scholarly journals DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

2020 ◽  
Author(s):  
Akihiro Kuno ◽  
Yoshihisa Ikeda ◽  
Shinya Ayabe ◽  
Kanako Kato ◽  
Kotaro Sakamoto ◽  
...  
Keyword(s):  
Genome Editing ◽  
Point Mutations ◽  
Comprehensive Analysis ◽  
Target Site ◽  
Analysis Software ◽  
Accurate Identification ◽  
Site Analysis ◽  
Genotyping Method ◽  
Cell Clones ◽  
Nanopore Sequencer

AbstractGenome editing induces various on-target mutations. Accurate identification of mutations in founder mice and cell clones is essential to perform reliable genome editing experiments. However, no genotyping method allows the comprehensive analysis of diverse mutations. We developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify diverse mutations, including point mutations, deletions, inversions, and knock-in. Our genotyping method with DAJIN can handle approximately 100 samples within a day and may become a new standard for validating genome editing outcomes.

scholarly journals Easy quantification of template-directed CRISPR/Cas9 editing

2017 ◽  
Author(s):  
Eva K. Brinkman ◽  
Arne Nedergaard Kousholt ◽  
Tim Harmsen ◽  
Christ Leemans ◽  
Tao Chen ◽  
...  
Keyword(s):  
Success Rate ◽  
Genome Editing ◽  
Point Mutations ◽  
Target Site

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.

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 Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

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.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

scholarly journals Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1841
Author(s):  
Silvia Panozzo ◽  
Elisa Mascanzoni ◽  
Laura Scarabel ◽  
Andrea Milani ◽  
Giliardi Dalazen ◽  
...  
Keyword(s):  
Cropping Systems ◽  
Point Mutations ◽  
Common Species ◽  
Target Site ◽  
The Other ◽  
Gene Copy ◽  
Resistance Level ◽  
Resistance Evolution ◽  
Gene Copies ◽  
Als Inhibitors

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

scholarly journals FIREVAT: finding reliable variants without artifacts in human cancer samples using etiologically relevant mutational signatures

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Hyunbin Kim ◽  
Andy Jinseok Lee ◽  
Jongkeun Lee ◽  
Hyonho Chun ◽  
Young Seok Ju ◽  
...  
Keyword(s):  
Data Storage ◽  
Human Cancer ◽  
Point Mutations ◽  
Variant Calling ◽  
Human Tumor ◽  
Cancer Genome ◽  
The Cancer Genome Atlas ◽  
Precision Oncology ◽  
Mutational Signatures ◽  
Accurate Identification

Abstract Background Accurate identification of real somatic variants is a primary part of cancer genome studies and precision oncology. However, artifacts introduced in various steps of sequencing obfuscate confidence in variant calling. Current computational approaches to variant filtering involve intensive interrogation of Binary Alignment Map (BAM) files and require massive computing power, data storage, and manual labor. Recently, mutational signatures associated with sequencing artifacts have been extracted by the Pan-cancer Analysis of Whole Genomes (PCAWG) study. These spectrums can be used to evaluate refinement quality of a given set of somatic mutations. Results Here we introduce a novel variant refinement software, FIREVAT (FInding REliable Variants without ArTifacts), which uses known spectrums of sequencing artifacts extracted from one of the largest publicly available catalogs of human tumor samples. FIREVAT performs a quick and efficient variant refinement that accurately removes artifacts and greatly improves the precision and specificity of somatic calls. We validated FIREVAT refinement performance using orthogonal sequencing datasets totaling 384 tumor samples with respect to ground truth. Our novel method achieved the highest level of performance compared to existing filtering approaches. Application of FIREVAT on additional 308 The Cancer Genome Atlas (TCGA) samples demonstrated that FIREVAT refinement leads to identification of more biologically and clinically relevant mutational signatures as well as enrichment of sequence contexts associated with experimental errors. FIREVAT only requires a Variant Call Format file (VCF) and generates a comprehensive report of the variant refinement processes and outcomes for the user. Conclusions In summary, FIREVAT facilitates a novel refinement strategy using mutational signatures to distinguish artifactual point mutations called in human cancer samples. We anticipate that FIREVAT results will further contribute to precision oncology efforts that rely on accurate identification of variants, especially in the context of analyzing mutational signatures that bear prognostic and therapeutic significance. FIREVAT is freely available at https://github.com/cgab-ncc/FIREVAT

scholarly journals A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bo Li ◽  
Naixia Ren ◽  
Lele Yang ◽  
Junhao Liu ◽  
Qilai Huang
Keyword(s):  
Single Cell ◽  
Genome Editing ◽  
Cell Clone ◽  
Editing Efficiency ◽  
Base Editing ◽  
Single Cell Clone ◽  
Cell Clones ◽  
Nucleotide Mismatch

AbstractCRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Sign in / Sign up

Export Citation Format

Share Document