Development Of Cotton Base Editors And Application For Plant Architecture Improvement

Cotton is the most important cash crops and source of natural fiber.In recent years,China’s cotton production has shifted to Xinjiang,where climatic conditions and a high degree of mechanization have dictated that cotton varieties need special characteristics,such as early maturity,compactness and concentrated flocculation.The plant florigenin（FT）and anti-florigenin（TFL1）hormone systems synergistically regulate nutritional and reproductive growth and ultimately determine plant architecture.However,the application of the florigenin and anti-florigenin system in cotton breeding is not yet mature enough.Knockout of the Gh TFL1 by CRISPR/Cas9 has resulted in early flowering phynotype with extreme dwarfism mutants,which are not compatible with production applications.Base editing based on the CRISPR system can achieve targeted base mutations in target genes without creating DNA double-strand breaks nor requiring the addition of DNA repair templates.In order to rapidly identify the function of nucleotide variants in the Gh TFL1 gene and identify agronomically important mutants,this study established optimized multiple base editors in cotton,and used efficient base editor to perform high-density base mutations in Gh TFL1,so as to obtain a large number of intermediate mutant phenotypes.Of which,new genetic loci controlling ideal plant architecture of cotton were identified.In addition,to realize the rapid directional evolution of Gh TFL1 gene,new cotton germplasm with moderate plant height,short fruit branches,compact plant type and short growth period can be created.Based on the Gh TFL1 base mutation material,we found that Gh LSH6 is involved in the regulation of the florigenin and anti-florigenin system which provides important genetic information to study and develop an ideal plant architecture.The main results were as follows:1.Four adenine deaminases,Tad A6.3,Tad A7.8,Tad A7.9 and Tad A7.10,were screened for optimization in cotton.The four adenine deaminases were fused to two Cas9 variants（d Cas9 and n Cas9）to establish eight adenine base editors（ABE）.The A-to-G base editing was successfully induced in cotton endogenous genes.Comprehensive analysis showed that Gh ABE7.10n had the best editing activity with an editing efficiency of 64.9%.In addition,Gh ABE7.10n and Gh ABE7.10d were evaluated the editing specificity at DNA and RNA levels by 50X whole genome sequencing（WGS）and whole transcriptome sequencing（WTS）.At the DNA level,Gh ABE7.10 exhibited high specificity and did not produce off-target editing.At the RNA level,145 SNVs were generated caused by overexpression of Tad A7.10,which is negligible in plant gene function studies.Gh ABE7.10n edited materials were isolated and screened in the progeny,and one transgene-free line showing 28.2%editing efficiency was identified indicating that the mutations produced by Gh ABE7.10n can be faithfully inherited from T0 parental plants to T1 progenies.2.Based on the effective editing of the ABE system in cotton,the cotton adenine base editing system Gh ABE8e was further optimized based on the highly active Tad A8e to address the problems of narrow editing window and low efficiency of Gh ABE7.10n.The editing activity and characteristics were evaluated,and results showed that the editing efficiency of Gh ABE8e was 99.7%,with an average of 90.2%,which was 4-fold higher than that of Gh ABE7.10n.Gh ABE8e showed an activity window of A4-A10 and could edit three genomic target loci simultaneously.WGS and WTS analyses were performed on cotton material obtained by Gh ABE8e editing to comprehensively assess the specificity of Gh ABE8e at the DNA and RNA levels.WGS and WTS analyses showed that Gh ABE8e did not produce sg RNA-dependent off-target at both the DNA and RNA levels in cotton,and that the deaminase-induced SNVs at the DNA level and RNA level SNVs only accounted for 0.1%and 0.3%of the total SNVs.Similarly,analysis of transgene-free material from Gh ABE8e edited progeny confirm that the mutations produced by Gh ABE8e can be faithfully inherited from T0 parental to T1 progenies.3.In response to the reported CRISPR-Cas12a system that recognizes PAM as TTTV,the Gh ABE7.10-d Cas12a and Gh ABE8e-d Cas12a base editors were constructed based on Tad A7.10 and the highly active Tad A8e to extend the range