Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.

Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9(CRISPR-associated protein9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na^+ /K^+ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na^+ efflux but also of K^+ and Ca^(2+) influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.

Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties

In rice, amylose content （AC） is controlled by a single dominant Waxy gene. We used Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）/CRISPR-associated 9 （Casg） to introduce a loss-of-function mutation into the Waxy gene in two widely cultivated elite japonica varieties. Our results show that mutations in the Waxy gene reduce AC and convert the rice into glutinous ones without affecting other desirable agronomic traits, offering an effective and easy strategy to improve glutinosity in elite varieties. Importantly, we successfully removed the transgenes from the progeny. Our study provides an example of generating improved crops with potential for commercialization, by editing a gene of interest directly in elite crop varieties.

Targeted Mutagenesis of Duplicated Genes in Caenorhabditis elegans Using CRISPR-Cas9

CRISPR （clustered regularly interspaced short palindromic repeats）-Cas9-based genome editing has revolutionized func- tional genomics in many biological research fields. The specificity and potency of CR1SPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo （Hsu et al., 2014）. Gene duplication is a key driver of evolu- tionary novelty （Taylor and Raes, 2004）. However, duplicated genes with near-identical sequences and functional redun- dancy have posed challenges for genetic analysis （Woollard, 2005）. The functions of duplicated genes can be assessed by simultaneous knockdown using homology-based methods such as RNA interference （RNAi） （Tischler et al., 2006）, Generation of double or triple mutants is an alternative way to assess functional redundancy of duplicated genes, However, generation of such compound mutants by forward or reverse genetic methods is time consuming.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.