Development Of Rapid And User-friendly Open-source CRISPR/Cas9 System

The CRISPR/Cas9 system has been a powerful tool for gene function analysis and crops genetic improvement.It has brought an unprecedented revolution to life sciences.At present,the CRISPR system is developing rapidly.About a dozen different CRISPR systems with different function have been developed,but the most widely used in plants are still the CRISPR/spCas9 system.Although several CRISPR/spCas9 systems have been established for plants,they are not perfect enough and lack an easy-to-use open-source design,which hinders the development of the CRISPR/spCas9 system to some extent.Therefore,this study designed an open-source CRISPR/spCas9 system for dicots and monocots using standard bio-brick assembly techniques based on isocaudarner.This system can be used not only for the progress of the CRISPR/spCas9 system,but also for other CRISPR systems.The main results of this study are as follows:1)This study designed an assembly strategy combining Hind III and KpnI with XhoI-SalI and Xba I-Spe I-Avr II isocaudarner groups as bio-brick sites(suitable for Dicots);an assembly strategy combining HindIII and KpnI with Acc5I-Pfl23 II,AscI-Mlu I,Avr II-SpeI isocaudarner groups as bio-brick site(suitable for monocots).2)According to the above standard bio-brick assembly strategy in this study,the plant binary vector backbones capable of accommodating standard bio-bricks: pHN,pHNII,pHNM were constructed and their ability to transfect plants by Agrobacterium was demonstrated.Based on the standard bio-brick assembly strategy in this study,a series of basic bio-brick elements were produced,which were further assembled into complete bio-brick units with expressive function in plants.After cloning functional genes in these bio-brick units,a series of bio-brick vectors with specific functions were obtained.3)In this study,four CRISPR/spCas9 systems suitable for dicots and monocots were constructed using functional bio-brick vectors: pHNCas9(dicotyledon),pHNCas9HTAtU3b(dicotyledon),pHNIICas9HTAtU61(dicotyledon),pHNM Cas9HTOsU6a(monocotyledonous).4)In this study,four assembly strategies of CRISRP target sites(CRISPR strategy I-IV)and four primer design aids(primer design aid I-IV)were designed based on the above four CRISPR/spCas9 vectors.Those primer design aid are able to provide target site's GC content,base number,primer name,target site-Specific primer sequence,complete sgRNA sequences of specified target-site.CRISPR strategy I(based on pHNCas9HTAtU3b)and primer design aid I are suitable for high-throughput assembly of single target site without PCR.CRISPR strategy II(based on pHNCas9)and primer design aid II are suitable for one-pot cloning of 2-8 sgRNA expression cassettes in pHNCas9 by Golden Gate cloning technology.CRISPR strategy III(based on pHNIICas9HTAtU61)and primer design aid III are suitable for high-throughput assembly of 1-4 target sites without PCR.CRISPR strategy IV(based on pHNMCas9HTOsU6a)and primer design aid IV are suitable for high-throughput assembly of 1-4 target sites without PCR.The above CRISPR target site assembly strategies are very suited for kit development.5)In this study,single target site knockout tests were performed on three fruit ripening related genes of SlEIN2(Solyc09g007870.2),SlERFE1(Solyc09g075420.2),SlARF2B(Solyc12g042070.1)in tomato using CRISPR Kit I.A multi-site knockout test was performed on tomato SlGRAS8(Solyc02g085600.1).A multi-gene knockout test was performed on tomato SlACS2(Solyc01g095080.2)and SlACS4(Solyc05g050010.2).The results showed that the standardized bio-brick system in this study can successfully complete single-site editing,multi-site editing,and multi-gene editing in tomato.In this study,a variety of mutation types were analyzed in the T1 plant of SlGRAS8 mutant;the SlGRAS8 mutant phenotype and genotype have good genetic stability and the system has extremely low off-target effects(based on T1 generation and T4 generation results).6)Based on the editing efficiency of different lines,the potential factors affecting editing,such as GC content,Cas9 and sgRNA expression,and secondary structure of sgRNA,were analyzed.The results indicate that the secondary structure of sgRNA has a clear effect on the editing efficiency of CRISPR/spCas9 system in tomato: when the target site consecutive pair with sgRNA backbone more than 6 base,the editing efficiency of the CRISPR system is significantly reduced;and sgRNA may loss editing ability if the position of the continuous pairing affects the formation of sgRNA stem loop 1.7)How to improve the high-throughput performance of the CRISPR system in the plant regeneration stage and screening non-transgenic mutants from the offspring is a main bottleneck in the high-throughput gene function analysis by the CRISPR system in the plant.In order to address this problem,this study first proposed the concept of visual CRISPR and conducted preliminary verification.The results showed that some of the light purple shoots(MYB75 overexpression-induced anthocyanin accumulation)were observed with the visualized CRISPR plant expression vector pHNCas9: OEMYB75 when explants transferred to the differentiation medium for about 30 days.This result implies that the CRISPR vector containing OEAt MYB75 BB may be important for the early screening of T0 positive seedlings and the high throughput screening non-transgenic plants in progeny.