Scalable and versatile genome editing using linear DNAs with micro-homology to Cas9 sites in C. elegans

نویسندگان

  • Alexandre Paix
  • Yuemeng Wang
  • Harold E Smith
  • Chih-Yung S Lee
  • Deepika Calidas
  • Tu Lu
  • Jarrett Smith
  • Helen Schmidt
  • Mike W Krause
چکیده

Homology-directed repair (HDR) of double-stranded DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone non-homologous end joining (NHEJ) in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in C. elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (~30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag or delete any gene in the C. elegans genome, without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline. Highlights:-Short (30-60 bp) homology arms immediately flanking Cas9 cuts are sufficient to integrate both small and large edits in the C. elegans genome.-Scalable and versatile method for seamless editing of the C. elegans genome.

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تاریخ انتشار 2014