Synthetic Chimeric Nucleases Function for Efficient Genome Editing: Article No. 5524

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

Research output: Contribution to journalArticlepeer-review

20 Scopus Citations

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.
Original languageAmerican English
Number of pages11
JournalNature Communications
Volume10
DOIs
StatePublished - 2019

NREL Publication Number

  • NREL/JA-2700-75556

Keywords

  • Cas12a
  • CRISPR
  • genetic techniques
  • genome editing
  • high-throughput screening
  • synthetic biology

Fingerprint

Dive into the research topics of 'Synthetic Chimeric Nucleases Function for Efficient Genome Editing: Article No. 5524'. Together they form a unique fingerprint.

Cite this