(TIPS)) to rice 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which
conferred resistance to glyphosate. They reported intron-mediated site-specific
gene replacement and insertion (endogenous exon 2 with a new exon containing
the TIPS substitutions in endogenous OsEPSPS gene) approach to engender
mutations using the non-homologous end joining pathway CRISPR-Cas9 system.
Butler et al. (2016) used a geminivirus replicon (GVR) to deliver sequence-specific
nucleases targeting the potato ACETOLACTATE SYNTHASE1 (ALS1—a key target
for important herbicides including chlorsulfuron and bispyribac sodium) gene and
repair templates designed to fit in herbicide-impeding point mutations within the
ALS1 locus. These point mutations were able to support a reduced herbicide susceptibility phenotype of potato. Sun et al. (2016) generated homozygous herbicideresistant rice plants by using two gRNAs and providing repair templates from
plasmid and free double-stranded DNAs to substitute two distinct amino acid
residues in the ALS1. Shimatani et al. (2017) designed a fusion of CRISPR-Cas9
and activation-induced cytidine deaminase (Target-AID) for point mutagenesis. In
rice, they encouraged multiple herbicide (Imazamox)-resistance point mutations in
C287T gene, resulted in an A96V amino acid substitution. While in tomato they
produced marker-free plants with homozygous heritable DNA substitutions,
signifying the possibility of base editing for improving crop’s stress resistance.
Nekrasov et al. (2017) generated a tomato variety resistant to the powdery mildew
fungal pathogen Oidium neolycopersici (‘Tomelo’) using the CRISPR/Cas9 technology. They used whole-genome sequencing to illustrate that Tomelo is
non-transgenic and only carries a 48 bp deletion that is identical to naturally existing
mutations. By utilising RNAi (RNA interference) and CRISPR-Cas9 strategies, Li
et al. (2017) demonstrated that two Arabidopsis glycosyltransferase genes—
UGT79B2 and UGT79B3 are regulated by CBF1 (CRT/DRE-binding factor 1, also
named DREB1B) and confer abiotic stress tolerance via modulating anthocyanin
accumulation. Five pCas9/CsLOB1sgRNA constructs were designed by Peng et al.
(2017) to modify the effector binding element (EBEPthA4) of the CsLOB1 promoter, so as to enhance citrus canker resistance in Wanjincheng orange. Earlier in
2016, canker-resistant citrus varieties via Cas9/sgRNA were also generated by Jia
et al. (2016). They modified the PthA4 effectors binding elements (EBEs) in the
CsLOB1 Promoter of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene
in Duncan grapefruit. Hahn et al. (2017) designed a generic vector system to be
exploited to clone any sgRNA sequence in a plant T-DNA vector containing a
ubiquitously expressed Cas9 gene. With this vector, they explored BIALAPHOS
RESISTANCE (BAR) and GLABROUS1 (GL1), where BAR, as a positive selection
marker, confers resistance to glufosinate and GL1 is required for the formation of
protective trichomes. Shi et al. (2017) employed a CRISPR-Cas technology to insert
native maize GOS2 promoter into the 5
0 -untranslated region of the native ARGOS8
gene (negative regulator of ethylene responses). The ARGOS8 variants had elevated
levels of ARGOS8 transcripts relative to the native allele and compared to the WT,
these variations generated drought-tolerant crops. Thus, CRISPR-Cas9 system
assures its enormous involvement in exploration of the gene regulatory networks
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
457
conferred resistance to glyphosate. They reported intron-mediated site-specific
gene replacement and insertion (endogenous exon 2 with a new exon containing
the TIPS substitutions in endogenous OsEPSPS gene) approach to engender
mutations using the non-homologous end joining pathway CRISPR-Cas9 system.
Butler et al. (2016) used a geminivirus replicon (GVR) to deliver sequence-specific
nucleases targeting the potato ACETOLACTATE SYNTHASE1 (ALS1—a key target
for important herbicides including chlorsulfuron and bispyribac sodium) gene and
repair templates designed to fit in herbicide-impeding point mutations within the
ALS1 locus. These point mutations were able to support a reduced herbicide susceptibility phenotype of potato. Sun et al. (2016) generated homozygous herbicideresistant rice plants by using two gRNAs and providing repair templates from
plasmid and free double-stranded DNAs to substitute two distinct amino acid
residues in the ALS1. Shimatani et al. (2017) designed a fusion of CRISPR-Cas9
and activation-induced cytidine deaminase (Target-AID) for point mutagenesis. In
rice, they encouraged multiple herbicide (Imazamox)-resistance point mutations in
C287T gene, resulted in an A96V amino acid substitution. While in tomato they
produced marker-free plants with homozygous heritable DNA substitutions,
signifying the possibility of base editing for improving crop’s stress resistance.
Nekrasov et al. (2017) generated a tomato variety resistant to the powdery mildew
fungal pathogen Oidium neolycopersici (‘Tomelo’) using the CRISPR/Cas9 technology. They used whole-genome sequencing to illustrate that Tomelo is
non-transgenic and only carries a 48 bp deletion that is identical to naturally existing
mutations. By utilising RNAi (RNA interference) and CRISPR-Cas9 strategies, Li
et al. (2017) demonstrated that two Arabidopsis glycosyltransferase genes—
UGT79B2 and UGT79B3 are regulated by CBF1 (CRT/DRE-binding factor 1, also
named DREB1B) and confer abiotic stress tolerance via modulating anthocyanin
accumulation. Five pCas9/CsLOB1sgRNA constructs were designed by Peng et al.
(2017) to modify the effector binding element (EBEPthA4) of the CsLOB1 promoter, so as to enhance citrus canker resistance in Wanjincheng orange. Earlier in
2016, canker-resistant citrus varieties via Cas9/sgRNA were also generated by Jia
et al. (2016). They modified the PthA4 effectors binding elements (EBEs) in the
CsLOB1 Promoter of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene
in Duncan grapefruit. Hahn et al. (2017) designed a generic vector system to be
exploited to clone any sgRNA sequence in a plant T-DNA vector containing a
ubiquitously expressed Cas9 gene. With this vector, they explored BIALAPHOS
RESISTANCE (BAR) and GLABROUS1 (GL1), where BAR, as a positive selection
marker, confers resistance to glufosinate and GL1 is required for the formation of
protective trichomes. Shi et al. (2017) employed a CRISPR-Cas technology to insert
native maize GOS2 promoter into the 5
0 -untranslated region of the native ARGOS8
gene (negative regulator of ethylene responses). The ARGOS8 variants had elevated
levels of ARGOS8 transcripts relative to the native allele and compared to the WT,
these variations generated drought-tolerant crops. Thus, CRISPR-Cas9 system
assures its enormous involvement in exploration of the gene regulatory networks
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
457