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

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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Attempts to implement CRISPR/Cas9 for genome editing in the oomycete Phytophthora infestans

10.1101/274829 ◽

2018 ◽

Cited By ~ 1

Author(s):

Johan van den Hoogen ◽

Francine Govers

Keyword(s):

Molecular Biology ◽

Phytophthora Infestans ◽

Genome Editing ◽

Target Gene ◽

Phytophthora Sojae ◽

Targeted Mutagenesis ◽

Future Directions ◽

Ribonucleoprotein Complexes ◽

Late Blight Pathogen ◽

Experimental Effort

AbstractFew techniques have revolutionized the molecular biology field as much as genome editing using CRISPR/Cas9. Recently, a CRISPR/Cas9 system has been developed for the oomycete Phytophthora sojae, and since then it has been employed in two other Phytophthora spp. Here, we report our progress on efforts to establish the system in the potato late blight pathogen Phytophthora infestans. Using the original constructs as developed for P. sojae, we did not obtain any transformants displaying a mutagenized target gene. We made several modifications to the CRISPR/Cas9 system to pinpoint the reason for failure and also explored the delivery of pre-assembled ribonucleoprotein complexes. With this report we summarize an extensive experimental effort pursuing the application of a CRISPR/Cas9 system for targeted mutagenesis in P. infestans and we conclude with suggestions for future directions.

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

Scientific Reports ◽

10.1038/s41598-019-55681-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Raviraj Banakar ◽

Alan L. Eggenberger ◽

Keunsub Lee ◽

David A. Wright ◽

Karthik Murugan ◽

...

Keyword(s):

High Frequency ◽

Genome Engineering ◽

Selectable Marker ◽

Marker Gene ◽

Targeted Mutagenesis ◽

Screening Methods ◽

Chromosomal Dna ◽

Target Sites ◽

Rnp Complex ◽

Biolistic Delivery

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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ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

Molecular Diagnostics and Biosafety – 2020. Russian national scientific and practical conference with international participation (October, 6–8, 2020): Conference Proceedings ◽

10.36233/978-5-9900432-9-9-98 ◽

2020 ◽

Author(s):

Y.V. Mikhaylova ◽

◽

M.A. Tyumentseva ◽

A.A. Shelenkov ◽

Y.G. Yanushevich ◽

...

Keyword(s):

High Sensitivity ◽

Correct Choice ◽

Target Sequence ◽

Dna Breaks ◽

Gene Encoding ◽

Guide Rna ◽

Guide Rnas ◽

Ribonucleoprotein Complexes ◽

Chemokine Receptor Ccr5 ◽

Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

Scientific Reports ◽

10.1038/s41598-021-89546-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuu Asano ◽

Kensuke Yamashita ◽

Aoi Hasegawa ◽

Takanori Ogasawara ◽

Hoshie Iriki ◽

...

Keyword(s):

Genome Editing ◽

Dictyostelium Discoideum ◽

Streptococcus Pyogenes ◽

Nucleotide Substitution ◽

Point Mutations ◽

Targeted Mutagenesis ◽

Single Amino Acid ◽

Specific Gene ◽

Knock Out ◽

Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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Rapid and marker-free gene replacement in citric acid-producing Aspergillus tubingensis (A. niger) WU-2223L by the CRISPR/Cas9 system-based genome editing technique using DNA fragments encoding sgRNAs

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2021.01.011 ◽

2021 ◽

Author(s):

Isato Yoshioka ◽

Kohtaro Kirimura

Keyword(s):

Citric Acid ◽

Genome Editing ◽

Gene Replacement ◽

Dna Fragments ◽

Aspergillus Tubingensis ◽

Marker Free ◽

Free Gene

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First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02103-5 ◽

2021 ◽

Author(s):

Daria Nitarska ◽

Robert Boehm ◽

Thomas Debener ◽

Rares Calin Lucaciu ◽

Heidi Halbwirth

Keyword(s):

Transgenic Plants ◽

Genome Editing ◽

Ornamental Plants ◽

Targeted Mutagenesis ◽

Cloning And Expression ◽

Euphorbia Pulcherrima ◽

Loss Of Function ◽

Wild Type ◽

Promising Tool ◽

Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

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CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

Scientific Reports ◽

10.1038/s41598-021-95547-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yoshihiko Nanasato ◽

Masafumi Mikami ◽

Norihiro Futamura ◽

Masaki Endo ◽

Mitsuru Nishiguchi ◽

...

Keyword(s):

Genome Editing ◽

Cryptomeria Japonica ◽

Fluorescent Protein ◽

Japanese Cedar ◽

Targeted Mutagenesis ◽

Embryogenic Tissue ◽

Breeding Period ◽

Single Mutation ◽

Knock Out ◽

Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

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