Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells

2016 ◽  
pp. 153-162 ◽  
Author(s):  
Zhenyu He ◽  
Kehkooi Kee
Keyword(s):  
Genome Editing ◽  
Human Cells ◽  
Targeted Mutagenesis ◽  
A Genome

scholarly journals Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

2019 ◽  
Vol 20 (5) ◽  
pp. 1155 ◽  
Author(s):  
Guillaume Bernard ◽  
David Gagneul ◽  
Harmony Alves Dos Santos ◽  
Audrey Etienne ◽  
Jean-Louis Hilbert ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mutation Frequency ◽  
Function Analysis ◽  
Expression System ◽  
Crop Improvement ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
A Genome ◽  
Transformation Methods ◽  
Biallelic Mutations

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

scholarly journals Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

2021 ◽  
Vol 22 (2) ◽  
pp. 480
Author(s):  
Jongrae Kim ◽  
Kwang Suk Chang ◽  
Sangmuk Lee ◽  
EonSeon Jin
Keyword(s):  
Genome Editing ◽  
Chlorella Vulgaris ◽  
Negative Selection ◽  
Screening Strategy ◽  
Feed Additive ◽  
Cell Factory ◽  
Dna Transformation ◽  
Research Groups ◽  
A Genome ◽  
Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

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

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.

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

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.

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

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.

CRISPR-Cpf1-mediated genome editing and gene regulation in human cells

2019 ◽  
Vol 37 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Tianwen Li ◽  
Linwen Zhu ◽  
Bingxiu Xiao ◽  
Zhaohui Gong ◽  
Qi Liao ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Human Cells

A genome-editing off switch

2016 ◽  
Vol 18 (2) ◽  
pp. 69-69
Author(s):  
Ross Cloney
Keyword(s):  
Genome Editing ◽  
A Genome

scholarly journals Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Plant Methods ◽  
2013 ◽  
Vol 9 (1) ◽  
pp. 39 ◽  
Author(s):  
Khaoula Belhaj ◽  
Angela Chaparro-Garcia ◽  
Sophien Kamoun ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome

scholarly journals Timing Polymerase Pausing with TV-PRO-seq

2018 ◽  
Author(s):  
Jie Zhang ◽  
Massimo Cavallaro ◽  
Daniel Hebenstreit
Keyword(s):  
High Resolution ◽  
Human Cells ◽  
Rrna Genes ◽  
Dna Motifs ◽  
Single Base ◽  
Chromatin States ◽  
Genome Wide ◽  
A Genome ◽  
Single Base Resolution ◽  
Polymerase Pausing

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking.We present here an extension of PRO-seq, time variant PRO-seq (TV-PRO-seq), that allowed us to estimate genome-wide pausing times at single base resolution. Its application to human cells reveals that promoter proximal pausing is surprisingly short compared to other regions and displays an intricate pattern. We also find precisely conserved pausing profiles at tRNA and rRNA genes and identified DNA motifs associated with pausing time. Finally, we show how chromatin states reflect differences in pausing times.

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