In:
Plant Biotechnology Journal, Wiley, Vol. 17, No. 2 ( 2019-02), p. 362-372
Abstract:
CRISPR /Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA ( gRNA ) and LbCas12a‐ CRISPR RNA (cr RNA ) into maize inbred B104 embryos using Agrobacterium ‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE ‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNA s, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNA s. In conclusion, our results suggest that the CRISPR /Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR /Cas12a needs further optimization for improved editing efficiency.
Type of Medium:
Online Resource
ISSN:
1467-7644
,
1467-7652
DOI:
10.1111/pbi.2019.17.issue-2
Language:
English
Publisher:
Wiley
Publication Date:
2019
detail.hit.zdb_id:
2136367-5
detail.hit.zdb_id:
2105743-6
SSG:
12