Recent progress and future prospect of cotton genome editing

Recently, we successfully knock out several cotton genes by CRISPR-Cas 9 system with an average 65-85% efficiency. We also perform the whole genome sequencing to investigate the off-target in the CRISPR-Cas9 edited cotton plants. The results showed that of 2000+ potential off-targets sites (allowing ≤ 5 mismatches within the 20-bp sgRNA and 3-bp PAM sequences), the WGS data revealed that only a few （less than 10) are bona fide off-target mutations which suggested that CRISPR/Cas9 system is highly specific for the editing of genes of polyploid plant species.Then, we further developed a high-throughput genome editing system in cotton. A sgRNAs library (containing 1100 sgRNAs targeted to 600 independent genes ) was constructed and cloned into the CRISPR-Cas 9 vector. By this way, we can edit several hundred target genes in one transformation. This system need a very high efficient cotton genetic transformation system to generate thousands of regenerated plantlets by somatic embryogenesis. The data we obtained recently suggested that this system works pretty well in cotton. In addition, we start to work a new genome editing system in cotton by using a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method. Cpf1, a single-strand RNA-guided endonuclease of the class 2 CRISPR-Cas system that cleaves targeted DNA with features distinct from those of Cas9. For example, preferring a T-rich protospacer-adjencent motif (PAM) and cutting in staggered ends. This system has several advantages over the CRISPR-Cas 9 system including small Cas protein size, generating sticky end after cleaving the DNA, cutting the RNA target and lower off-target risk. Our data showed that CRISPR-Cpf1 system works very well with high efficiency and accuracy. At the same time, we developed a base editing system for cotton by fusion of nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) with activation-induced cytidine deaminase (AID). Guided by single guide (sg)RNAs, dCas9-AID will directly transfer cytidines or guanines (C or G) to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9- AIDx converted targeted cytidines (C) especifically to thymines (T), creating specific point mutations. By targeting GhCLA1 with dCas9-AID, we efficiently created known and new mutations showing various phenotype . Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution in cotton.