Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

Abstract Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

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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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Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

10.1101/2020.01.06.890244 ◽

2020 ◽

Author(s):

Rabinowitz Roy ◽

Abadi Shiran ◽

Almog Shiri ◽

Offen Daniel

Keyword(s):

Genome Editing ◽

Point Mutations ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Computational Tool ◽

Single Nucleotide ◽

Base Editing ◽

A Genome ◽

Wide Range ◽

Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

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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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Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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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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Adenoviral vector DNA for accurate genome editing with engineered nucleases

Nature Methods ◽

10.1038/nmeth.3075 ◽

2014 ◽

Vol 11 (10) ◽

pp. 1051-1057 ◽

Cited By ~ 93

Author(s):

Maarten Holkers ◽

Ignazio Maggio ◽

Sara F D Henriques ◽

Josephine M Janssen ◽

Toni Cathomen ◽

...

Keyword(s):

Genome Editing ◽

Adenoviral Vector ◽

Engineered Nucleases ◽

Vector Dna

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Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection inClostridium beijerinckiias an Example

ACS Synthetic Biology ◽

10.1021/acssynbio.6b00060 ◽

2016 ◽

Vol 5 (7) ◽

pp. 721-732 ◽

Cited By ~ 91

Author(s):

Yi Wang ◽

Zhong-Tian Zhang ◽

Seung-Oh Seo ◽

Patrick Lynn ◽

Ting Lu ◽

...

Keyword(s):

Genome Editing ◽

Bacterial Genome ◽

Mutant Selection ◽

Single Nucleotide ◽

Nucleotide Modification

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Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

10.1101/2021.09.14.460298 ◽

2021 ◽

Author(s):

Jeffrey C Medley ◽

Shilpa Hebbar ◽

Joel T Sydzyik ◽

Anna Y. Zinovyeva

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Paternal Genome ◽

Single Nucleotide ◽

C Elegans ◽

Homology Directed Repair ◽

Maternal Genome ◽

Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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