Study Of CRISPR/Cas9-mediated GhCLA1 And GhVP Gene Editing In Cotton

As an important resource of natural fbre,plant protein and edible oil,cotton has tremendous economic value and social value.The widely cultivated cotton cultivars are allotetraploid species.Although the allotetraploid genome of upland cotton is large and complicated,which bring the challenges for cotton genetic improvement,cotton investigators never stop updating their biotechnology arsenal to more effectively and accurately dissect cotton genes functions.Owing largely to its simplicity,efficiency,flexibility and unexpensive,the CRISPR/Cas9 system as a newest genome editing tool,has been recently proven to be effective for targeted gene editing in a wildly range of organisms.In this study,we targeted mutagenesis of GhCLA1 and GhVP gene in cotton(Gossypium hirsutum L.)and analyzed the gene editing status in cotton protoplasts and transgenic cotton plants using the CRISPR/Cas9 system.Evaluation using restriction-enzyme-PCR(RE-PCR)assay and sequence analysis detected off-target mutations in transgenic cotton plants.Specific plant expression vectors of CRISPR/Cas9 system for cotton were constructed and detected their effective of gene editing in cotton protoplasts.We also detected the effective of the CRISPR-Cas9 ribonucleoprotein complexes in targeting mutagenesis of GhVP gene in vitro and in vivo.The main results of this study are as follows:1.We examined the expression levels of GhCLA1 and GhVP gene by semi-quantitative PCR(RT-PCR)and found that their expression levels were high.This results indicated that GhCLA1 and GhVP gene can be as the target genes to detect the effective of CRISPR/Cas9 system in cotton.Five sgRNAs of GhCLA1 and GhVP gene were designed with the online software CRISPR-P and the upland cotton genome database,respectively.Finally,GhCLA1-sgRNA5 and GhVP-sgRNA4 were chose to target mutagenesis in cotton after detecting the effective of all ten sgRNAs in cotton protoplasts.2.The constructed plant expression vectors of CRISPR/Cas9 system were transferred into cotton protoplasts and genomic DNA was isolated.To detect mutations in cotton protoplasts,the genomic DNA was digested with restriction enzymes(PstI for GhCLA1-sgRNA5 and Hinf I for Gh VP-sgRNA4).After digestion,the target genes were amplified with gene-specific primers and high fidelity DNA polymerase,and the PCR fragments were ligated to a vector for sequencing.Mutations in these two genes were detected in cotton protoplasts.Most of the mutations were nucleotide substitutions,with one nucleotide insertion and one substitution found in GhCLA1 gene and one deletion found in GhVP gene.3.The above two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation and we verified the presence of the Cas9 gene in transgenic plants;22 lines for the GhCLA1-sgRNA5 target and 18 lines for the GhVP-sgRNA4 target were obtained.Then,we identified the target gene mutations in these transgenic lines by RE-PCR assay.PCR products of these two genes were digested with a specific restriction enzyme,and undigested bands were observed.The undigested bands from the RE-PCR assay were cloned and sequenced to confirm the mutations.Sequence analysis indicated that most mutations were nucleotide deletions,while one mutation of GhCLA1 gene was a nucleotide insertion.The mutation rates of these two genes were 47.6–81.8% by directly sequencing the PCR products of the target genes in each transgenic plants.4.We analyzed the potential off-targets of GhCLA1-sgRNA5 and GhVP-sgRNA4 in transgenic cotton plants which contained the verified GhCLA1 or GhVP gene mutations.After searching on the CRISPR-P website and the G.hirsutum genome database with the GhCLA1-sgRNA5 and GhVP-sgRNA4 target sequences,22 potential off-target sequences of GhCLA1-sgRNA5 and 19 sequences of GhVP-sgRNA4 with the PAM motif were identified.These identified off-target sites were verified by RE-PCR assay and sequence analysis.We managed to assay 30 of the identified off-target loci,and none of these potential off-target loci showed evidence of a CRISPR/Cas9 system-induced mutation.These results indicated that the CRISPR/Cas9 system had high specificity for targeted mutagenesis in cotton.5.Specific plant expression vectors of CRISPR/Cas9 system for cotton were constructed and detected their effective of gene editing in cotton protoplasts.Each of these vectors contains an upland cotton codon-optimized Cas9 gene and a cotton U6 promoter.We examined the effective of cotton U6 gene promoters GhU6-1P,GhU6-2P,GhU6-3P and GhU6-6P in cotton protoplasts with the GhVP gene as a target.After detected the mutations,we found that most of the mutations induced by Gh U6-1P,GhU6-2P and GhU6-3P were nucleotide deletions,with a few mutations were nucleotide substitution/insertion.There was no mutation induced by GhU6-6P found in GhVP gene.6.We also detected the effective of the CRISPR-Cas9 ribonucleoprotein complexes(RNP)in targeting mutagenesis of GhVP gene in vitro and in vivo.Firstly,we examined the effective of the CRISPR-Cas9 ribonucleoprotein complexes in inducing DSB in vitro.The PCR fragments of GhVP gene were digested completely by the RNP complex.Then the RNP complex was delivered into cotton protoplasts and results showed that most of the mutations were nucleotide substitutions,with a few mutations were nucleotide deletions/insertions.