The potential applications of site‑directed mutagenesis for crop improvement: a review

Bezie Yilkal,Tilahun Tadesse,Atnaf Mulugeta,Mengistie Taye5 한국작물학회 2021 Journal of crop science and biotechnology Vol.24 No.3

The search for technologies for crop improvement has been a continuous practice to address the food insecurity to the growing human population with an ever-decreasing arable land and dynamic climate change around the world. Considering the potential technologies for crop improvement could close the rooms of poverty in developing countries in particular and around the globe at large. This review aimed to assess the site-directed mutation creation methods and show the potential tools for future crop improvement programs. Site-directed mutagenesis was found to be an efcient process to create targeted mutation on cereal crops, horticultural crops, oilseed crops, and others. Agronomic traits such as yield, quality, and stress tolerance have been improved using site-directed mutagenesis. Besides, selectable marker elimination was also reported from transgenic crops by targeted mutation. Most of the reports on site-directed mutagenesis is focusing on cereal crops (58.339%) followed by horticultural crops (22.92%). Among the four mutagenic tools that have been reported, the CRISPR/ Ca9 technology was found to be frequently used (66.67%) followed by TALENs. This tool is potential, since it is efcient in creating targeted mutagenesis and less likely of-target efect, so it is repeatedly used in diferent research works. TALENs were used usually to knockout genes with bad traits. Moreover, the mutation created by mutagenic tools was found to be efcient, and the mutated traits proved to be heritable to generations. Hence, site-directed mutagenesis by the CRISPR/Cas9 system is advisable for agricultural development, thereby ensuring food sustainability around the world.

Site-directed mutagenesis in <i>Arabidopsis thaliana</i> using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles

Hyun, Youbong,Kim, Jungeun,Cho, Seung Woo,Choi, Yeonhee,Kim, Jin-Soo,Coupland, George Springer Berlin Heidelberg 2015 Planta Vol.241 No.1

<P><B><B><I>Main conclusion</I></B></B></P><P><B>Dividing tissue-targeted site-directed mutagenesis using RGEN of CRISPR/Cas system produces heritable mutations in</B><B><I>Arabidopsis thaliana.</I></B></P><P><B>Abstract</B></P><P>Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the <I>Arabidopsis</I> nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the <I>single guide RNA</I> (<I>sgRNA</I>) and Cas9 DNA endonuclease were expressed from the <I>U6 snRNA</I> and <I>INCURVATA2</I> promoters, respectively. After <I>Agrobacterium</I>-mediated introduction of T-DNAs encoding RGENs that targets <I>FLOWERING LOCUS T</I> (<I>FT</I>) and <I>SQUAMOSA PROMOTER BINDING PROTEIN</I>-<I>LIKE 4</I> genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the <I>FT</I> gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of <I>FT</I> including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in <I>A. thaliana</I>.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1007/s00425-014-2180-5) contains supplementary material, which is available to authorized users.</P>

Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1

Jong-Uk Koh,Hyun-Young Cho,공광훈 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.5

In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. KmGSH, KmDCNB and KmEPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.

Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1

Koh, Jong-Uk,Cho, Hyun-Young,Kong, Kwang-Hoon Korean Chemical Society 2007 Bulletin of the Korean Chemical Society Vol.28 No.5

In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. Km GSH, Km DCNB and Km EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.

Site-Directed Mutagenesis of Two Cysteines (155, 202) in Catechol 1,2-dioxygenase $I_1$ of Acinetobacter lwoffii K24

Kim, Seung-Il,Kim, Soo-Jung,Leem, Sun-Hee,Oh, Kye-Heon,Kim, Soo-Hyun,Park, Young-Mok Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.2

Catechol 1,2-dioxygenase $I_1$ ($CDI_1$) is the first enzyme of the $\beta$-ketoadipate pathway in Acinetobacter lowffii K24. $CDI_1$ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, $AgNO_3$. Two mutants, $CDI_1$ C155V and $CDI_1$ C202V, were obtained by site-directed mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys$\rightarrow$Val) were characterized by peptide mapping and amino acid sequencing. Interestingly, $CDI_1$ C155V was inhibited by $AgNO_3$, whereas $CDI_1$ C202V was not inhibited. This suggests that $Cys^{202}$ is the sole inhibition site by $AgNO_3$ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated $CDI_1s$ suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of $CDI_1$.

