Construction Of Rice Defense Related Mutants Using Crispr Technology

Plant diseases severely affect the quality and yield of crops and threaten the world food security.Rice is one of the most important food crops in the world.Sophisticate genetic tools and mutant resources are needed to understand the molecular mechanism by which rice defend against disease.In recent years,the emergence of CRISPR-Cas9 genome editing technology presents revolutionary tools for life sciences.Besides Cas9,many Cas nucleases,particularly Cas12a,have been discovered and engineered for genome editing,which has greatly expanded the scope of genome editing.In addition,a variety of genetic manipulation tools based on CRISPR-Cas systems have been developed,including transcriptional regulation,epigenetic modification,base editing and so on.In this study,we aim to optimize and apply CRISPR technologies to accelerate the functional characterization of rice defense related genes.We established efficient transcriptional activation systems and Cas12a-based multiplex genome editing tools in rice.More importantly,we developed a new strategy to create arrayed CRISPR library and generated mutant library covering rice receptor-like kinase（RLK）genes.First,we optimized and validated the efficiencies of d Cas9-mediated transcription activation of target genes.Three transcriptional activation strategies were tested here,including d Cas9-VP160,d Cas9-SNAC1-activation-domain（d Cas9-SA）and Synergistic Activation Mediator using d Cas9-MCP-VP160.In rice protoplasts,d Cas9-VP160 displayed highest efficiency（up to 22.8-fold）to activate target gene expression.We then optimized d Cas9-VP160 vectors using the intronic polycistronic t RNA-g RNA（in PTG）which can simultaneously express sg RNAs（single guide RNAs）and d Cas9-VP160 using one PolⅡpromoter.Transcriptional activation efficiency of in PTG-d Cas9-VP160 system was 2.3～4.1-fold higher than conventional rice sno RNA U3 promoter（Os U3p）expressed sg RNAs.Second,Fn Cas12a and Lb Cas12a based multiplex gene editing tools were established and tested in rice.We optimized and compared four Cas12a vectors for multiplex gene editing,including vectors expressing cr RNAs with Os U3p（p32Lb and p32Fn）and intron（p33Lb and p33Fn）.Furthermore,multiple cr RNAs could be expressed using cr RNA array and PTC（Polycistronic t RNA-cr RNA）for these vectors.We compared the editing efficiencies of these vectors with two cr RNA expressing strategies in rice protoplasts and stable transgenic plants.The results showed that all systems were capable editing multi sites in rice and p32Fn vector with cr RNA arrays showed highest efficiency.Furthermore,the targeted gene mutagenesis efficiencies of Cas9,Fn Cas12a,Lb Cas12a,and Cas9-NG were compared in stable transgenic rice.The classical Cas9 showed much higher gene editing frequency（92.6%）and biallelic mutation frequency（85.1%）than others.Therefore,CRISPR-Cas9 was used for the high-throughput genetic screening system.Third,we designed and validated a FLASH gene editing pipeline to create arrayed CRISPR library for high throughput gene editing.Previously,pooled transformation of CRISPR library was used to generate a population of plant mutants.However,determining the sg RNA in transformants is tedious and costly in pooled CRISPR library approach.To overcome the limitations,we designed a method which uses PCR fragment-length-tag to proxy sg RNA distinguishing（FLASH）for arrayed CRISPR library construction.In FLASH gene editing pipeline,a set of 12 CRISPR-Cas9 vectors carrying DNA fragments of different lengths（FLASH tags）are constructed.After assembling the sg RNA to one of these FLASH tag indexed vectors,unique linkages are established between each sg RNAs and FLASH tags within a set of 12 constructs.In FLASH gene editing pipeline,a group of 12 constructs were mixed to transform plant.As a result,the sg RNAs are surrogated by FLASH tags which can be easily read out using PCR and conventional gel electrophoresis.This pipeline can be used to construct arrayed CRISPR library in plants.At last,we used FLASH gene editing pipeline to generate an arrayed CRISPR library to edit all 1072 RLK genes in rice.With 12 Cas9/sg RNA vectors as a group,Cas9-g RNA constructs targeting these RLKs were assembled individually in Cas9 vectors indexed by FLASH tags and then divided into 89 groups for transformation.One-shot transformation of each group obtained 5039 T₀ lines（RLK mutant library）and sg RNAs in all lines were assigned according to the FLASH tag detection results.This RLK mutant library covers 89%（955/1072）target genes and 74.3%（710/955）of target genes have 3 more independent T₀lines.Among the 391 randomly genotyped target genes,92.1%（360/391）have been edited.Furthermore,we detected low frequency of unintended editing events of transient T-DNA expression,which can be readily tracked using the FLASH gene editing pipeline.In a pilot screen of rice defense related genes using this RLK mutant library,mutants of 14 RLK genes were phenotyped after Magnaporthe oryzae inoculation and 8 RLK genes were identified as positive regulators of rice blast resistance.In summary,this study first optimized and compared the efficiencies of CRISPR-Cas systems for targeted gene mutation and activation.Based on the comparisons result,the FLASH gene editing pipeline using a most efficient Cas nuclease was established for arrayed library screening in rice.Our data demonstrated that FLASH gene editing pipeline is an efficient,robust,and flexible method for arrayed CRISPR library construction,which render CRISPR screening to be a routine method for plant research.These methods and RLK mutant resources would accelerate functional genomics and crop breeding.