DNA-free genome editing is a novel and speedy technology in biological sciences that became a method of choice as it is a means of precise genomic modification without disturbance in genome of an organism. It opened the possibility to generate genetic modified organism called as non-GMO in classical biology and biotechnology
(Malzahn et al., 2017; Wolter and Puchta, 2017;
Mao et al., 2019). Conventional methods of gene editing include RNAi, zinc finger, TALENs
etc. Breakthrough in gene editing occurs with the avent of RNA-guided endoDNAses followed by identification of Cas9 system incurred from immune system of bacterial paved novel path of targeted gene modifications. CRISPR/Cashas revolutionized the world of gene editing with surprising success in crop improvement through gene editing and alteration
(Arora and Narula, 2017).
CRISPR-Cas9 can be extended as clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9
. CRISPR is DNA sequences occur in the genomes of prokaryotic organism in reply of infection of phages that invades bacteria
. Cas genes are essential for function of CRISPR and provide immunity in response to attack of viruses and plasmids in bacteria and archaebacteria
(Barrangou and Marraffini, 2014;
Sorek et al., 2013; Barrangou, 2013). CRISPR with Cas9 enzymes assemble as CRISPR-Cas9 system which is widely utilized to edit genome of organism
(Barrangou et al., 2007). CRISPR is transcribed into pre-crRNA and cas genes becomes active and functional to express as cas proteins which help in processing of pre-crRNA into mature crRNA. Target nucleic acid is recognized and destroyed combinedly by crRNA and cas proteins
(Koonin and Krupovic, 2014;
Rath et al., 2015; Kumari et al., 2021).
CRISPR-Cas9 genome editing needs single guide RNA (sg RNA) that guide the Cas9 endonuclease to specific region of the genomic DNA, resulting in double stranded nicks in the target DNA. The CRISPR-Cas9 technique cleaves specific nucleotides
via complementary sequence with Cas9 protein and sgRNA
(Peng et al., 2016). Cas9 protein composed of two nucleic acid binding site like a large recognition (REC) lobe and a small nuclease (NUC) lobe that are linked by a helix bridge. REC controls Cas9 specific function and NUC integrate two nucleases, RuvC and HNH and protospacer adjacent motif (PAM). The presence of PAM flanking the target sites is required for target recognition and R-loop formation
(Jinek, et al., 2012; Nishimasu et al., 2014; Anders et al., 2014; Jiang and Doudna, 2017).
Cas9 have endonuclease activity to produce double-strand breaks (DSBs) in target DNA during bacterial immune response
(Mali et al., 2013; Bao et al., 2019). DSBs can be repaired by non-homologous end joining (NHEJ) and homology directed repair (HDR) process. NHEJ uses DNA ligase IV to re-join the broken ends results in insertion or deletion mutations (indels) and can resulting in frameshift or introduction of a premature stop codon. HDR repairs the DSBs based on a homologous complementary template and results in a perfect repair. HDR is generally used for gene knock-in in plants
(Schiml et al., 2014). A transgenic DNA can be generated by providing a donor DNA in Trans and the double strand break will be repaired by the host cell. This pathway is useful in generating loss-of-function/ knockout of the gene of interest
(Costa et al., 2017) (Fig 1).
This technique has been applied in many species with diverse goal Gene manipulation through DNA-free CRISPR/Cas system have been targeted in commercial crops in recent years.
viz.,
Nicotiana benthaminiana,
Capsicum annuum, wheat, maize, rice, potato, soybean, banana,
brassicaceae, lettuce, tobacco
etc. (Andersson et al., 2018; Murovec et al., 2018; Gonzalez et al., 2019; Hu et al., 2019; Park et al., 2019; Toda et al., 2019; Kim et al., 2020; Ma et al., 2020; SantAna et al., 2020; Wu et al., 2020). Various approaches of Cas9/gRNA delivery have been utilized to attain editing
via DNA-free system as for example, CRISPR/Cas delivering as ribonucleoprotein, virus-mediated delivery of CRISPR/Cas,
Agrobacterium tumefaciens delivery for Cas9.
CRISPR/Cas delivery as ribonucleoprotein
Foremost important method for DNA-free gene editing in plants are assembly of CRISPR-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing. They are easy, precise and convincing technique for genome editing in crop plant which involves interaction between Cas9 and gRNA. Cas9 is expressed and purified in bacteria known as
Escherichia coli and gRNA is generated
via transcription or synthesized chemically. Ribonucleoprotein assembly and nanoparticles are acquired for transformation process
(Park and Choe, 2019;
Wang et al., 2019). Ribonucleoprotein and nanoparticles assembly are inserted in plant tissues
via protoplast fusion or particle gun methods. Sometimes, PEG- mediated transfer and lipofections and electroporation are also been utilized
(Liang et al., 2018; Liu et al., 2020).
