High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

AbstractAn important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

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Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

In Vitro Cellular & Developmental Biology - Plant ◽

10.1007/s11627-019-10042-2 ◽

2020 ◽

Vol 56 (3) ◽

pp. 265-279 ◽

Cited By ~ 3

Author(s):

George Hoerster ◽

Ning Wang ◽

Larisa Ryan ◽

Emily Wu ◽

Ajith Anand ◽

...

Keyword(s):

Somatic Embryo ◽

High Frequency ◽

Selectable Marker ◽

Baby Boom ◽

Marker Gene ◽

Single Copy ◽

System A ◽

Maize Transformation ◽

Embryo Formation ◽

Wide Range

AbstractThe use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltppro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as “altruistic transformation.”

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Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize

Frontiers in Genome Editing ◽

10.3389/fgeed.2021.670529 ◽

2021 ◽

Vol 3 ◽

Author(s):

Shujie Dong ◽

Yinping Lucy Qin ◽

Christopher A. Vakulskas ◽

Michael A. Collingwood ◽

Mariam Marand ◽

...

Keyword(s):

Genome Editing ◽

Marker Gene ◽

Gene Cassette ◽

Gene Replacement ◽

Callus Cultures ◽

Targeted Mutagenesis ◽

Target Sequence ◽

Ribonucleoprotein Complexes ◽

Biolistic Delivery ◽

Maize Embryos

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

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Molecular analysis of transgenic rapeseed plants obtained by direct transfer of two separate plasmids containing, respectively, the cauliflower mosaic virus coat protein gene and a selectable marker gene

Plant Science ◽

10.1016/0168-9452(93)90141-l ◽

1993 ◽

Vol 91 (2) ◽

pp. 181-193 ◽

Cited By ~ 21

Author(s):

C. Hervé ◽

D. Rouan ◽

P. Guerche ◽

M.-H. Montané ◽

P. Yot

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Selectable Marker ◽

Marker Gene ◽

Cauliflower Mosaic Virus ◽

Direct Transfer ◽

Selectable Marker Gene ◽

Virus Coat ◽

Transgenic Rapeseed

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Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka

Development Growth & Differentiation ◽

10.1111/dgd.12104 ◽

2013 ◽

Vol 56 (1) ◽

pp. 98-107 ◽

Cited By ~ 48

Author(s):

Satoshi Ansai ◽

Keiji Inohaya ◽

Yasutoshi Yoshiura ◽

Manfred Schartl ◽

Norihito Uemura ◽

...

Keyword(s):

Targeted Mutagenesis ◽

Design Evaluation ◽

Screening Methods ◽

Transcription Activator

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The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4011-4019.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4011-4019

Author(s):

J A Nelson ◽

P B Savereide ◽

P A Lefebvre

Keyword(s):

Chlamydomonas Reinhardtii ◽

Selectable Marker ◽

Marker Gene ◽

Small Subunit ◽

Direct Selection ◽

Nuclear Transformation ◽

Ribulose Bisphosphate Carboxylase ◽

Bisphosphate Carboxylase ◽

Dominant Selectable Marker ◽

Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

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