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Synthetic chimeric nucleases function for efficient genome editing
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DOI：10.1038/s41467-019-13500-y.png)
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Abstract
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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.
Keywords:
GUIDED SCHEMA RECOMBINATION
CRISPR-CAS9 NUCLEASES
WIDE SPECIFICITIES
CPF1
CAS9
ENDONUCLEASE
EVOLUTION
MUTAGENESIS
DIVERSITY
DESIGN
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