**11. CRISPR Cas system for crop improvement in cotton**

In the last decade, there is a revolution in the field of genome modification and continuous advancement in the targeted genome modification technologies. Genome editing tools like zinc-finger nucleases (ZFN), transcription activator-like effectors nucleases (TALENs) were extensively used before the advent of CRISPR Cas9 technology. These ZFN and TALENs technologies didn't become as popular as that of CRISPR Cas9 due to low efficiency, low specificity, low engineering feasibility and low design simplicity. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) system is the latest gene-editing technology, which has the power to alter the DNA or the code of life. Scientists predict that CRISPR has enormous potential for the next green revolution by 2050. To date, the CRISPR/Cas9 system has been successfully applied to efficient genome editing not only in model plants species but also in crop plant species. Many institutions and several groups all over the world are studying the feasibilities of the CRISPR/Cas9 system in cotton (*Gossypium hirsutum* L.). Successful use of CRISPR/ Cas9 system in cotton still relies on Agrobacterium-mediated transformation and tissue culture, a genotype-dependent and low-efficiency process, but it provides a powerful tool for cotton functional genomics as it seems to be more efficient than RNA interference (RNAi) and virus-induced gene silencing (VIGS) in terms of knocking out the function of target genes [168]. The applications of the CRISPR/ Cas9 system in cotton is been presented in **Table 6**. Chen and coworkers [169] demonstrated for the first time that the CRISPR/Cas9 can be used for advanced functional genomic research in cotton through targeted mutagenesis of two endogenous genes [Cloroplastos alterados 1 (GhCLA1) and vacuolar H + -pyrophosphatase (GhVP)]. Many factors influence the efficiency of the CRISPR/Cas9 system to obtain high mutation rates. [174] studied the sgRNA expression and mutagenesis efficiency by taking the endogenous U6 promoter over the existing one (Arabidopsis AtU6-29 promoter). Improved mutagenesis efficiency (4 to 6 times) was obtained by the use of an endogenous U6 promoter to drive the sgRNA expression. This study provided a fast and effective method to validate sgRNA mutagenesis efficiency in cotton using CRISPR/Cas9. Gao and coworkers [168] analyzed the nature of mutations induced by the CRISPR/Cas9 system through transient expression study of two genes Translation elongation factor 1 (GhEF1) and Phytoene desaturase (GhPDS) in cotton. The CRISPR/Cas9 system has been used for multiple sites targeting and simultaneously editing of multiple genes. Wang and his colleagues [171] successfully utilized the CRISPR/Cas9 system in allotetraploid cotton and accomplished multiple sites genome editing by targeting the exogenously transformed gene Discosoma red fluorescent protein2 (DsRed2) and an endogenous gene Cloroplastos alterados 1 (GhCLA1).

CRISPR/Cas9 has been used to edit a couple of agronomically important cotton genes, such as the genes involved in fiber development (GhMYB25-like A and GhMYB25-like D) [170] and a gene encoding arginase (ARG) for the increased lateral root formation [160]. Zhu and his colleagues [172] demonstrated the high


**Table 6.**

*The applications of the CRISPR/Cas9 system in cotton (*Gossypium hirsutum *L.).*

#### *Plant Breeding - Current and Future Views*

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editing efficiency of the CRISPR/Cas9 system in cotton by targeting-ALARP, a gene encoding an alanine-rich protein that is preferentially expressed in cotton fibers. CRISPR/Cas9 knockout of the Cotton Gland Formation (CGF3) gene resulted in a glandless phenotype in cotton. Gao and coworkers [168] confirmed the important role of Cotton Gland Pigmentation 1 (CGP1) in gland biology through CRISPR knockout of CGP1. Decreased accumulation of gossypol and of related terpenoids was observed in the CRISPR knockout plants. The above successful studies indicate that the CRISPR Cas9 system can further be effectively utilized in the functional genomics of cotton research. However, there are some limitations of the CRISPR/Cas9 system, including off-target effects, difficulties in PAM (protospacer adjacent motif) sequence selection for fewer potential target sites, and difficulties in generating homozygous mutations in the offspring [175–177]. Therefore, there is a lot of scope for the modification of the CRISPR Cas9 system or finding new alternative CRISPR systems. Zeng and co-workers [178], for the first time, established an efficient CRISPR/LbCpf1 system to expand the scope of genome editing in allotetraploid cotton by targeting the cotton endogenous gene Cloroplastos alterados (GhCLA). In addition to CRISPR/Cas9 & CRISPR/LbCpf1 system, a new effector with a single nuclease domain, a relatively small size, with low-frequency off-target effects, and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1) [179]. CRISPR/Cas12b is a heat-induced system which requires a temperature ranging between 40 and 55°C for effective cleavage, when the temperature is lower than 40°C, cleavage cannot be accomplished [180, 181]. Recently the manipulation of the Cloroplastos alterados (GhCLA) gene in cotton plants using AacCas12b has been successfully established with no off-target effects. This system is ideal for plant species that can tolerate temperatures above 40°C, such as cotton that can grow well at temperatures reaching 45°C [171]. Some researchers are deactivating one or both of the Cas9's cutting domains and fusing new enzymes onto the protein. Cas9 can then be used to transport those enzymes to a specific DNA sequence. In one example, the Cas9 is fused to an enzyme, a deaminase, which mutates specific DNA bases eventually replacing cytidine with thymidine. [182] developed a new base editor system (GhBE3) consisting of a cytidine deaminase domain fused with nCas9 and uracil glycosylase inhibitor (UGI), for use in allotetraploid cotton, and obtained high base-editing efficiency with no detectable off-target effects. From all the above studies, it is indicated that CRISPR/Cas9 and its alternatives are potential gene-editing tools which would be superior to RNAi for cotton functional genomics. In future, this technology will have much scope for targeting tolerance to sucking pests, increased fiber yield and improved fiber quality traits in cotton.
