Development And Optimization Of CRISPR/Cas System Mediate Genome Editing Technology In Rice

Solving the problems coming from plant breeding and cultivating are must-do so as to control food production independently and ensure food security.Rice（Oryza sativa L.）,one of the major food crops,needs to be improved on its various kinds of valuable agronomic traits modified by some correlated genes and excellent rice seed bank for ensuring food security and promoting agricultural development in China.The establishment of genome editing technologies mediated by the CRISPR/Cas system is a milestone in the history of plant biotechnology.Through this system,it is possible to introduce precise and predictable genome modifications into plants offering more possibilities to obtain desired traits.Although the CRISPR/Cas-based genome editing tools is powerful for genetic improvement on crop traits,it is still have limitations in specific applications,such as the absence of suitable PAM sequences and low efficiency of base editing at some loci,etc.while arbitrary editing cannot be accomplished at any position at the whole genome level.Therefore,continuous exploration and optimization of plant CRISPR/Cas systems are needed to improve the application scope and editing efficiency of genome editing technologies.Here,we established and optimized a CRISPR/Cas12a-based rice genome editing technology,and optimized the CRISPR/Cas9n-mediated adenine base editors（ABEs）and cytosine base editors（ABEs）using Rad51DBD,which provides a good technical support for more CRISPR/Cas-derived tools can be developed and used for gene function research and molecular crop breeding in the future.The main results were listed as follows:To obtain an efficient CRISPR/Cas12a-mediated rice genome editing system,the transient expression vectors p UC19:Cas12a were first constructed to express Lb Cas12a and As Cas12a proteins,respectively,and p HZ4 and p HZ11 to express cr RNAs corresponding to 23 nt of Lb Cas12a and As Cas12a proteins,respectively.The ability of the above two Cas12a proteins to edit the target rice endogenous gene Os CERK1 was examined by the rice protoplast transient expression system,and the PCR/RE analysis and sequencing results showed that both As Cas12a and Lb Cas12a could cause deletion at the predicted target site sequence with a deletion fragment size of 6～12 bp.This proves that the CRISPR/Cas12a system with TTTV as PAM has editing activity in rice.Further,the editing effect of rice Lb Cas12a and As Cas12a was examined using Agrobacterium-mediated genetic transformation technique at different cr RNA lengths and conformations（3’addition of UUUUUAUUUUU（U4AU4）sequence of cr RNA）and different Cas12a mutants and other optimized parameters,and the results showed that The editing activity of Lb Cas12a in rice was significantly higher than that of As Cas12a,and the editing efficiency was best when the cr RNA was 23 nt,reaching 86.67%,and the addition of U₄AU₄ sequence did not improve the editing efficiency of Cas12a in rice.The above proved that Lb Cas12a was more suitable for rice genome editing.In order to construct the CRISPR/Cas12a ABE system for introducing A-to-G conversion at AT-rich regions in the rice genome,Tad A8e,an evolved version of t RNA adenosine deaminase derived from Escherichia coli,was fused with d Lb Cas12a named Tad A8e-d Lb Cas12a and introduced into p Ubi:r BE58 vector was obtained to construct a novel rice adenine base editor 58（Tada8E-DLBCas12A）.The results showed that r BE58 could achieve the targeted site base A to G targeted substitution in rice with an efficiency of 33.33%,and the base editing window was located between+8 and+14 bp downstream of TTTV PAM.In addition,no indel（base insertion or deletion）and unintended base mutations were detected.This suggests that CRISPR/Lb Cas12a and Tad A8e-mediated r BE58 can perform single-base editing in the rice genome,expanding the richness and targeting range of the rice base editing toolbox.To enhance the efficiency of rice base editors,the rice codon optimized RAD51DBD was integrated to optimize the existing base editors r BE9,r BE25 and r BE46b and obtained new base editors r BE9c,r BE25c and r BE46c.We compared and tested the editing efficiency by targeting the rice endogenous genes,it was found that the editing efficiency was mostly improved in the tested genes,but to a lesser extent.This suggests that enhancing the editing efficiency by using Rad51DBD to enhance the binding ability of deaminase to the target site and prolong the resulting action time is a feasible strategy to optimize the single-base editing system.