Targeted Editing Of Maize Photoperiod Sensitive Genes By CRISPR/Cas9 Technology

Corn is the main food crop widely grown around the world.Maize originated from the tropical southwestern part of Mexico and was domesticated from teosinte.It is a short-day crop which is sensitive to photoperiod and limits the application of tropical germplasm resources.The gene editing technology based on CRISPR/Cas system provides a possibility to rapidly improve the sensitivity of corn to photoperiod and improve the flowering stage of maize.In this study,the three genes of photoperiod sensitive genes ZmCCT10,ZmCCT9 and ZmGhd7 were used as targeting genes,and the above three genes were modified by CRISPR/Cas9 technology and embryo-specific DsRed fluorescence screening system,and the target genes of four corn materials were improved.The main results are as follows:1.Four maize materials,KN5585,CML312SR,LCL-1 and LCL-2,were used as receptors.Among them,KN5585 was a stable transforming receptor,and CML312SR,LCL-1 and LCL-2 were pre-modified late maturity receptors.The sequencing results of the four receptor material target regions indicated that the target region sequences of the three target genes ZmCCT10,ZmCCT9 and ZmGhd7genes were conserved.This result lays the foundation for sequence analysis for the design of subsequent site-directed mutagenesis.2.This study designed a single-molecule guide RNA（sgRNA）to edit the ZmCCT10,ZmCCT9 and ZmGhd7 genes,and constructed a stably transformed gene-editing targeted gene knockout vector CCT-CPD with Ubi Promoter driven Cas9 expression cassette,U6 promoter driven sgRNA expression cassette,CaMV 35S（enhanced）promoter driven Bar screen marker expression cassette and P3896promoter driven DsRed fluorescent selection marker expression cassette（embryo specific expression）.3.The maize inbred line KN5585 was used as a receptor,and three expression vectors of CCT-CPD,CPD-2 and CPD-3 were simultaneously transformed by Agrobacterium transformation method（this study only introduces CCT-CPD expression vector）.There were obtained stability 42 transforming receptors,and 25 transformants containing CCT-CPD expression vector,the proportion was 59.5%,of which 23 transformants contained only CCT-CPD expression vector,accounting for 54.8%of positive transformants,accounting for CCT-CPD transformants 92.0%.The transgenic elements of the transformants obtained by transforming the expression vector using the Agrobacterium transformation method can be stably inherited by molecular identification and fluorescence identification of the T₀generation and the corresponding T₁ generation positive plants.4.The identification methods of receptor materials are as follows:（1）Sanger sequencing method,using Sanger sequencing method to identify mutations of three genes;（2）PCR/RE method,different from ZmCCT9 and ZmGhd7 genes,The target of ZmCCT10 gene has a recognition site for common restriction endonucleases Bst XⅠnear 3 bases upstream of PAM.Before sequencing to identify genotypes,it can be quickly screened for unmutated（wild type）,heterozygous mutations andhomozygous or biallelic mutations by enzyme digestion;（3）Fluorescent screening,transformant screening system constructed using embryo-specific promoter and DsRed fluorescent protein combined with hand-held fluorescent lamps for easy harvesting of corn seeds（KN5585 selfing seeds and the F₁ obtained by crossing the late maturing material with the KN5585-positive strain）were screened for the negative seeds and the positive seeds.5.The sgRNA designed is identical to the target site of ZmGhd7 gene,and has a base difference with the target sites of ZmCCT10 and ZmCCT9 genes.After analysis,the forms and mutations of the mutation types of these three genes are found.The rates are different:（1）17 samples have the target mutation of ZmCCT10 gene,and the mutation site is 3-4 bases upstream of PAM,mainly in the form of+1 bp mutation.The results were consistent that the mutation type and enzyme of these mutants,that is,two samples were heterozygous mutations,15 samples were homozygous or biallelic mutations,and the mutation rate of ZmCCT10 gene was 68.0%（17/25）;（2）ZmCCT9 occurred in 3 samples.The target mutation of the gene,the mutation type is mainly the large fragment knockout,the mutation rate of ZmCCT9 gene is 12.0%（3/25）;（3）11 samples have the target mutation of ZmGhd7 gene,knock out with large fragment and the mutant form of+（1-2）bp or-（1-2）bp exists,and the mutation rate of ZmGhd7 gene is 44.0%（11/25）,and the editing activity is currently more obvious in the T₀ generation.6.The seeds obtained by self-crossing or cross-testing of T₀ generation obtained positive and negative seeds through the fluorescence screening system,and heterozygous mutants were identified in the negative materials,which facilitated the subsequent rapid acquisition of homozygous transgenics mutant.7.F₁ was obtained by hybridization,ie,late maturing material（♀,female parent）and KN5585positive strain（♂,male parent）.A late-maturing material containing effective gene editing elements（seeds with DsRed fluorescence expression in embryo）was obtained by fluorescence screening system.In summary,the combination of CRISPR/Cas9 technology and embryo-specifically expressed DsRed fluorescence screening system can effectively target the editing of three genes and rapidly obtain the corresponding mutants.