**6. Genome editing: a revolutionary tool in breeders' toolkit**

Genome editing has enabled breeders to precisely add or delete any DNA sequence in the genome and has shown enormous potential to revolutionize the crop improvement in this very decade [70, 77]. Some approaches like transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have been in the game for more than 2 decades. However, type II clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system from *Streptococcus pyogenes* [78] developed in last decade has been most versatile tool in breeder's toolkit to introduce desirable or novel traits and accelerate development of climate smart crop varieties.

Usually, a custom-made guide RNA (gRNA) along with Cas9 nuclease is delivered into plant protoplast, where Cas9 produces double strand break (DSB) 3 bp upstream of the NGG motif (protospacer adjacent motif-PAM sequence) [78]. Cellular repair machinery through non-homologous end joining (NHEJ) can lead to frameshift mutation causing a knock-out. Otherwise, a donor DNA template can be provided for precise genetic knock-in through homologous recombination (HR). CRISPR/Cas9 was initially used to disrupt genes related to disease susceptibility in crops such as OsERF922 gene disruption in rice for blast resistance [79] and loss of function in susceptibility gene TaMLO for powdery mildew resistance in wheat [80]. Genome editing has also been used to tackle some abiotic stresses in staple crops like a promoter of a gene AGROS8 was replaced with a stronger one to impart drought tolerance in maize [81].

Due to changing climates, it may be quite beneficial for the farmers to have early maturing varieties, which enables plants to complete crucial developmental periods before the onset of a stress. It has been achieved by disrupting a flower repressing gene SP5G to develop early maturing tomato varieties [82]. For instance, developing climate rice to grow in diverse climates, generally desirable traits are cold, heat and drought tolerance at seedling and reproductive stages [83]. Secondary characters like root and flag leaf traits can be useful to generate cultivars with improved drought and heat tolerance [84]. Here, CRISPR tools could prove to be of great value for exploration of the candidate genes from CWR (*O. officinalis*, *O. nivara* and *O. glaberrima*) for abiotic stress resistance [85].

Genome editing has also huge potential to accelerate the domestication of novel crops form CWR or minor crops with valuable traits for coping with extreme climatic events. This would allow the editing of key genes for domestication in potential new crops for rapid enhancement of currently limited gene pools to maximize the use of germplasm adapted to climate change. Also, multiplexing of CRISPR

systems for simultaneous editing of multiple genetic loci can boost the speed and efficiency manifolds. However, there are a number of shortcomings in this approach including off target effects [86], low efficiency of HR, restrictive PAM sequences and regulatory concerns, which paved the way for advent of more sophisticated technologies like DNA free genome editing, base editing and prime editing.

#### **6.1 DNA free genome editing (DFGE)**

Conventional genome editing using recombinant DNA (rDNA) leads to random host genome integration and can generate undesirable genetic changes or DNA damage [87], along with concerns over genetically modified organism (GMO) regulations with introduction of foreign DNA [88]. DFGE takes care of such critical issues along with reduced risk of off-targets. Initially, it was successfully deployed in rice and tobacco with transfection of protoplast with CRISPR-Cas9 ribonucleoprotein (RNP) [89]. Also, a particle bombardment mediated DFGE approach has been developed in wheat and maize [90, 91].

#### **6.2 Base editing**

It is evident that a single base change can cause variation in the elite traits [92], so there required an efficient technique to cause precise and efficient point mutations in plants. CRISPR-Cas9 driven base editing is new approach which accurately transform one DNA base to another without repair template [93]. E.g. Cytidine deaminases convert cytosine (C) to uracil (U), which is treated as thymine (T) in subsequent DNA repair and replication, thus creating C•G to T•A substitution. It has been utilized in wheat, maize and tomato [94] and can be quite useful for gene functional analysis and therefore can assist breeding for better stress adapted varieties.

#### **6.3 Prime editing**

Another latest milestone in this genome engineering era called prime editing allows introduction of all known 12 base to base conversions in addition to mutations such as insertions and deletions using prime editing guide RNA (pegRNA) [95]. This promising approach opening up numerous possibilities for effectively targeting and modifying desirable genome sequences to accelerate functional genomics and introduction of genes for adaptation to diverse climates can boost breeding for climate smart crop varieties in near future [96].

In this rejuvenated plant mutagenesis breeding era, genome editing can be used in functional genomics for the identification of candidate genes for climate related agronomic, physiological and phonological traits, which can be exploited for crop improvement in adaptation to changing climate. Despite having enormous potential and real world applications of genome editing technologies, the regulatory and ethical concerns may limit it, as happened in a few European countries. In the nutshell, genome editing in complementation with conventional plant breeding can be adopted to develop and deploy climate smart crop varieties in the farmers' fields.
