Gao Caixia

Research progress of genome editing and derivative technologies in plants.

Genome editing technologies using engineered nucleases have been widely used in many model organisms. Genome editing with sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways, which can be employed to achieve targeted genome modifications such as gene mutations, insertions, replacements or chromosome rearrangements. There are three major SSNs─zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. In contrast to ZFN and TALEN, which require substantial protein engineering to each DNA target, the CRISPR/Cas9 system requires only a change in the guide RNA. For this reason, the CRISPR/Cas9 system is a simple, inexpensive and versatile tool for genome engineering. Furthermore, a modified version of the CRISPR/Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression, such as activation, depression and epigenetic regulation. In this review, we summarize the development and applications of genome editing technologies for basic research and biotechnology, as well as highlight challenges and future directions, with particular emphasis on plants.

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China; State Key Laboratory of Plant Cell and Chromosome Engineering, and Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China

Manipulating mRNA splicing by base editing in plants

Precursor-mRNAs (pre-mRNA) can be processed into one or more mature mRNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intron-mediated enhancement (IME), whereas alternative splicing (AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB1.1 (encoding a type 2C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly, altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.

Evaluation of temperature and emissivity separation method using the hyperspectral data for contrast emissivity surfaces

Land surface temperature and emissivity separation (TES) is a key parameter in the physical processes of land surface energy and water balance at regional and global scales. Various methods have been proposed to retrieve the temperature and emissivity for the high emissivity (close to 1) materials. This work addressed the iterative spectrally smooth temperature-emissivity separation method (ISSTES) proposed by Borel (1998) for retrieval of temperature and emissivity from the simulated hyperspectral thermal infrared (TIR) data for contrast (high- and low-) emissivity materials. The results show that small modeling errors less than 0.3 K for temperature and 0.01 for contrast materials are shown in ISSTES algorithm. A sensitivity analysis is carried out and the experimental results show that the instrumental noise, the atmospheric downwelling radiance and the atmospheric transmittance have a great influence on the retrieval accuracy, especially for low-emissivity materials.