Site-directed Mutagenesis of the Evolutionarily Conserved Tyr8 Residue in Rice Phi-class Glutathione S-transferase F3

Jo, Hyun-Joo,Pack, Mi-Jin,Seo, Jin-Young,Lim, Jin-Kyung,Kong, Kwang-Hoon Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.9

To elucidate the role of the evolutionarily conserved Tyr8 residue in rice Phi-class GSTF3, this amino acid was replaced with alanine and phenylalanine by site-directed mutagenesis, respectively. The replacement of Tyr8 with Ala significantly affected the catalytic activity and the kinetic parameters, whereas the substitutions of Tyr8 with Phe had almost no effect. The Y8A mutant resulted in approximately 90-100% decrease of the specific activity. Moreover, the Y8A mutant resulted approximately in 2-fold increase of $K_m$, approximately 60-80% decrease of $k_{cat}$, and approximately 6.5-fold decrease in $k_{cat}/K_m$. From the pH/log $k_{cat}/K_m$ plot, $pK_a$ values of the GSH in the wild-type enzyme-GSH complex, Y8A-GSH complex and Y8F-GSH complex were estimated to be approximately 6.8, 8.5 and 6.9, respectively. From these results, we suggest that the evolutionarily conserved Tyr8 residue in OsGSTF3 seems to influence the structural stability of the active site of OsGSTF3 rather than directly its catalytic activity.

Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis

Jung, I.P.,Cho, J.H.,Koo, B.S.,Yoon, M.Y. IPC Science and Technology Press ; Elsevier Scienc 2015 Enzyme and microbial technology Vol.78 No.-

Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC<SUB>50</SUB> than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.

유전자 재조합 탄저 성분백신 개발 연구(Ⅱ) : 방어항원 안정화를 위한 유전자 결실 돌연변이주 제조

오희복,성원근,박영미아,한지선 국립보건연구원 1999 국립보건원보 Vol.36 No.-

Molecular Modeling and Site Directed Mutagenesis of the O-Methyltransferase, SOMT-9 Reveal Amino Acids Important for Its Reaction and Regioselectivity

Park, So-Hyun,Kim, Bong-Gyu,Lee, Sun-Hee,Lim, Yoong-Ho,Cheong, You-Hoon,Ahn, Joong-Hoon Korean Chemical Society 2007 Bulletin of the Korean Chemical Society Vol.28 No.12

SOMT-9 is an O-methyltransferase that utilizes quercetin to produce 3'-methoxy quercetin. In order to determine which amino acids of SOMT-9 are important for this reaction and its regioselectivity, molecular docking experiments followed by site directed mutagenesis were performed. Molecular modeling and molecular docking experiments identified several amino acid residues involved in metal binding, AdoMet binding, and substrate binding. Site-directed mutagenesis showed that Asp188 is critical for metal binding and that Lys165 assists other metal binding residues in maintaining quercetin in the proper position during the reaction. In addition, Tyr207 was shown to play an important role in the determination of the regioselectivity and Met60 was shown to be involved in formation of the hydrophobic pocket necessary for substrate binding. The molecular modeling and docking experiments discussed in this study could be applicable to future research including prediction of substrate binding and regioselectivity of an enzyme.

Molecular Modeling and Site Directed Mutagenesis of the O-Methyltransferase, SOMT-9 Reveal Amino Acids Important for Its Reaction and Regioselectivity

So Hyun Park,Bong Gyu Kim,Sun Hee Lee,Yoongho Lim,Youhoon Cheong,Joong-Hoon Ahn* 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.12

SOMT-9 is an O-methyltransferase that utilizes quercetin to produce 3'-methoxy quercetin. In order to determine which amino acids of SOMT-9 are important for this reaction and its regioselectivity, molecular docking experiments followed by site directed mutagenesis were performed. Molecular modeling and molecular docking experiments identified several amino acid residues involved in metal binding, AdoMet binding, and substrate binding. Site-directed mutagenesis showed that Asp188 is critical for metal binding and that Lys165 assists other metal binding residues in maintaining quercetin in the proper position during the reaction. In addition, Tyr207 was shown to play an important role in the determination of the regioselectivity and Met60 was shown to be involved in formation of the hydrophobic pocket necessary for substrate binding. The molecular modeling and docking experiments discussed in this study could be applicable to future research including prediction of substrate binding and regioselectivity of an enzyme.