Abstract
The CRISPR/Cas9 system has been widely utilized in plant biotechnology as a gene editing tool. However, a conventional design with ubiquitously expressed CRISPR/Cas9 was observed to cause large numbers of somatic mutations that complicated the identification of heritable mutations. We constructed a pollen-specific CRISPR/Cas9 (PSC) system using pollen-specific promoters of maize Profilin 1 and Profilin 3 (pZmPRO1 and pZmPRO3) to drive Cas9 expression, and the bZIP transcription factor Opaque2 (O2) was employed as the target gene. The maize ubiquitin promoter (pZmUbi)-driven CRISPR/Cas9 (UC) system was employed as a control. We generated transgenic plants for the PSC and UC systems and analyzed three independent events for each system. We found that the pZmPRO1 PSC system generated no target gene mutations in the T0 generation but successfully generated 0–90% target gene mutations in the T1 generation. A total of 31 of 33 mutations in the T1 generation could be inherited in the T2 generation. In addition, 88.9–97.3% of T2 mutations were from the T1 generation. The UC system generated mutations in the T0 generation, and 0%, 50% and 92.9% of T1 mutations were from the T0 generation. Our results demonstrate that the PSC system provided stable, heritable mutants in the next generation, and this approach might also be applied in other crops using germinal cell-specific CRISPR/Cas9 systems to facilitate plant breeding.
Highlights
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system is a form of prokaryotic adaptive immune response.Cas9 and guide RNAs form a complex that recognizes a specific DNA sequence consisting of a base-pairing sequence to the spacer region of the gRNA and a protospacer adjacent motif (PAM), and this causes a DNA double-strand break (DSB) in the target site [1,2,3]
ZmPRO1–3 were first visible in maturing pollen grains (10 mm) and significantly accumulated in anthers dissected from 10 mm spikelets and mature pollen at dehiscence, suggesting that they were anther- or pollen-specific
The high-throughput tracking of mutations (Hi-TOM) results are consistent with the editing efficiency based on the PCR and Sanger sequencing data (Table S6). These results demonstrate that the pollen-specific CRISPR/Cas9 (PSC) system could improve the production of heritable mutations
Summary
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system is a form of prokaryotic adaptive immune response.Cas and guide RNAs (gRNAs) form a complex that recognizes a specific DNA sequence consisting of a base-pairing sequence to the spacer region of the gRNA and a protospacer adjacent motif (PAM), and this causes a DNA double-strand break (DSB) in the target site [1,2,3]. The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system is a form of prokaryotic adaptive immune response. The CRISPR/Cas system has been demonstrated to be a powerful and efficient genome editing tool and has been widely utilized in a variety of organisms and cell types [4,5,6,7,8,9,10]. A number of crop traits such as disease resistance [19], flower color regulation [20], herbicide resistance [21] and stress resistance [22] can be improved by the CRISPR/Cas system
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