Journal: bioRxiv

Article Title: A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

doi: 10.1101/2025.04.18.649274

Figure Lengend Snippet: (A) A plasmid is constructed containing a tRNA-gRNA cassette driven by the U6 promoter, along with a transcription unit for Cas9 expression. (B) After transformation into the plant, the primary RNA transcript is cleaved by endogenous tRNA-processing enzymes, RNase P and RNase Z, resulting in functional guide RNAs for genome editing

Article Snippet: Finally, the L1 tRNA-gRNA construct is transferred into the L2 pCsA (Addgene #136067 ( )) acceptor vector through another round of Loop assembly , linking the tRNA-gRNA unit with the transcription unit for Cas9 expression (Addgene #136135 ( )) and a transcription unit containing the desired selection marker.

Techniques: Plasmid Preparation, Construct, Expressing, Transformation Assay, Functional Assay

Journal: bioRxiv

Article Title: A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

doi: 10.1101/2025.04.18.649274

Figure Lengend Snippet: (A) Top: The first primer includes a portion of the tRNA sequence along with a BbsI recognition site and four-base overhang sequences for Loop assembly. The middle primers contain BbsI recognition sites and four-base overlapping overhang sequences for Loop assembly, which can correspond to any four consecutive nucleotides (highlighted with colored rectangles) within the 20-nucleotide gRNA sequence (shown with numbers from 1 to 20). Forward middle primers also contain a portion of the gRNA scaffold sequence, whereas reverse middle primers contain a portion of the tRNA sequence. The final primer includes part of the tRNA sequence, the full reverse-complement of the last gRNA, and a BbsI recognition site plus four-base overhang sequences for Loop assembly. Overhangs for cloning into the acceptor vectors shown with pink letters. Bottom: The pGTR plasmid should be used as the template for the PCR reactions. The primer combinations used for amplification are as follows: (1) G-primer-F & gRNA a Primer R, (2) gRNA a Primer F & gRNA b Primer R, and (3) gRNA b Primer F & gRNA c Primer R, n: any nucleotide (please see Supplemental Information for a detailed explanation and an example of primer design). (B) After PCR amplification and gel extraction, all fragments are combined with the OP-074 or OP-075 vector (Sauret-Gueto et al., 2020) in a Loop Assembly/Type IIS cloning reaction (see Supplemental Information for a detailed protocol of Loop Assembly L1 cloning). LacZ: lacZα cassette for blue-white screening of colonies (negative blue colonies contain undigested L1 vectors, while positive white colonies contain tRNA-gRNA parts inserted into the L1 vectors. (C) Finally, the tRNA-gRNA-OP-074 or OP-075 vector is combined with the OP-073 vector, which contains the Mp EF1a::Cas9 transcription unit, an appropriate L1 vector for plant selection, in a Loop Assembly/Type IIS cloning reaction (see Supplemental Information for a detailed protocol of Loop Assembly L2 cloning).

Article Snippet: Finally, the L1 tRNA-gRNA construct is transferred into the L2 pCsA (Addgene #136067 ( )) acceptor vector through another round of Loop assembly , linking the tRNA-gRNA unit with the transcription unit for Cas9 expression (Addgene #136135 ( )) and a transcription unit containing the desired selection marker.

Techniques: Sequencing, Cloning, Plasmid Preparation, Amplification, Gel Extraction, Selection

Journal: bioRxiv

Article Title: A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

doi: 10.1101/2025.04.18.649274

Figure Lengend Snippet: (A) Left: Schematic representation of the plant transformation vector for simultaneous delivery of one or three gRNAs and Cas9 into M. polymorpha plants by Agrobacterium -mediated transformation, with or without the use of tRNAs. Right: The three phenotypic classes of regenerating M. polymorpha plants: wild-type, chimeric, and mutant. Scale bar: 400 μm. (B) Comparison of the number of transformants with wild-type, chimeric, or mutant phenotypes. Graphs show values from triplicate experiments (dots) and their average (bars). Error bars represent the SEM; n = 3. A pairwise t-test was conducted to compare the number of chimeric mutants (generated using either the U6 tRNA-gRNA vector or U6 tRNA-gRNA vector) against the number of plants generated using the control vector (U6 gRNA). ** for p < 0.01, while no significant difference (n.s) was noted for p > 0.05. A similar pairwise test was performed for the fully mutated plants. (C-D) Sequence analysis of Mp glk mutant lines. Schematic representation of Mp GLK gene structure showing exons as blue rectangles, untranslated regions (UTRs) as grey rectangles, and introns as grey lines. The position of the gRNA used for CRISPR/Cas9 gene editing is indicated with an arrow. The wild-type M. polymorpha Cam-1 sequence is shown at the top, with the 20-bp gRNA target sequence highlighted in grey. Mutations are highlighted in red. (E) Gel electrophoresis images of PCR-based genotyping of Mp glk mutants obtained using the vector that allows the expression of three gRNAs. The positions of primers used are shown with red arrowheads. The expected size of PCR products when large deletions occurred is approximately ∼600-800 bp (lanes 1-3). The expected size of PCR products for wild-type or mutants with small deletions is 1648 bp (lanes 4, 5, 6 and WT).

