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1.
Cell ; 187(13): 3249-3261.e14, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38781968

RESUMO

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , DNA , Edição de Genes , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/metabolismo , DNA/genética , Edição de Genes/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Domínios Proteicos , Genoma Humano , Modelos Moleculares , Estrutura Terciária de Proteína , Conformação de Ácido Nucleico , Biocatálise , Magnésio/química , Magnésio/metabolismo
2.
Nat Commun ; 15(1): 1727, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409124

RESUMO

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Encéfalo/metabolismo
3.
bioRxiv ; 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38014175

RESUMO

Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) has the potential to enable high-efficiency in vivo genome editing with low toxicity and an easily manufactured technology, if RNP efficacy can be maintained during LNP production. In this study, we engineered a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) using directed evolution to generate iGeoCas9 evolved variants capable of robust genome editing of cells and organs. iGeoCas9s were significantly better at editing cells than wild-type GeoCas9, with genome editing levels >100X greater than those induced by the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell lines and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue-selective LNP formulations, we observed genome editing of 35‒56% efficiency in the liver or lungs of mice that received intravenous injections of iGeoCas9 RNP:LNPs. In particular, iGeoCas9 complexed to acid-degradable LNPs edited lung tissue in vivo with an average of 35% efficiency, a significant improvement over editing efficiencies observed previously using viral or non-viral delivery strategies. These results show that thermostable Cas9 RNP:LNP complexes are a powerful alternative to mRNA:LNP delivery vehicles, expanding the therapeutic potential of genome editing.

4.
bioRxiv ; 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38014180

RESUMO

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects 1 . However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identified potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins identified a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibited substantially improved editing efficacy compared to other constructs. We found that self-deliverable Cas9 RNPs generated robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo .

5.
bioRxiv ; 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38168238

RESUMO

Thermostable CRISPR-Cas9 enzymes could improve genome editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold higher genome editing levels. Cryo-EM structures of the wildtype and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome editing activity.

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