Abstract
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.
Highlights
CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors
A broad genetic and functional diversity of CRISPR-Cas immune systems have been identified across bacteria and archaea[14]
To elucidate functional protospacer adjacent motif (PAM) sequences, we developed a high-throughput in vivo screen with two features: applicability across PAM-dependent CRISPR-Cas systems and the generation of a distinct signal for functional PAMs
Summary
CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. 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. There is intense interest in identifying and rapidly characterizing nuclease systems that can be exploited to broaden and improve overall editing capabilities[2,14] To this end, Cas12a-type nucleases have emerged as suitable alternatives to Cas[9] nucleases, where several nucleases
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