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1.
Mol Cell ; 76(6): 922-937.e7, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31604602

RESUMO

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.


Assuntos
Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/metabolismo , Streptococcus thermophilus/enzimologia , Proteínas Virais/metabolismo , Regulação Alostérica , Bacteriófagos/genética , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/ultraestrutura , DNA/genética , DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Células K562 , Cinética , Mutação , Ligação Proteica , Conformação Proteica , Streptococcus thermophilus/genética , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/ultraestrutura
2.
Genome Res ; 30(1): 107-117, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31900288

RESUMO

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Streptococcus thermophilus/enzimologia , Streptococcus thermophilus/genética , Animais , Proteína 9 Associada à CRISPR/química , Linhagem Celular , Células Cultivadas , Clivagem do DNA , Humanos , Mamíferos , Camundongos , Camundongos Knockout , Relação Estrutura-Atividade , Especificidade por Substrato
3.
bioRxiv ; 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36711952

RESUMO

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

4.
Cell Genom ; 3(5): 100304, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37228746

RESUMO

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we substantially improve the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of the genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

5.
Nat Commun ; 13(1): 3512, 2022 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-35717416

RESUMO

Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Linhagem Celular , Fusão Gênica , Peptídeos/genética
6.
bioRxiv ; 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32869031

RESUMO

The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1.Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

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