Dataset Information

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

ABSTRACT: Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.

SUBMITTER: Jiang W

PROVIDER: S-EPMC6082914 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

Jiang Wen W Feng Songjie S Huang Shisheng S Yu Wenxia W Li Guanglei G Yang Guang G Liu Yajing Y Zhang Yu Y Zhang Lei L Hou Yu Y Chen Jia J Chen Jieping J Huang Xingxu X

Cell research 20180606 8

Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased ...[more]

PMID: 29875396

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Project description:Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of the bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we demonstrate the genome engineering of Corynebacterium glutamicum as a GC-rich model industrial bacterium by generating premature termination codons (PTCs) in desired genes using high-fidelity cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single-gene-inactivated strains for three genes (ldh, idsA, and pyc) with high base editing efficiencies of average 95.6% (n = 45, C6 position) and the highest success rate of up to 100% for PTCs and ultimately developed a strain with five genes (ldh, actA, ackA, pqo, and pta) that were inactivated sequentially for enhancing succinate production. Although these mutant strains showed the desired phenotypes, whole-genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To lower the undesirable mutations, high-fidelity CBEs (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) was employed for single or multiplexed genome editing in C. glutamicum, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a CRISPR-assisted bacterial genome engineering tool with an average high efficiency of 90.5% (n = 76, C5 or C6 position) at the desired targets. IMPORTANCE Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.

Dataset Information

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

Publications

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

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