Functional Characterization And Whole Genome Sequencing For Off-target Analysis Of CRISPR/Cas9-edited Cotton Plants Of GhMYB44,GhAP2,and GhARC Genes

Cotton has been considered as one of the most essential and crucial crops all around the world.It is the foundation of global economy and backbone of the textile industry due to its massive value of fibre and other by-products which are also part of paper,livestock feed,fertilizers,seed oil,and other consumer products.Moreover,it serves as an ideal plant for various kinds of biological studies,such as polyploidization,genome evolution,and single-celled biological processes.MYB transcription factors comprise an MYB domain consisting of up to four imperfect repeats of a 52 amino acid motif.MYB transcription factors play various vital roles in guard cell signal transduction.The At MYB44 is a multifaceted transcription factor which mainly plays a crucial role in association with the ethylenesignaling pathway in Arabidopsis thaliana.The APETALA2/Ethylene-Responsive Factor(AP2/ERF)is a superfamily of plant-specific transcription factors(TFs)which plays a vital role in the regulation of diverse plant responses,from plant organ development to response to various environmental and biotic stresses.The NB-ARC domain is a type of STAND(Signal Transduction ATPases with Numerous Domains)protein which regulates programmed cell death,and it binds to ATP for hydrolyzation and induces downstream resistance signaling.Genome editing(GE)has modernized the world by editing genomes of multiple living organisms.Various kinds of GE tools have been explored for editing from simple to complex genomes,and the most recent and exciting one is the CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)which has revolutionized the world of science from its applications.CRISPR/Cas9 is an RNA guided endonuclease which targets the DNA explicitly through nucleotide base pairing.Due to its high efficiency,easy to use and accuracy,the CRISPR/Cas9 system has been considered and accepted as one of the most powerful technologies for GE in all kinds of living organisms including crops,fruits,and vegetables for gene modifications,knockouts,insertions,activation and the repression of target genes as compared to other traditional random mutagenesis methods.However,ourunderstanding of Cas9 specificity is very limited in Cas9-edited plants.To study the functions of Gh MYB44,Gh AP2,and Gh ARC genes under insect stress conditions,the CRISPR/Cas9 was used to knockout these genes.To check the corresponding results,the Gh MYB44 was overexpressed in cotton using overexpression technique.The subcellular localization revealed that Gh MYB44 is nuclear-localized.The expression analysis revealed that Gh MYB44,Gh AP2 and Gh ARC response to insect stress.The editing of Gh MYB44,Gh AP2,and Gh ARC genes by CRISPR/Cas9 into the cotton genome was confirmed by PCR,RT-PCR,Sanger sequencing,Bar Codes,T7E1,and Hi-TOM.Mutations were observed in the form of additions and deletions of the nucleotides with an 80–100% frequency of gene editing.Similarly,for the overexpression lines of Gh MYB44,its integration was confirmed by PCR,RT-PCR,and Southern blot.All knockout lines of Gh MYB44,Gh AP2,and Gh ARC genes displayed smaller size of flowers,short height,and pale green leaf color than WT,while overexpression lines showed large-sized flowers,and higher height than CRISPR/Cas9 mutated lines while overexpression lines were observed to be shorter than the WT.Furthermore,to identify on-and off-target mutation in an edited crop,we described whole genome sequencing(WGS)of 14 Cas9-edited cotton plants targeted to three genes,three negative(Ne)control and 3 wild-types(WT)plants.In total,4,188 – 6,404 unique single nucleotide polymorphisms(SNPs)and 312 – 745insertions/deletions(indels)were detected in 14 Cas9-edited plants compared with WT,negative and cotton reference genome sequences.Since the majority of these variations lack a protospacer-adjacent motif(PAM),we demonstrated that the most variations following Cas9-edited are due either to somaclonal variation or/and preexisting/natural variation from maternal plants,but not off-target effects.Of a total of4,413 potential off-target sites(allowing ≤ 5 mismatches within the 20-bp sg RNA and3-bp PAM sequences),the WGS data revealed that only 4 are bona fide off-target indel mutations,validated by Sanger sequencing.Moreover,inherent genetic variation of WT can generate novel off-target sites and destroy PAMs,which suggested great care should be taken to design sg RNA for the minimizing of off-target effect.Thesefindings indicated that the CRISPR/Cas9 system is highly specific for cotton plants.This study will expand our knowledge in understanding of insect stress response mechanism.