Establishment Of Genotype Independent Genome Editing System In Cotton

Cotton,the main fiber crop,is also an important source of oil and protein.Genome editing technology can be used to precisely edit the cotton gene,further improving cotton yield,resistance and quality.However,efficient somatic regeneration and genetic transformation systems are necessary for cotton genome editing.The genotype-dependent of cotton somatic regeneration are evident,with only few kinds of upland cotton are capable of obtaining somatic regeneration plants.Therefore,establishing a genotype-independent cotton genome editing system is of great significance for the transition of cotton from conventional breeding to molecular design breeding.This study involved using distant hybridization between upland and sea-island cotton followed by rounds of backcrossing with the sea-island cotton used as the recurrent parent.The gland formation-related gene PGF and the key gene for chlorophyll synthesis CLA were selected as the target genes.The study aimed to verify whether the CRISPR/Cpf1 system in upland cotton can edit the corresponding alleles in sea-island cotton"Hai-7124".The CRISPR/Cpf1 element was also delivered to the upland cotton genotype"TM-1",the non-regenerative Xinjiang upland cotton cultivars"Xin Lu Zao58"and"ZB1092",and the diploid Asian cotton cultivar"An Hei Fei Xi Xiao Hei Zi".In this study,we also used CRISPR/Cas9 multi-genes editing system to target three genes:PGF,FAD2,and PEPC simultaneously in two cotton species:the upland cotton"TM-1"and the sea-island cotton"Hai-7124"to investigate the efficiency of multi-gene editing.Moreover,we attempted to use mobile elements reported in plants in combination with the TRV virus delivery system to construct six delivery vectors to examine whether they could induce heritable genome editing in the injected plants.Our main results are as follows:1.we successfully edited the PGF gene in sea-island cotton using the CRISPR/Cpf1 system transferred from upland cotton（Hai-7124×PGF（Cpf1）-Jin668）,with editing efficiency at the cr RNA1 site ranging from 39.76%to 98.27%and at the cr RNA2 site from 1.53%to 75.52%.We observed novel types of mutations in each generation,with the highest proportion reaching 25.34%.Experimental results targeting the key gene CLA for chlorophyll synthesis revealed similar conclusions（Hai-7124×CLA（Cpf1）-Jin668）,with editing efficiency ranging from 51.98%to89.24%and with the highest proportion reaching 18.36%for n novel types of mutations in each generation.In addition,off-target detection of 10 potential off-target sites for the PGF and CLA genes did not reveal any off-target effects.2.In the backcross progeny,we discovered bolls with an oval shape,which is a typical characteristic of upland cotton,but with a deep green color,a characteristic of sea-island cotton.Measurement of average boll weight and fiber length revealed that the fiber length（30.43 mm）of these bolls was similar to that of sea-island cotton（31.57 mm）and much longer than that of upland cotton（27.07 mm）,while the average boll weight（3.82 g）was similar to that of upland cotton（4.27 g）and much higher than that of sea-island cotton（3.11 g）.Seven individual plants from the BC₂F₁generation were selected for 10×whole-genome resequencing based on their similarity to sea-island cotton.The BC₂F₁generation monoclonal lines"2A-4","2B-3","2B-12",and"2L-5",which are relatively similar to sea island cotton,exhibit a total of 3.66-6.38 million SNPs and 0.47-0.69 million In Dels when compared to the reference genome of sea-island cotton Hai-7124.When compared to the reference genome of upland cotton TM-1,they exhibit a total of 14.17-16.63 million SNPs and3.13-3.61 million In Dels.The BC2F1 generation monoclonal lines"2P-2"and"2H-1",which exhibit some morphological characteristics similar to sea-island cotton,exhibit a total of 7.62 and 9.70 million SNPs and 1.18 and 9.06 million In Dels,respectively,when compared to the reference genome of sea-island cotton Hai-7124.When compared to the reference genome of upland cotton TM-1,they exhibit a total of14.03-16.40 million SNPs and 3.36-3.64 million In Dels.The BC2F1 individual"2E-8",which lacks the typical morphology of island cotton,exhibits a total of 11.69million SNPs and 1.38 million In Dels when compared to the reference genome of sea-island cotton Hai-7124.When compared to the reference genome of upland cotton TM-1,they exhibit a total of 12.47 million SNPs and 3.18 million In Dels.3.Using the gland formation key gene PGF as target gene,the CRISPR/Cpf1system was successfully delivered into the genome of upland cotton genotype TM-1（TM-1×PGF（Cpf1）-Jin668）,upland cotton cultivar"Xin Lu Zao58"（Xin Lu Zao58×PGF（Cpf1）-Jin668）,and"ZB1092"from Xinjiang（ZB1092×PGF（Cpf1）-Jin668）.Selection for glandless plants in the hybrid offspring revealed a proportion of 43.48%,83.33%,and 38.89%in the hybrid F₁generation.The editing efficiency at the cr RNA1 sites ranging from 45.02%to 98.94%,and at the cr RNA2site from 10.47%to 63.59%.The proportion of the novel types of mutations reached a maximum of 20.94%.plants that almost glandless and containing CRISPR/Cpf1system were also selected from the hybrid offspring of the diploid Asian cotton cultivar"An Hei Fei Xi Xiao Hei Zi"（An Hei Fei Xi Xiao Hei Zi×PGF（Cpf1）-Jin668）.The editing efficiency at the cr RNA1 sites ranging from 81.10%to 91.06%,and that at the cr RNA2 sites ranging from 45.88%to 48.41%.4.Cotton genes PGF,FAD2,and PEPC were selected as target genes simultaneously,and glandless plants were successfully selected from the hybrids of upland cotton TM-1 and sae-island cotton Hai-7124（TM-1×PFP（Cas9）-Jin668,Hai-7124×PFP（Cas9）-Jin668）,which accounting for 58.82%and 42.11%of the total hybrids,respectively.Multi-gene editing events were detected in each glandless plant.The editing efficiency of PGF,FAD2,and PEPC ranged from 71.46%to 98.47%,64.03%to 99.29%,and 42.12%to 97.72%.5.A TRV virus-based sg RNA delivery system was used by constructing 6vectors,in which mobile elements(At FT,Atm FT,t RNA^met,t RNA^ile)was added to the3’end of the sg RNA to facilitate its movement within the plants.Using this system,sg RNA was successfully delivered to cotton plants that contain Cpf1/Cas9 elements,resulting in successful gene editing with the highest efficiency of up to 24.26%.The PEBV promoter combined with t RNAile showed a higher delivery efficiency than other mobile elements in this study.In summary,this experiment illustrated that the complete genome editing system can be transmitted to upland cotton,sea-island cotton,and Asian cotton through hybridization and backcrossing.Transmitted genome editing system can efficiently edit three genes simultaneously.Theoretically,all cotton varieties can obtain the genome editing system through traditional backcross breeding methods and achieve endogenous genes editing.Additionally,this study preliminarily verified the feasibility of the sg RNA delivery system based on TRV virus and mobile elements,which provides experimental support for the future direct transmission of the complete gene editing system through virus-mediated delivery.