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1.
Nat Biotechnol ; 38(5): 582-585, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393904

RESUMO

Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.

2.
Nat Biotechnol ; 38(5): 620-628, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32042165

RESUMO

Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

3.
Nat Biomed Eng ; 4(1): 125-130, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31740768

RESUMO

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.


Assuntos
Adenina/metabolismo , Edição de Genes/métodos , Tirosinemias/genética , Animais , Modelos Animais de Doenças , Feminino , Células HEK293 , Hepatócitos/metabolismo , Humanos , Hidrolases/genética , Fígado/metabolismo , Mutação Puntual , RNA/administração & dosagem
4.
Sci Adv ; 5(5): eaax5717, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31086823

RESUMO

Adenine base editors (ABEs) enable precise and efficient conversion of target A•T base pairs to G•C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.


Assuntos
Adenina/metabolismo , Edição de RNA , Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Inosina/metabolismo , Mutação , Transcriptoma
5.
Nat Biotechnol ; 36(9): 843-846, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29813047

RESUMO

Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.


Assuntos
Adenina/metabolismo , Citidina/genética , DNA/genética , Sistemas CRISPR-Cas , Códon , Edição de Genes , Células HEK293 , Humanos
7.
Angew Chem Int Ed Engl ; 55(5): 1733-6, 2016 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-26692420

RESUMO

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized (129) Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]-Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn(2+) in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive (129) Xe NMR spectroscopy.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Xenônio/química , Cristalografia por Raios X , Limite de Detecção
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