of plant genome targeting.Gh ABE7.10-d Cas12a and Gh ABE8e-d Cas12a in the cotton genome showed that both could perform A-to-G base editing at the cotton A/T enrichment site.However,Gh ABE7.10-d Cas12a had lower editing activity with editing efficiencies ranging from 0.2%-0.5%,while Gh ABE8e-d Cas12a showed editing efficiency as high as 1.5%,effectively expanding the range of ABE editing.4.Based on the first-generation cytosine base editor in cotton（Cytosine Base Editing,CBE;Gh BE3）developed by our team,the cytosine deaminase r APOBEC1 was further optimized to enhance the editing efficiency and accuracy of CBE in cotton.A new Gh YE1-BE3-FNLS system was constructed after introducing point mutations in r APOBEC1,and targets were designed for cotton endogenous genes to detect their editing activity.The results showed that Gh YE1-BE3-FNLS could achieve C-to-T editing at the target site with an efficiency of 49.5%,which is an improvement compared to the editing efficiency of Gh BE3（up to 30.7%） previously developed by our group.5.To further enrich the precision editing system,the dual-base editor（Du BE）Gh CABE,Gh ACBE and Gh A3A-ABE were constructed based on the highly active Tad A8e and the optimized two cytosine deaminases YE1-r APOBEC1 and APOBEC3A in cotton,and their editing activities in the cotton genome were evaluated.The results showed that Gh A3A-ABE could achieve simultaneous editing of A and C in the same allele,and the editing efficiency of the A locus can reach 39.2%.Both Gh ACBE and Gh CABE produced one type of editing,A-to-G and T-to-G,respectively.6.According to promoter and gene coding region of Gh TFL1 gene,high-density sg RNAs were designed.Based on the highly active Gh ABE8e,the directional evolution of Gh TFL1 gene in cotton was studied.Through phenotypic identification,Ghtfl1^L86 showed shortened fruit branches and cluster flowering at the top of main stem,thus forming a determinate growth phenotype.The offspring showed a more obvious reduction in plant height,and a zero-type fruit branch with fruiting branches borne directly on the main stem.Ghtfl1^K53G+S78G exhibits a phenotype of two bells at one fruiting node,but still maintains shoot-indeterminate state of the inflorescence.The corresponding progeny also exhibited a shortened fruiting branch,changing from a dichotomous fruiting branch to a monotonous fruiting branch,and flowering in clusters at the top of the main stem.Further transcriptome analysis of Jin668,Ghtfl1^L86P and the CRISPR/Cas9 knockdown material of Gh TFL1 showed that both L86P base editing and CRISPR/Cas9 knockout of Gh TFL1（Ghtfl1）could resulted in upregulation of Gh AP1 gene expression and downregulation of Gh14-3-3 gene expression.It was further demonstrated by Y2H,LCI and Bi FC experiments that both Gh TFL1 and Ghtfl1^L86P could interact with Gh AP1 and Gh14-3-3,but the strength of the interaction between Ghtfl1^L86P and Gh AP1 and Gh14-3-3 was reduced,indicating that the L86P mutation only affected the spatial conformation of Gh TFL1 protein and did not cause loss of protein function.In addition,transcriptome data also screened for the transcription factor Gh LSH6 showing consistent down-regulated expression in Ghtfl1^L86P and Ghtfl1 material.The interaction between Gh TFL1 and Gh LSH6 was verified by Y2H,Bi FC,LCI and Pull-down.Overexpressing the Gh LSH6 gene in cotton exhibited vigorous nutritional growth,while CRISPR/Cas9 knockout of Gh LSH6 did not affect growth and development.This indicates that Gh LSH6 is involved in Gh TFL1-mediated processes that promote cotton nutritional growth,but has functional redundancy with other homologous proteins.Based on the above research results,the Ghtfl1^L86P and Ghtfl1^K53G+S78G mutant materials screened by directed evolution of Gh TFL1 through high-density base editing are of great value in cotton strain improvement and mechanization production.In summary,this study established and optimized multiple base editors for cotton,and used efficient base editor to perform high-density base mutations in Gh TFL1,from which new genetic loci were identified,creating new cotton germplasms with moderate height,shortened fruiting branches,compact architecture and shortened fertility,and achieving rapid targeted evolution of the Gh TFL1 gene.The rapid evolution of the Gh TFL1 gene will be of great value for cotton strain improvement and mechanization.