Manipulation of genetic recombination by editing the transcriptional regulatory regions of a meiotic gene in hybrid rice

Dear Editor,Genetic breeding involves the recombination and selection of various valuable genes.Meiotic crossover(CO)promotes the generation of new allelic combinations on chromosomes,which is essential for breeding elite varieties(Wijnker and de Jong,2008).An increase in CO promotes genetic diversity,whereas a decrease can rapidly stabilize excellent traits(Mercier et al.,2015).Furthermore,the complete elimination of CO facilitates heterotic fixation during apomixis(Wang et al.,2019).

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9） system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction（PCR）/restriction enzyme（RE） assay, T7 endonuclease I（T7EI） assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting（HRM） analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR（ACT-PCR）, for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

Erratum to: Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The same figure was misused for the PCR/RE assay results of Gn1a and GW2 fragments in Figure 3,and the arrows in the graphicsal result of GW2 were not on the tape.The corrected Figure 3 is as follows.