Delivery of CRISPR/Cas as virus-like particles
Utilizing virus like particle for DNA-free genome editing by means of CRISPR/Cas system in plants has major limitations. Positive-strand RNA and DNA viruses possess limited capacity for foreign DNA insert replication renders to deletion and loss of sequence due course of replication. Furthermore, editing of small genome like CRISPR gRNA, zinc-finger nuclease, meganuclease
etc. is easy and convenient with viral vectors. However, sequence size restrictions would pose viral infection with this large system difficult as size ofCRISPR/Cas system is more than 5.0 kb
(Marton et al., 2010; Honig et al., 2015; Cody and Scholthof, 2019;
Ariga et al., 2020; Liu and Zhang, 2020). Utilization of virus vectors for insertion of CRISPR/Cas9 in plants was applied in
Nicotiana benthamiana and potato Virus X was employed to deliver Cas9 and gRNA to attain productive DNA-free genome edited plants. Likewise, in wheat, barley stripe mosaic virus was used to deliver guide RNA
(Hu et al., 2019; Ariga et al., 2020; Ma et al., 2020).
Agrobacterium based delivery of CRISPR/Cas
Agrobacterium based delivery of CRISPR/Cas cassette is method of choice of transformation in plant species. This method has been widely implicated in numerous plant species where leaf, flower, callus were used as a targeted explants usingT-DNA as integral part of Ti-plasmid consisting of Cas9 and the gRNA
(Gelvin, 2003;
Sandhya et al., 2020). Agrobacterium contamination could be applied for generation of Cas9 and gRNA without antibiotics which prevents production of DNA-free plants and integration of T-DNA in genome of plant species. Transgenic-free transgenic plant in tobacco was procured by targeting phytoene desaturase (PDS) gene using this strategy
(Chen et al., 2018; He and Zhao, 2020). This method has advantages as compared to CRISPR/Cas9 ribonucleo proteins and particle gun, though it has limitations to not applicable to all plant species. Acetolactate synthase gene was edited by cytidine editor by Agrobacterium infection in potato. Similarly, in tomato chlorsulfuron resistant plants were produced by modification in acetolactate synthase gene
via point mutation through Agrobacterium mediated delivery
(Danilo et al., 2019; Veillet et al., 2019).
Crop improvement through DNA-free genome editing via CRISPR/Cas
Rice
Several examples for trait improvement after utilization of CRISPR based genome editing tools are illustrated here. For example, rice genes, phytoene desaturase, betaine aldehyde dehydrogenase and mitogen activated protein kinase conferring stimuli for various abiotic stress were modified by CRISPR/Cas9 mediated genomic modification. Genes, OsDERF1, OsPMS3, OsEPSPS, OsMSH1, OsMYB5 responsible for drought tolerance were edited through inducing targeted mutation in rice. Disease susceptibility gene, OsSWEET13 has been knockout leads to bacterial blight resistance Indica rice. Annexin gene has been deactivated by CRISPR knocked out to confer cold stress in rice
(Shen et al., 2017; Kumari and Kumawat, 2021).
Multiplexed plant genome editing and transcriptional regulation has been demonstrated in
Arabidopsis and rice and made easier by CRISPR/Cas9.Knock-out of OsSEC3A gene increases salicylic acid content which caused resistance against blast disease in rice. Grain weight (GW) were upgraded by disruption of GW2, GW5 and GW6 genes, negative regulators of grain shape in rice is proof that grain weight is affected by grain shape. Grain size 3 (GS3) gene was knocked off in japonica varieties of rice pertains to increased grain length in T1 lines compared to wild type using CRISPR-QTL editing tool
(Xu et al., 2016; Yuyu et al., 2020).
Low cesium rice plants were formulated by inactivation of the K+ transporter OsHAK 1 with the CRISPR/Cas9 system and OsPRX2 for potassium deficiency tolerance.
OsRR22,
O.
sativa response regulator 22, gene was knocked out to improve salt tolerance in rice the gene using the CRISPR/Cas9-targeted mutagenesis. SD1 and photosensitivity 5 (SE5) genes were targeted to produce semi-dwarf elite lines in rice. Genes (GS3, GW2 and GN1A) controls plant architecture, seed size, yield and erect panicle were targeted by bringing out knockouts by CRISPR/Cas9.Cooking and eating quality determines market value and consumer’s preference in rice. Wx gene essential for amylose synthesis was mutated applying CRISPR/Cas9 system to produce high amylose content in rice accessions. Mutant’s series with fine-tuned amylose contents was created by specific base alteration of Wx genes in rice. Targeted mutagenesis of starch branching enzyme SBEIIb leads to generation of high-amylose rice
(Lacchini et al., 2020; Xu et al., 2020).
Likewise, aromatic rice was generated from unscented variety, ASD16 by targeting the OsBADH2 through CRISPR/Cas9. Knockoff of Vacuolar Iron Transporter genes, OsVIT2 to increase Fe distribution in embryo and endosperm. Sulfur metabolisms molecular switch reduces arsenic and enhance selenium in rice. Knock into the carotenoid biosynthetic pathway and integration of CrtI and PSY genes into the target spot by CRISPR/Cas9 resulted into high β carotene in rice. GABA-rich rice was created which contains seven-fold GABA by truncating the C-terminal of the OsGAD3 by means of CRISPR/Cas9 approach
(Akama et al., 2020; Ashokkumar et al., 2020; Dong et al., 2020; Chen et al., 2021; Sun et al., 2021).
