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A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

ABSTRACT: A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

PROVIDER: PRJNA1198967 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31735700_1.fastq.gz	Fastqsanger.gz
	SRR31735700_2.fastq.gz	Fastqsanger.gz
	SRR31735701_1.fastq.gz	Fastqsanger.gz
	SRR31735701_2.fastq.gz	Fastqsanger.gz
	SRR31735702_1.fastq.gz	Fastqsanger.gz

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A comprehensive benchmark for multiple highly efficient base editors with maximal targeting scope

Project description:As the toolbox of base editors (BEs) expands, selecting appropriate BE and guide RNA (gRNA) to achieve optimal editing efficiency and outcome for a given target becomes challenging. Here we construct a set of 10 adenine and cytosine BEs with high activity and maximal targeting scope, and comprehensively evaluate their editing profiles and properties head-to-head with 34,040 BE-gRNA-target combinations using genomically integrated long targets and tiling gRNA strategies. A deep learning model BEEP (Base Editing Efficiency Predictor) is built for predicting the editing efficiency and outcomes. Guided by BEEP, we experimentally install 3,558 disease-associated single nucleotide variants (SNVs), including 20.1% of target sites that would not be edited due to known protospacer adjacent motif (PAM) restriction, and predicted candidate BE-gRNA-target combinations for modeling 1,752,651 ClinVar SNVs. We also identify several cancer-associated SNVs that drive the resistance to BRAF inhibitors in melanoma. These efforts benchmark the performance and illuminate the capabilities of multiple highly useful BEs for interrogating functional SNVs.

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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.

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Project description:As the toolbox of base editors (BEs) expands, selecting appropriate BE and guide RNA (gRNA) to achieve optimal editing efficiency and outcome for a given target becomes challenging. Here, we construct a set of 10 adenine and cytosine BEs with high activity and broad targeting scope, and comprehensively evaluate their editing profiles and properties head-to-head with 34,040 BE-gRNA-target combinations using genomically integrated long targets and tiling gRNA strategies. Interestingly, we observe widespread non-canonical protospacer adjacent motifs (PAMs) for these BEs. Using this large-scale benchmark data, we build a deep learning model, named BEEP (Base Editing Efficiency Predictor), for predicting the editing efficiency and outcome of these BEs. Guided by BEEP, we experimentally test and validate the installment of 3,558 disease-associated single nucleotide variants (SNVs) via BEs, including 20.1% of target sites that would be generally considered as "uneditable", due to the lack of canonical PAMs. We further predict candidate BE-gRNA-target combinations for modeling 1,752,651 ClinVar SNVs. We also identify several cancer-associated SNVs that drive the resistance to BRAF inhibitors in melanoma. These efforts benchmark the performance and illuminate the capabilities of multiple highly useful BEs for interrogating functional SNVs. A practical webserver (http://beep.weililab.org/) is freely accessible to guide the selection of optimal BEs and gRNAs for a given target.

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