Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells DOI Open Access
Júlia Daher Carneiro Marsiglia,

Kia Vaalavirta,

Estefany Knight

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 13, 2023

Abstract Promiscuous editing by CRISPR/Cas systems within the human genome is a major challenge that must be addressed prior to applying these therapeutically. In bacteria, have evolved in co-evolutionary arms race with infectious phage viruses contain inhibitory anti-CRISPR proteins their genomes. Here, we harness outcome of this engineer an AcrIIA4 protein increase precision CRISPR/Cas-based targeting. We developed approach specifically leveraged (1) language models, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements cells. single experiment, ∼10,000 variants were tested identify lead eliminated detectable off-target events while retaining on-target activity. The candidates further focused round screening included high-fidelity version Cas9 as benchmark. Finally, arrayed experiments using delivered ribonucleoprotein conducted demonstrated gene across two independent genomic loci reduction frequency translocation between site. Thus, language-model-guided high-throughput effective way efficiently precision, which could used improve fidelity editing-based therapeutics reduce genotoxicity.

Language: Английский

Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells DOI Open Access
Júlia Daher Carneiro Marsiglia,

Kia Vaalavirta,

Estefany Knight

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 13, 2023

Abstract Promiscuous editing by CRISPR/Cas systems within the human genome is a major challenge that must be addressed prior to applying these therapeutically. In bacteria, have evolved in co-evolutionary arms race with infectious phage viruses contain inhibitory anti-CRISPR proteins their genomes. Here, we harness outcome of this engineer an AcrIIA4 protein increase precision CRISPR/Cas-based targeting. We developed approach specifically leveraged (1) language models, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements cells. single experiment, ∼10,000 variants were tested identify lead eliminated detectable off-target events while retaining on-target activity. The candidates further focused round screening included high-fidelity version Cas9 as benchmark. Finally, arrayed experiments using delivered ribonucleoprotein conducted demonstrated gene across two independent genomic loci reduction frequency translocation between site. Thus, language-model-guided high-throughput effective way efficiently precision, which could used improve fidelity editing-based therapeutics reduce genotoxicity.

Language: Английский

Citations

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