Wheat
CRISPR/Cas9 genome editing for improvement of trait are applied in wheat
viz., three genes knockout by CRISPR/Cas9 conferred powdery mildew resistance. Switching of the three homologs of
TaEDR1 gene leads to creation of
TaEDR1 lines in wheat having resistance to powdery mildew
(Gil-Humanes et al., 2017; Zhang et al., 2017). Similarly
, two genes in protoplasts were focussed to confer resistance to head blight caused by
Fusarium graminearum (Ansari et al., 2020). Many genes were targeted by CRISPR/Cas9 technology for enhancing yield and protein content in wheat
(Wang et al., 2018; Hillary and Ceasar, 2019). Knockoff of
TaGW7 gene provides grain width enhancement and weight in wheat
(Wang et al., 2019). Likewise, gene editing using CRISPR/Cas9 has been implicated to reduce gluten content in wheat
(Jouanin et al., 2020; Liu et al., 2021).
Maize
Gene editing of PSY1 gene in maize was done through CRISPR/Cas9 resulted into mutant (psy1) with white kernels and albino seedlings. ZmTMS5, thermo-sensitive genic male sterile gene liable for male sterility in maize was selected for CRISPR/Cas9 genome editing. ARGOS, genes upgrade drought tolerance in transgenic maize because of their role in negative regulation of ethylene response and signal transduction in ethylene production pathway. Knockout of the Wx gene generated twelve elite inbred lines with waxy mutants in maize by CRISPR/Cas9
(Ansari et al., 2020;
Gao et al., 2020).
Tomato
CRISPR/Cas9 system have huge role for lengthening shelf life in tomatoes. CRISPR/Cas9 targeted mutagenesis in ALC gene was used to prolong shelf life in tomato lines. Disruption of ripening inhibitor gene, RNA recognition motif-containing gene confirms their role in fruit ripening in tomato by CRISPR gene editing. Fruit yield increases by gene knock out of flowering repressor, SP5G gene, seedless fruit by somatic mutations in the parthenocarpy related gene, SlIAA9, increased shelf life by replacement of the dominant ALC (Alcobaca) gene in tomato by CRISPR gene editing. Yellow and purple tomato was created by mutation in gene, PSY1 involves in carotene synthesis. Knockout of genes involved in carotenoid metabolic pathway leads to generation of lycopene rich tomato by CRISPR/Cas9
(Li et al., 2018: Vu et al., 2020; Wang et al., 2020; Chattopadhyay et al., 2021).
Soybean
CRISPR/Cas9 induced genome editing was first studied in soybean by
Cai et al., (2015) by editing two genes (GmFEI2 and GmSHR).
Bao et al., (2020) described construction of CRISPR/Cas9 plasmid for soybean gene editing. CRISPR/Cas9 mediated base editing tool to induce single base substitution in soybean was developed by
Cai et al., (2020). Two genes, GmIPK1 and GmIPK2 codes for enzyme related to phytic acid biosynthesis pathway were edited by two components CRISPR/Cas9 tool
(Carrijo et al., 2021).
Arabidopsis
CRISPR/Cas9 system of genome editing was firstly applied in
Arabidopsis by
Feng et al., (2013) where three genes, brassinosteroid insensitive1, jasmonate-zim-domain protein 1 and gibberellic acid insensitive were edited simultaneously. Similarly,
Mao et al., (2013) utilized CRISPR/Cas9 targeted genome editing of albinism genesCHLI1 and CHLI2 with AFLP marker. CRISPR/Cas9 system was utilized to provide TuMV resistance and induced germline mutation in
Arabidopsis (LeBlanc et al., 2018; Zhang et al., 2018).
Potato
Potato and their excellent nutritive value like starch, vitamin C, potassium, fibre, vitamins B, copper, tryptophan
etc. help in control many of the deadly diseases. It is important crop worldwide which need recent research attention through implications of biotechnological approaches. Waxy type of potato was developed through mutation of granule-bound starch synthase gene and multi-allele mutation was also created by knocking off Acetolactate Synthase1 gene
(Butler et al., 2016; Andersson et al., 2017).
Cotton
Targeted genome editing utilizing CRISPR/Cas9 system was first applied in cotton by
Janga et al., (2017). Later, CRISPR induced gene truncation in two copies of Gh14-3-3d gene was used to produce
Verticillium resistant in upland cotton germplasm
(Zhang et al., 2018).
Rare species
In recent days, CRISPR/Cas9 technique has also been utilized for improvement of tree with heterozygous genome like hybrid poplar and resistant to cotton leaf curl multan virus in
Nicotiana benthamiana, by targeting CLCuMuV genome. In, wild strawberry (
Fragaria vesca) TAA1 genes (responsible for auxin biosynthesis and ARF8 (regulates auxin response factor 8) were edited to generate faster growth plant. These studies demonstrate the use of CRISPR/Cas9 genome editing for gene editing in wild species and creating new variants of wild species rather better in overall plant phenotype essential for the commercial cultivation
(Zhou et al., 2018; Yin et al., 2019; Wang et al., 2020a) (Table 1).
