Computational Tools and Resources for CRISPR/Cas Genome Editing

The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)nucleases and their functional variants,as well as in developing precise CRISPR/Cas-derived genome editors.The programmable and robust features of the genome editors provide an effective RNAguided platform for fundamental life science research and subsequent applications in diverse scenarios,including biomedical innovation and targeted crop improvement.One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts,which strongly depends on the efficiency and specificity of single guide RNA(sgRNA)-directed recognition of targeted DNA sequences.Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction.In this review,we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes.Secondly,we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on-and off-target mutations.Thirdly,we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization.Lastly,we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.

A zinc finger protein, interacted with cyclophilin,affects root development via IAA pathway in rice

The plant hormone auxin plays a crucial role in lateral root development. To better understand the rnolecular mechanisms underlying lateral root formation, an auxin-responsive gene OsCYP2 （Oso2g0121300） was characterized from rice. Compared to the wild type, OsCYP2-RNAi （RNA interference） lines exhibited distinctive defects in lateral root development. Yeast two-hybrid and glutathione S-transferase puIl-down results confirmed that OsCYP2 interacted with a C2HC-type zinc finger protein （OsZFP, O501g0252900） which is located in the rice nucleus. T2OsZFP-RNAi lines had significantly fewer lateral roots than did wild-type plants, which suggests a role for OsCYP2 and OsZFP in regulating lateral root development.Quantitative real-time polymerase chain reaction showed that the expression of certain Aux/IAA （auxin/indole-3- acetic acid） genes was altered in OsCYP2- and OsZFP-RNAi lines in response to IAA. These findings imply that OsCYP2 and OsZFP participate in IAA signal pathways controlling lateral root development. More importantly, OslAA11 showed functional redundancy not only in OsCYP2-RNAi lines but also in OsZFP-RNAi lines, which provides important clues for the elucidation of mechanisms controlling lateral root development in response to auxin.

Effects of ZJ0273 on barley and growth recovery of herbicide-stressed seedlings through application of branched-chain amino acids

In this study, we evaluated the effect of the herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate(ZJ0273) on barley growth and explored the potential to trigger growth recovery through the application of branched-chain amino acids(BCAAs). Barley plants were foliar-sprayed with various concentrations of ZJ0273(100, 500, or 1000 mg/L) at the four-leaf stage. Increasing either the herbicide concentration or measurement time after herbicide treatment significantly impaired plant morphological parameters such as plant height and biomass, and affected physiological indexes, i.e. maximal photochemical efficiency(Fv/Fm), quantum yield of photosystem II(ФPSII), net photosynthetic rate(Pn), and chlorophyll meter value(soil and plant analyzer development(SPAD)). Cellular injury of herbicide-treated plants was also evidenced by increased levels of reactive oxygen species(ROS) and antioxidative enzyme activity. Elevated levels of herbicide significantly reduced the activity of acetolactate synthase(ALS)-a keyD, enzyme in the biosynthesis of BCAAs. In a separate experiment, growth recovery in herbicide-stressed barley plants ? was studied using various concentrations of BCAAs(10, 50, 100, and 200 mg/L). Increasing BCAA concentration in growth media significantly increased the biomass of herbicide-stressed barley seedlings, but had no significant effect on non-stressed plants. Further, BCAAs(100 mg/L) significantly down-regulated ROS and consequently antioxidant enzyme levels in herbicide-stressed plants. Our results showed that exogenous application of BCAAs could reverse the inhibitory effects of ZJ0273 by restoring protein biosynthesis in barley seedlings.