Highly efficient CRISPR-SaKKH tools for plant multiplex cytosine base editing

Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.

An efficient culture procedure for micro-propagation of rice regeneration plants

In this paper,we describe briefly an efficientculture procedure for micro-propagation of riceregeneration plants.The procedure consists ofthe following steps: 1.Preparation of materials Calli wereinduced from mature seeds of an indica rice lineG67 by culturing the husked and disinfectedseeds on agar medium,consisting of Nbasalelements,3％ sucrose,1000mg/L proline,and 32mg/L 2,4-D.After one month＇s in- duction culture and 3 wk subculture,compactand nodular calli were picked out and trans- ferred into liquid medium for suspension cul-tures.The liquid medium contained the same

A preventive control strategy for static voltage stability based on an efficient power plant model of electric vehicles

With the increasing integration of wind farms and electric vehicles(EVs)in power systems,voltage stability is becoming more and more serious.Based on vehicle-to-grid(V2G),an efficient power plant model of EVs(E-EPP)was developed to estimate EV charging load with available corresponding response capacity under different charging strategies.A preventive control strategy based on E-EPP was proposed to maintain the static voltage stability margin(VSM)of power system above a predefined security level.Two control modes were used including the disconnection of EV charging load(‘V1G’mode)and the discharge of stored battery energy back to power grid(‘V2G’mode).A modified IEEE 14-bus system with high penetration of wind power and EVs was used to verify the effectiveness of preventive control strategy.Simulation results showed that the proposed strategy can not only improve the static voltage stability of power system with considerable wind generation,but also guarantee the travelling comfort for EV owners.

Rapid and efficient method for isolating plant high molecular weight(HMW)DNA with high purity

PREPARATION of HMW DNA (Megabase-size) is the basis for construction of genomic library with large DNA inserts such as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC), and for long-range physical mapping. It can also be used for the macro-study of repeat sequences. Since HMW DNA during preparation is inclined to be sheared physically and digested by internal nucleases, it is very difficult to prepare the HMW DNA. Initially, plant HMW DNA was prepared by embedding protoplasts in the low melting-point (LMP) agarose; however, it had several disadvantages: (ⅰ) Culture of protoplasts was time-consuming, costly and tedious. ( ⅱ ) It was only used successfully for limited

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Since the discovery that nucleases of the bacterial CRISPR(clustered regularly interspaced palindromic repeat)-associated(Cas) system can be used as easily programmable tools for genome engineering,their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction,resulting in mutations by non-homologous recombinatr e-tion. Strategies for performing such experiments à from Rthe design of guide RNA to the use of different transformation technologies à are evaluated. Furtherweive-more, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNAbinding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.

How to model the effect of mechanical erosion control practices at a catchment scale?

Conservation agriculture practices are a crucial factor in mitigating and controlling erosion by water.To address water erosion estimates,most environmental models use the USLE,RUSLE,and MUSLE models.Management practices that affect soil erosion by modifying the flow pattern,such as contour farming,strip farming,or terracing,are represented within these models as a support practice(P)factor.However,due to the difficulty in accurately mapping the P-factor,many studies choose to ignore it,using only the default value P-factor 1 which represents the absence of sowing at the level or cultivation in strips.This study proposes a methodology that evaluates the current P-factor based on the angle between the crop row orientation and the elevation contour lines.The method was tested in four areas under soybean crop fields in southern Brazil,totaling 25 km^(2).The reason for choosing four areas is to select different characteristics of rural properties and topographic conditions.The ideal values of the P-factor are expected to be between 0.5 and 0.6;however,in our case,a P-factor greater than 0.8 was obtained in 60%of the area,indicating the low occurrence of contour farming reduces erosion rates.The results show that policymakers could potentially use this methodology(angle between the crop rows and contour lines)to run soil-erosion risk scenarios for a broader application of contour farming.This allows the P-factor to be quantified via a thematic map instead of assigning uniform P-factor values.With a detailed study of the P-factor on the slopes,there is a better understanding of where to target support practices to reduce erosion.

Effects of temperature and solvent concentration on the solvent crystallization of palm-based dihydroxystearic acid with isopropyl alcohol

Palm-based dihydroxystearic acid of 69.55% purity was produced in a 500-kg-per-batch operation pilot plant and purified through solvent crystallization in a custom fabricated simultaneous batch crystallizer unit. The effects of temperature and solvent concentration on yield, particle size distribution and purity were studied. The purity was higher, while the yield and particle size were lower and smaller, respectively, at higher temperature and solvent concentration. The solvent crystallization process efficiency was rated at 66-69% when carried out with 70-80% isopropyl alcohol at 20℃.