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 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.

scholarly journals Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility

2020 ◽  
Vol 2 ◽  
Author(s):  
Chengwei Zhang ◽  
Guiting Kang ◽  
Xinxiang Liu ◽  
Si Zhao ◽  
Shuang Yuan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plant Species ◽  
Streptococcus Pyogenes ◽  
Genome Engineering ◽  
Human Cells ◽  
Rice Plants ◽  
Potential Target ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Related Plant

The CRISPR-Cas9 system enables simple, rapid, and effective genome editing in many species. Nevertheless, the requirement of an NGG protospacer adjacent motif (PAM) for the widely used canonical Streptococcus pyogenes Cas9 (SpCas9) limits the potential target sites. The xCas9, an engineered SpCas9 variant, was developed to broaden the PAM compatibility to NG, GAA, and GAT PAMs in human cells. However, no knockout rice plants were generated for GAA PAM sites, and only one edited target with a GAT PAM was reported. In this study, we used tRNA and enhanced sgRNA (esgRNA) to develop an efficient CRISPR-xCas9 genome editing system able to mutate genes at NG, GAA, GAT, and even GAG PAM sites in rice. We also developed the corresponding xCas9-based cytosine base editor (CBE) that can edit the NG and GA PAM sites. These new editing tools will be useful for future rice research or breeding, and may also be applicable for other related plant species.

scholarly journals Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2099
Author(s):  
Yunxing Liu ◽  
Fang Liang ◽  
Zijiong Dong ◽  
Song Li ◽  
Jianmin Ye ◽  
...  
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Gene Editing ◽  
Zebrafish Genome ◽  
Human Cells ◽  
Ribonucleoprotein Complex ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

scholarly journals Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

2019 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Target Site ◽  
Gene Repression ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

scholarly journals CRISPR/Cas9-mediated Targeted Mutagenesis in Japanese Cedar (Cryptomeria japonica D. Don)

2021 ◽  
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

Abstract Cryptomeria 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 3.1–41.4% 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 102 of 103 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out endogenous C. japonica magnesium chelatase subunit I (CjChlI) using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in genome-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.

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

2015 ◽  
Vol 5 (3) ◽  
pp. 407-415 ◽  
Author(s):  
Léna Vouillot ◽  
Aurore Thélie ◽  
Nicolas Pollet
Keyword(s):  
Genome Editing ◽  
Mutation Detection ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Nucleotide ◽  
Heteroduplex Dna ◽  
Hit Rate ◽  
Engineered Nucleases ◽  
Mismatch Cleavage ◽  
Surveyor Nuclease

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.

scholarly journals A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum.

1994 ◽  
Vol 14 (9) ◽  
pp. 5840-5849 ◽  
Author(s):  
J A Powell-Coffman ◽  
G R Schnitzler ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Point Mutations ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Cis Acting ◽  
Minimal Sequences

In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium discoideum, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression on a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cyclic AMP. The CAEs have differential affinities for a specific developmentally induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-box-binding factor (GBF) and show that in vitro-transcribed and -translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation.

scholarly journals Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Science ◽  
2020 ◽  
Vol 368 (6488) ◽  
pp. 290-296 ◽  
Author(s):  
Russell T. Walton ◽  
Kathleen A. Christie ◽  
Madelynn N. Whittaker ◽  
Benjamin P. Kleinstiver
Keyword(s):  
High Resolution ◽  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Genetic Variants ◽  
Human Cells ◽  
Target Site ◽  
Protospacer Adjacent Motif ◽  
Wide Range ◽  
Almost All ◽  
Site Recognition

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

CRISPER/Cas in plant natural product research: therapeutics as anticancer and other drug candidates and recent patents

2021 ◽  
Vol 16 ◽  
Author(s):  
Abhijit Dey ◽  
Samapika Nandy
Keyword(s):  
Medicinal Plants ◽  
Natural Product ◽  
Genome Editing ◽  
Targeted Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Medical Conditions ◽  
Knock Out ◽  
Therapeutic Applications ◽  
Drug Candidates ◽  
Natural Product Research

Background: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a facile, highly efficient and selective site‐directed mutagenesis tool for RNA-guided genome-editing. CRISPR/Cas9 genome-editing strategy uses designed guide-RNAs that recognize a 3 base-pair protospacer adjacent motif (PAM) sequence in the target-DNA. CRISPR/Cas-editing tools have mainly been employed in crop plants in relation to yield and stress tolerance. However, the immense potential of this technology has not yet been fully utilized in medicinal plants in deciphering or modulating secondary metabolic pathways, producing therapeutically active phytochemicals against cancer and other diseases. Objective: The present review elucidates the use of CRISPR-Cas9 as a promising genome-editing tool in plants and plant-derived natural products with anticancer and other therapeutic applications. It also includes recent patents on the therapeutic applications of CRISPR-CAS systems implicated in cancer and other human medical conditions. Methods: Popular search engines such as PubMed, Scopus, Google Scholar, Google Patents, Medline, ScienceDirect, SpringerLink, EMBASE, Mendeley etc., were searched in order to retrieve literature using relevant keywords viz. CRISPER/Cas, plant natural product research, anticancer, therapeutics etc., either singly or in various combinations. Results: Retrieved citations and further cross-referencing among the literature have produced a total number of 71 publications and 3 patents cited in this work. Information presented in this review aims to support further biotechnological and clinical strategies to be carried using CRISPER/Cas mediated optimization of natural plant products against cancer and an array of other human medical conditions. Conclusion: Off late, knock-in and knock-out, point mutation, controlled tuning of gene-expression, and targeted mutagenesis have been enabled the versatile CRISPR/Cas-editing device to engineer medicinal plants’ genomes. In addition, by combining CRISPR/Cas-editing tools with next-generation sequencing (NGS) and various tools of system biology, many medicinal plants have been engineered genetically to optimize the production of valuable bioactive compounds of industrial significance.

scholarly journals A Highly Efficient Identification of Mutants Generated by CRISPR/Cas9 Using the Non‑Functional DsRed Assisted Selection in Aspergillus Oryzae

2021 ◽  
Author(s):  
Junxia Fan ◽  
Huanxin Zhang ◽  
Yuzhen Li ◽  
Ziming Chen ◽  
Tianming Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Reporter Gene ◽  
Aspergillus Oryzae ◽  
Target Gene ◽  
Kojic Acid ◽  
Acid Synthesis ◽  
Start Codon ◽  
Knock Out ◽  
Protospacer Adjacent Motif ◽  
Cut Method

Abstract The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon “ATG” of DsRed, yielding the non‑functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

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