A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

A Simple CRISPR/Cas9 System for Multiplex Genome Editing in Rice

Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.

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.

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the edits.Here,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation experiment.The duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to Xa23SW11.The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 generation.In addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 gen-eration.We performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of genes.The editing rates were dependent on the activity of pegRNA and/or ngRNA.For instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing experiments.Thesefindings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.