The CRISPR/Cas9 gene editing method has been successfully applied to modify genomes in many organisms1. However, several critical issues remain unresolved and have become major hurdles for its broad applications1. First, editing efficiency varies widely at different genetic loci and some targeted sites are resistant to editing for unknown reasons. Even for the same gene, editing efficiency differs greatly at different positions. Second, generation of undesirable insertions or deletions (InDels) at the target sites constitutes a major issue in precise genome editing and gene therapy. Third, one-step homozygosity in precise gene editing is extremely rare and is highly desirable for genetic and functional analyses, especially in systems where genetic manipulations are not possible or are tedious and time-consuming, such as cultured cells and mammals.

A Cas9-based technique has been established to generate precise nucleotide changes in the C. elegans genome using single-stranded oligonucleotides as donor templates2,3,4,5,6,7,8. In this system, the eft-3 gene promoter is used to drive Cas9 protein expression in C. elegans germline9,10. A SV40 Large T-antigen nuclear localization signal (NLS) is added to the C-terminus of Cas9 to usher it to the nucleus. Precise nucleotide alterations were achieved at multiple target sites with varying efficiencies, but not at some other loci tested2,3,4,5,6,7.

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