Functional Validation of cas9/guideRNA Constructs for Site-directed Mutagenesis of Triticale ABA8’OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8’-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.

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Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

International Journal of Molecular Sciences ◽

10.3390/ijms22137038 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7038

Author(s):

Krzysztof Michalski ◽

Christian Hertig ◽

Dariusz R. Mańkowski ◽

Jochen Kumlehn ◽

Janusz Zimny ◽

...

Keyword(s):

Nuclease Activity ◽

Site Directed Mutagenesis ◽

Plant Genome ◽

Directed Mutagenesis ◽

Specific Pcr ◽

Induction Frequency ◽

Cleavage Assay ◽

Living Organisms ◽

Genome Information ◽

Molecular Biological Approach

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

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TEM-80, a Novel Inhibitor-Resistant β-Lactamase in a Clinical Isolate of Enterobacter cloacae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.5.1183-1189.2002 ◽

2002 ◽

Vol 46 (5) ◽

pp. 1183-1189 ◽

Cited By ~ 10

Author(s):

Corinne Arpin ◽

Roger Labia ◽

Véronique Dubois ◽

Patrick Noury ◽

Muriel Souquet ◽

...

Keyword(s):

Promoter Region ◽

Gene Sequencing ◽

Pcr Amplification ◽

Enterobacter Cloacae ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Specific Pcr ◽

Resistant Phenotype ◽

Inhibitor Resistance

ABSTRACT Enterobacter cloacae Ecl261 was isolated with Escherichia coli Ec257 from the urine of a patient living in a nursing home. Both isolates were resistant to ticarcillin (MICs, 1,024 μg/ml), without significant potentiation of its activity by 2 μg of clavulanate per ml (MICs, 512 μg/ml), and susceptible to naturally active cephalosporins. This inhibitor-resistant phenotype was conferred in both strains by similar conjugative plasmids of 40 kb (Ecl261) and 30 kb (Ec257), which also conveyed resistance to sulfonamides and trimethoprim. Clinical and transconjugant strains produced a β-lactamase with a pI of 5.2 which belonged to the TEM family, as indicated by specific PCR amplification. Compared with TEM-1, this enzyme exhibited lower catalytic efficiencies (14- and 120-fold less for amoxicillin and ticarcillin, respectively), and higher concentrations of β-lactamase inhibitors were required to yield a 50% reduction in benzylpenicillin hydrolysis (750-, 82-, and 50-fold higher concentrations for clavulanate, sulbactam, and tazobactam, respectively). Gene sequencing revealed four nucleotide differences with the nucleotide sequence of bla TEM-1A. The first replacement (T32C), located in the promoter region, was described as being responsible for the increase in the level of β-lactamase production. The three other changes led to amino acid substitutions that define a new inhibitor-resistant TEM (IRT) β-lactamase, TEM-80 (alternate name, IRT-24). Two of them, Met69Leu and Asn276Asp, have previously been related to inhibitor resistance. The additional mutation, Ile127Val, was demonstrated by site-directed mutagenesis to have a very weak effect, at least alone, on the IRT phenotype. This is the first description of an IRT β-lactamase in E. cloacae. The horizontal transfer of bla TEM-80 may have occurred either from Ec257 to Ecl261 or in the reverse order.

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Site-directed mutagenesis in a conserved motif of Epstein–Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases

Journal of General Virology ◽

10.1099/vir.0.18739-0 ◽

2003 ◽

Vol 84 (3) ◽

pp. 677-686 ◽

Cited By ~ 11

Author(s):

Ming-Tsan Liu ◽

Hsien-Ping Hu ◽

Tsuey-Ying Hsu ◽

Jen-Yang Chen

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Nuclease Activity ◽

Restriction Endonucleases ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Type Ii ◽

Conserved Motif ◽

Barr Virus ◽

Epstein Barr

Sequence alignment of human herpesvirus DNases revealed that they share several conserved regions. One of these, the conserved motif D203…E225XK227 (D…EXK) in the sequence of Epstein–Barr virus (EBV) DNase, has a striking similarity to the catalytic sites of some other nucleases, including type II restriction endonucleases, λ exonuclease and MutH. The predicted secondary structures of these three residues were shown to resemble the three catalytic residues of type II restriction endonucleases. Site-directed mutagenesis was carried out to replace each of the acidic residues near the motif by residues with different properties. All substitutions of D203, E225 and K227 were shown to cause significant reductions in nuclease activity. Six other acidic residues, within the conserved regions, were also replaced by Asn or Gln. Five of these six variants retained nuclease activity and mutant D195N alone lost nuclease activity. The four charged residues, D195, D203, E225 and K227, of EBV DNase were found to be important for nuclease activity. Biochemical analysis indicated that the preference for divalent cations was altered from Mg2+ to Mn2+ for mutant E225D. The DNA-binding abilities of D203E, E225D and E225Q were shown to be similar to that of wild-type. However, K227 mutants were found to have variable DNA-binding abilities: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability. Comparison of the biochemical properties of the corresponding substitutions among EBV DNase and type II restriction enzymes indicated that the D…EXK motif is most likely the putative catalytic centre of EBV DNase.

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Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus

Biochemical Journal ◽

10.1042/bj2950313 ◽

1993 ◽

Vol 295 (1) ◽

pp. 313-315 ◽

Cited By ~ 47

Author(s):

A A Fryer ◽

L Zhao ◽

J Alldersea ◽

W R Pearson ◽

R C Strange

Keyword(s):

Pcr Primers ◽

Single Step ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Glutathione S Transferase ◽

Specific Pcr ◽

Target Dna ◽

Allele Specific ◽

Pcr Method ◽

Allele Specific Pcr

We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms was confirmed by comparison with, firstly, an established PCR method that distinguishes GSTM1 *0 homozygotes from individuals with the other GSTM1 genotypes and, secondly, GSTM1 phenotypes determined using chromatofocusing.

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