibilidade e Especificidade
3.
Ophthalmic Genet ; 42(4): 365-374, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33821751

RESUMO

BACKGROUND: Precision genome engineering, with targeted therapy towards patient-specific mutations is predicted to be the future of personalized medicine. Ophthalmology is in the frontiers of development of targeted therapy since the eye is an accessible organ and has the ease of both delivery as well as monitoring effects of therapy. MATERIALS AND METHODS: We reviewed literature using keywords CRISPR, precision medicine, genomic editing, retinal dystrophies, retinitis pigmentosa, Usher syndrome, Stargardt's Disease. Further, we collated data on current clinical trials. RESULTS: There is growing evidence on the role of genomic editing in retinal dystrophies, the various methods used, and stage of development of different therapies have been summarized in this paper. CONCLUSIONS: The CRISPR-Cas9 system has revolutionized genome editing, and opened avenues in drug discovery. It is important to understand the role of this system along with its applicability in the field of ophthalmology. In this review article, we briefly describe its methodology, the strategies of employing it for making genetic perturbations, and explore its applications in inherited retinal dystrophies.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Genoma Humano/genética , Distrofias Retinianas/genética , Terapia Genética , Medicina Genômica , Humanos
4.
Proc Natl Acad Sci U S A ; 116(42): 20959-20968, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570623

RESUMO

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , Francisella/enzimologia , Edição de Genes , Animais , Proteína 9 Associada à CRISPR/genética , Catálise , DNA/química , DNA/metabolismo , Francisella/genética , Genoma , Humanos , Cinética , Especificidade por Substrato
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