Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice.

Abstract

The transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are two current genome editing technologies. Here, we compare and analyze the characteristics of the targeted mutations mediated by these two systems, such as efficiency, type, position, time, and genetic patterns. Both the TALEN and CRISPR/Cas9 systems can induce site-specific mutations in T0 rice plants effectively, but CRISPR/Cas9 is more effective. The major mutation type in both systems is the short insertion/deletion(InDel) mutation within 10 base pairs: deletions ranging from 1 to 10 bps are more often in TALEN, and 1bp insertions are more often in CRISPR/Cas9. Moreover, double-strand breaks (DSBs) generated by CRISPR/Cas9 are more precise than TALEN. In addition, DSBs could be repaired by the homologous recombination at a low frequency, causing DNA fragment duplication mutations. In some cases, the DNA fragments between the two close targets are deleted or inverted, and the mutation efficiency does not positively correlatewith the mutation efficiency of each target. Mutagenesis mediated by the TALEN or CRISPR/Cas9 system can occur as early as in transformed callus cells, and less frequently in somatic cells. Consequently, four different mutation types are formed, including homozygous, heterozygous, bi-allelic and chimeric mutations, with bi-allelic mutations having the highest rate and chimeric mutations having the lowest rate. All, except chimeric mutations, can descend stably into the next generation.