Functional Analysis Of P-CR Region Mutation Of Rice OsCESA4 And OsCESA7 Using CRISPR/Cas9

Genetic modification of plant cell walls has been considered for largely enhanced biomass enzymatic saccharification in bioenergy crops.In this study,we identified total seven types of non-frameshift mutants of Os Ces A4 and Os CEs A7 genes using CRISPR/Cas9-editing technology,and then characterized four types of mutants by observing plant growth,detecting Os Ces As transcription levels and examining cell wall composition and cellulose features.Finally,this study determined biomass enzymatic saccharification under acid and alkali pretreatments in the mutants.The major results were described below:1.This study initially selected four T₁ non-frameshift homozygous mutants of OsCESA7?OsCESA7-PCR-1-2/L337del?;OsCESA7-PCR-2-6/E416del;OsCESA7-PCR-2-8/E415_Q421del;OsCESA7-PCR-4-1/F385_V386del),one heterozygosis mutants?OsCESA7-PCR-4-4/F385del?and two T0 non-frameshift heterozygosis mutants of OsCESA4?OsCESA4-PCR-5-16 /E420 del and E420_G422del?and OsCESA4-PCR-5-17?L418_G419del?.2.Based on DNA sequence analysis,OsCESA7-PCR-1-2 mutant showed Leu?AA337/amino acid location?loss of CESA7;OsCESA7-PCR-2-6 mutant had Glu loss?AA416?;OsCESA7-PCR-2-8 mutant lost total seven amino acids?Glu?Glu?Gly?Trp?Val?Met?Gln?from AA 415;OsCESA7-PCR-4-1mutant lost two amino acids?Phe,Val?from AA 385.3.Compared to control cultivar?NPB?,three T₁ OsCESA7 non-frameshift homozygous mutants?OsCESA7-PCR-1-2,OsCESA7-PCR-2-6 and OsCESA7-PCR-2-8?exhibited brittle culm phenotypes,with reduced mechanical strength and seed setting rates.4.As a further comparison with NBP,three brittle culm mutants showed significantly reduced cellulose levels and features.For instance,the mutants had reduced cellulose contents by 7.4%-18.5%,cellulose degree of polymerization?DP?by 22%-26%,and crystallinity index?Cr I?by 22%-39%.In addition,the secondary cell wall thickness of sclerenchyma cells were reduced by 41% and 68% in OsCESA7-PCR-2-6 and OsCESA7-PCR-2-8,respectively.5.Under 2.0% H2SO4 or 0.5% Na OH pretreatment,the brittle culm mutants exhibited much enhanced biomass enzymatic saccharification,compared to NPB.In particular,the mutants showed either increased hexoses yields by 11%-27% released from enzymatic hydrolysis after 2.0% H2SO4 pretreatment,or total sugars yields by 9.0%-13% released from both pretreatment and enzymatic hydrolysis.In addition,the OsCESA7-PCR-2-6 and OsCESA7-PCR-2-8 mutants showed increased hexoses yields by 8.5% and 9.5% upon 0.5% Na OH pretreatment.6.Despite the OsCESA7-PCR-4-1 mutant and NPB showed a similarity of plant height,cellulose contents and Ces A protein levels,the mutant had either increased cellulose DP by 5.0% or reduced Cr I by 9.6%,leading to decreased biomass saccharification by 6.8% and 9.4% after 2.0% H2SO4 and 0.5% Na OH pretreatments.7.Frameshift mutation of OsCESA4 or OsCESA7 led to strong phenotypes of dwarfism and sterility in the mutants.