Construction And Application Of Efficient CRISPR-Cas Genome Editing Systems In Rice

Rice(Oryza sativa),one of the world's most important food crops,has emerged as a model plant for structural and functional genomics research.For functional genomics basic research and germplasm resources innovation and application in rice,to obtain reliable mutant materials is a key factor that determines project success.However,the limited rice natural mutant resources can not meet the actual requirements.Genome editing has become one of the frontiers in biological research.In recent years,CRISPR-Cas genome editing technology,derived from bacteria and archaea,has revolutionized the basic and applied research of biology and related subjects.The related achievements have been named as Science magazine's “Breakthrough of the Year” and Nature magazine's “What to expect in 2019: science in the new year”.Researchers have successively exploited and identified various CRISPR-Cas systems for genome editing from genome sequences of bacteria and archaea,such as CRISPR-Cas9,CRISPR-Cas12a(Cpf1),CRISPR-Cas12b(C2c1),which greatly expand the applicability of CRISPR-Cas system in the basic and applied research.The Streptococcus pyogenes Cas9(SpCas9)is the most widely used CRISPR-Cas system to date,a series of research work have been carried out in recent years.However,the editing efficiency,species suitability,multiplexing in plants still require further improvements.Furthermore,CRISPR-Cas12 a,a newly reported member of class 2 CRISPR-Cas system,has significant difference with Cas9 in protein structure,guide RNA composition,and cutting characteristics,and shows its potential use as a genome editing tool.Although previous studies have reported that CRISPR-Cas12 a can be used as an effective genome editing tool in human and animal cells,the editing efficiency,editing specificity,genetic stability,and scope of application in plant genome editing have not yet reliably confirmed.Based on the above research status,we carried out plenty of work focusing on the key issues of plant CRIPSR-Cas genome editing in this study.The main results as follows:1.We developed a single transcript unit CRISPR-Cas9(STU-CRISPR 1.0)system that process sgRNA with the cis-acting ribozyme to achieve effective genome editing in rice.We tested to generate genome edited plants by the STU CRISPR-Cas9 system expressing single sgRNA,mutation frequencies in T0 plants ranges from 53.8% to 100% with a majority of mutations being bi-allelic.When two sgRNAs were multiplexed in the STU CRISPR-Cas9 system,we found the frequency of simultaneous mutagenesis at both target sites approximated the frequency of mutagenesis at individual sites.In further work,we confirmed the feasibility of STU CRISPR-Cas9 system to achieve nickase-based editing and inducible editing.The results in Arabidopsis and tobacco indicated that the STU is broadly applicable to plant genome editing,and its efficiency is directly correlated with the Pol II promoter of choice.2.Based on the effective implementation of the STU-CRISPR 1.0 system,we further developed the STU-CRISPR 2.0 genome editing system.The results showed that both three STU CRISPR-Cas9 2.0 systems can effectively achieve editing at the target site.Among them,STU-Cas9-Csy4 and STU-Cas9-tRNA are more robust in genome editing than STU-Cas9-RZ.The STU-CRISPR 2.0 system also can effectively achieve Cas9 and Cas12 a mediated plant genome editing for 1-6 target sites.When multiplexing six sgRNAs with STU-Cas9-tRNA,18 out of 38 T0 lines contained mutations at all six target sites,and 10.5% of T0 lines carried biallelic mutations at all six target sites.In further work,we applied the STU-Cas9-tRNA system in base editing based on fusion of nCas9(Cas9_D10A)with two different cytidine deaminases,rAPOBEC1 and PmCDA1.Our data showed that STU nCas9::PmCDA1 system achieved 38.9%-68.8% C to T base editing efficiency in rice stable plants,and-1 bp C to T base editing event was also detected.3.According to the structure and functional characteristics of CRISPR-Cas12 a components,we achieved CRISPR-Cas12 a mediated efficient genome editing in rice with ZmUbi1 promoter driven RZ-crRNA-RZ and Cas12 a nuclease expression cassette,and revealed the main features : 1)The editing event is dominated by 6-13 bp deletion;2)The editing event mainly occurs in the 13-22 bp region distal to the 5'-TTTV-3' PAM site;3)CRISPR-Cas12 a nuclease has good editing specificity.In rice stable plants,the editing efficiency of LbCas12 a at four editing sites for OsPDS,OsDEP1,and OsROC5 was 100%,and almost all editing events were biallelic mutation,not chimeric.Cas12 a nuclease has temperature sensitivity in editing activity,but its indel mutation profiles will not be affected by temperatures.In addition,we further evaluated the applicability of STUCRISPR strategy in CRISPR-Cas12 a,and achieved multiplexed genome editing in rice by the STU-Cas12 a system.4.We conducted a large-scale WGS analysis for evaluating the specificity of CRISPR-Cas mediated plant genome editing by sequencing rice control plants of different types and Cas9 or Cas12a-edited T0 and T1 plants.Our results showed: 1)No significant difference was found between Cas9/Cas12 a backbone-transformed plants and tissue culture-only/Agrobacterium-infected plants on the numbers of variations and its genomewide distribution;2)The numbers of SNVs and indels among T0 edited plants didn't increase with the expression of Cas9/Cas12 a nuclease and sgRNA/crRNA;3)There was no significantly difference on the numbers of variations and its genome-wide distribution between those T0 edited plants with different T-DNA copy numbers and sgRNA numbers;4)There was no correlation between the numbers of SNVs or indels and the on-targeting efficiency by Cas9 or Cas12 a in these T0 plants 5)All on-target mutations by Cas9 or Cas12 a can be germline-transmitted to T1 plants;6)The number and genome distribution of the new mutations found in T1 plants were similar with the spontaneous mutations discovered in WT plants across two generations.5.Based on prime editing strategy,we developed three versions of plant prime editors and demonstrated their use for precise editing in rice.Our data showed: 1)The editing efficiencies of the PPE3-V01 system at five sites were quite low(0.05%-0.15%).The positive reads of the NGS data were validated for precise incorporation of designed edits and some reads revealed large deletions;2)The PPE3b-V01 system increased the maximum editing efficiency by 3 times compared with PPE3-V01,further realizing complex editing of multiple bases and discontinuous types at the target site;3)The PPE2-V02 and PPE3-V02 systems by systemic optimization had improved editing efficiency.Notably,PPE3-V02 resulted in the highest editing frequency of 1.55%.The PE editing efficiency of different sites was greatly impacted by the PBS-RT choices.The PPE2-V02 and PPE3-V02 systems significantly reduced the large deletion byproduct.