Dataset Information

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

ABSTRACT: The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.

SUBMITTER: Niyonzima N

PROVIDER: S-EPMC5914421 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

Niyonzima Nixon N Lambert Abigail R AR Werther Rachel R De Silva Feelixge Harshana H Roychoudhury Pavitra P Greninger Alexander L AL Stone Daniel D Stoddard Barry L BL Jerome Keith R KR

Protein engineering, design & selection : PEDS 20170701 7

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while bindi ...[more]

PMID: 28873986

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Project description:BackgroundThe CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).ResultsWe created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.ConclusionsOur discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Dataset Information

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

Publications

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

Similar Datasets

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