Analysis of var genes cloned from a Plasmodium falcivarum isolate in China

Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falciparum) isolate in Hainan Province,China.Methods:The sequences of var DBL1 regions were PCR-amplified,sequenced and the sequence characteristics was bioinformalically analysed.Recombinant proteins encoded by the var2csa genes were expressed and purified.The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays.Results:Fifty six unique DBI.a sequences were obtained,and the sequences represented similar diversity to the var genes of the genome parasite 3D7.There are two var2csa genes in the P.falciparum isolated from Hainan Province.Unlike in other falciparum parasites such as HB3,the two var2csa genes are more diverged.The receptor-binding capacity of DBL-5εand DBI.-6 e domains of HN var2CSA was studied.Conclusions:This work represented the diversity of rar genes of a P.falciparum isolate in China.

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Dear Editor,Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).Recently,the Liu group reported three optimization strategies for improving prime editing efficiency(Chen et al.,2021;Nelson et al.,2022).The first strategy is based on engineered prime editing guide RNAs(epegRNAs),which were generated by incorporating structured RNA motifs to the 3′terminus of pegRNAs.This strategy enhances pegRNA stability and prevents degradation of the 3′extension(Nelson et al.,2022).The second strategy is based on the optimized PE2 protein(PEmax),which harbors a SpCas9 variant with increased nuclease activity,an additional nuclear localization signal(NLS)sequences,and a new linker between nCas9 and reverse transcriptase(Chen et al.,2021).The third strategy is based on inhibition of DNA mismatch repair(MMR)in cells(Chen et al.,2021).In this work,we tested the optimized PEs generated with these three strategies in rice,demonstrating that the optimized PEs greatly improved prime editing efficiency in rice.We named the two optimized PEs ePE3max and ePE5max:the former is comprised of the PEmax protein,an epegRNA with evopreQ1 appended to its 3′end,and a nicking sgRNA;the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR.Using the two optimized PEs,we efficiently generated homozygous and heterozygous T173I,A174V,and P177S(TAP-IVS)mutation in EPSPS in rice,which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.

Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants.Agrobacterium-mediated genetic transformation(AMGT)is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process.In this article,we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents.We give an overview of the development of AMGT vectors including binary vector,superbinary vector,dual binary vector,and ternary vector systems.We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains.We focus in particular on the ternary vector system and the resources we developed.In summary,it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems,which are often integrated with morphogenic regulators.The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.