Synthetic chimeric nucleases function for efficient genome editing

Liu, R. M.; Liang, L. L.; Freed, E.; Chang, H.; Oh, E.; Liu, Z. Y.; Garst, A.; Eckert, Carrie A.; Gill, R. T.

doi:10.1038/s41467-019-13500-y

Title: Synthetic chimeric nucleases function for efficient genome editing

Journal Article · 03 December 2019 · Nature Communications

DOI: https://doi.org/10.1038/s41467-019-13500-y · OSTI ID:1580024

Liu, R. M. ^[1]; Liang, L. L. ^[1]; Freed, E. ^[1]; Chang, H. ^[1]; Oh, E. ^[1]; Liu, Z. Y. ^[1]; Garst, A. ^[2];

^[3]; Gill, R. T. ^[4]

Univ. of Colorado, Boulder, CO (United States)
Inscripta, Inc., Boulder, CO (United States)
Univ. of Colorado, Boulder, CO (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
Univ. of Colorado, Boulder, CO (United States); Danish Technical Univ., Lyngby (Denmark)

CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC36-08GO28308; SC0018368

OSTI ID:: 1580024

Report Number(s):: NREL/JA--2700-75556

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 10; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids Jiang, Wenyan; Maniv, Inbal; Arain, Fawaz PLoS Genetics, Vol. 9, Issue 9 https://doi.org/10.1371/journal.pgen.1003844	journal	September 2013
Deep scanning lysine metabolism in Escherichia coli Bassalo, Marcelo C.; Garst, Andrew D.; Choudhury, Alaksh Molecular Systems Biology, Vol. 14, Issue 11 https://doi.org/10.15252/msb.20188371	journal	November 2018
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation Jinek, Martin; Jiang, Fuguo; Taylor, David W. American Association for the Advancement of Science https://doi.org/10.5167/uzh-96990	text	January 2014
Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs Nillius, Dorothea; Müller, Joachim; Müller, Norbert Oxford University Press https://doi.org/10.7892/boris.12111	text	January 2011

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