Article Snippet: Finally, the L1 tRNA-gRNA construct is transferred into the L2 pCsA (Addgene #136067 ( )) acceptor vector through another round of Loop assembly , linking the tRNA-gRNA unit with the transcription unit for Cas9 expression (Addgene #136135 ( )) and a transcription unit containing the desired selection marker.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Comparison, Generated, Control, Sequencing, CRISPR, Nucleic Acid Electrophoresis, Expressing

Journal: bioRxiv

Article Title: A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

doi: 10.1101/2025.04.18.649274

Figure Lengend Snippet: (A) Schematic representation of the plant transformation vector for simultaneous delivery of three gRNAs and Cas9 into M. polymorpha plants by Agrobacterium -mediated transformation, with or without the use of tRNAs. (B) The three phenotypic classes of regenerating M. polymorpha plants: wild-type, chimeric, and mutant. Scale bar: 1 mm. (C) Comparison of the number of transformants with wild-type, chimeric, or mutant phenotypes. n = 100. (D) Sequence analysis of Mp glk mutant lines. Top: Schematic representation of Mp GLK gene structure. The position of the gRNA used for CRISPR/Cas9 gene editing is shown with an arrow. Bottom: Mutant genotyping analysis. The wild-type M. polymorpha Cam-1 sequence is shown at the top, with the 20-bp gRNA target sequence highlighted in grey. Mutations are highlighted in red. (E) Gel electrophoresis images of PCR-based genotyping of Mp glk mutants obtained using the vector that allows the expression of three gRNAs. Schematic representation of Mp GLK gene structure showing exons as blue rectangles, untranslated regions (UTRs) as grey rectangles, and introns as grey lines. Positions of primers used are shown with red arrowheads. The expected size of PCR products when large deletions occur is approximately 765 bp (lanes 1 and 2). The expected size of PCR products for wild-type or mutants with small deletions is 1648 bp (lanes 3, 4, 5 and WT).

Article Snippet: Finally, the L1 tRNA-gRNA construct is transferred into the L2 pCsA (Addgene #136067 ( )) acceptor vector through another round of Loop assembly , linking the tRNA-gRNA unit with the transcription unit for Cas9 expression (Addgene #136135 ( )) and a transcription unit containing the desired selection marker.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Comparison, Sequencing, CRISPR, Nucleic Acid Electrophoresis, Expressing

Journal: bioRxiv

Article Title: A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

doi: 10.1101/2025.04.18.649274

Figure Lengend Snippet: (A) Schematic representation of the plant transformation vector for delivery of a gRNA for targeting Mp RR-MYB5 into Mp gata4 or Mp glk mutant M. polymorpha plants by Agrobacterium - mediated transformation. mALS: Chlorsulfuron selection. (B-C) The three phenotypic classes of regenerating Mp gata4 and Mp glk mutant plants transformed with the L2 vector (A) for targeting Mp RR-MYB5 : Mp gata4 , chimeric, and mutant or Mp glk , chimeric, and mutant. Scale bar: 1 mm (D-E) Top: Schematic representation of Mp RR-MYB5 gene structure showing exons as blue rectangles, untranslated regions (UTRs) as grey rectangles and introns as grey lines. Positions of gRNAs used for CRISPR/Cas9 gene editing are shown as arrows. Middle: Comparison of the number of transformants with Mp glk,rr-myb5 or Mp gata4 , rr-myb5 chimeric or mutant phenotypes. n=100. Bottom: Sequence analysis of Mp glk,rr-myb5 and Mp gata4 , rr-myb5 double mutant lines. The wild-type M. polymorpha Cam-1 sequence is shown at the top, with the 20 bp gRNA target sequence highlighted with grey. Mutations are highlighted in red.

Article Snippet: Finally, the L1 tRNA-gRNA construct is transferred into the L2 pCsA (Addgene #136067 ( )) acceptor vector through another round of Loop assembly , linking the tRNA-gRNA unit with the transcription unit for Cas9 expression (Addgene #136135 ( )) and a transcription unit containing the desired selection marker.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Selection, CRISPR, Comparison, Sequencing

Journal: Diagnostic Pathology

Article Title: Atypical cellular neurothekeoma: a case report with a novel NF1 mutation

doi: 10.1186/s13000-024-01578-y

Figure Lengend Snippet: Methods and results of immunohistochemistry

Article Snippet: Cathepsin K (CK4) , Novocastra , RTU/Low pH/ Heat-induced/ 30 min , Polymer-based , +/-.

Techniques: Polymer