scholarly journals A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 458 ◽  
Author(s):  
François M. Seys ◽  
Peter Rowe ◽  
Edward L. Bolt ◽  
Christopher M. Humphreys ◽  
Nigel P. Minton
Keyword(s):  
Gold Standard ◽  
Mutant Allele ◽  
Target Sequence ◽  
Proof Of Concept ◽  
Wild Type ◽  
Guide Rna ◽  
Simple Strategy ◽  
Clostridium Autoethanogenum ◽  
Gene Knockouts

Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of ‘watermark’ sequences into the complementing genes.

scholarly journals A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

2020 ◽  
Author(s):  
François M. Seys ◽  
Peter Rowe ◽  
Edward L. Bolt ◽  
Christopher M. Humphreys ◽  
Nigel P. Minton
Keyword(s):  
Gold Standard ◽  
Mutant Allele ◽  
Target Sequence ◽  
Proof Of Concept ◽  
Wild Type ◽  
Guide Rna ◽  
Essential Step ◽  
Simple Strategy ◽  
Clostridium Autoethanogenum

Phenotypic complementation of gene knock-outs is an essential step in establishing function. Here we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9, but could be designed for any CRISPR/Cas system. For proof of concept, 9 bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by incorporation of ‘watermark’ sequences into the complementing genes.

scholarly journals Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Yaoge Jiao ◽  
Lifang Zhou ◽  
Rui Tao ◽  
Yanhong Wang ◽  
Yun Hu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Mammalian Cells ◽  
Fine Tuning ◽  
Random Sequences ◽  
Strand Breaks ◽  
Guide Rna ◽  
Simple Strategy ◽  
Protospacer Adjacent Motif ◽  
Efficient Integration

AbstractPrime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

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

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.

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae

2020 ◽  
Vol 85 (4) ◽  
pp. 895-901
Author(s):  
Takamitsu Amai ◽  
Tomoka Tsuji ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Nuclear Genome ◽  
Target Sequence ◽  
Guide Rna ◽  
Crispr System ◽  
Mitochondrial Target ◽  
Gene Treatment ◽  
Mtdna Deletion

ABSTRACT Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a “mito-CRISPR system” that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Mutator-Suppressible Alleles of rough sheath1 and liguleless3 in Maize Reveal Multiple Mechanisms for Suppression

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 437-446 ◽  
Author(s):  
Lisa Girard ◽  
Michael Freeling
Keyword(s):  
Untranslated Region ◽  
Mutant Allele ◽  
Negative Regulation ◽  
Wild Type ◽  
Knox Gene ◽  
Transcript Accumulation ◽  
Mu Suppression ◽  
Different Types ◽  
Rna Blot Analysis

Abstract Insertions of Mutator transposons into maize genes can generate suppressible alleles. Mu suppression is when, in the absence of Mu activity, the phenotype of a mutant allele reverts to that of its progenitor. Here we present the characterization of five dominant Mu-suppressible alleles of the knox (knotted1-like homeobox) genes liguleless3 and rough sheath1, which exhibit neomorphic phenotypes in the leaves. RNA blot analysis suggests that Mu suppression affects only the neomorphic aspect of the allele, not the wild-type aspect. Additionally, Mu suppression appears to be exerting its effects at the level of transcription or transcript accumulation. We show that truncated transcripts are produced by three alleles, implying a mechanism for Mu suppression of 5′ untranslated region insertion alleles distinct from that which has been described previously. Additionally, it is found that Mu suppression can be caused by at least three different types of Mutator elements. Evidence presented here suggests that whether an allele is suppressible or not may depend upon the site of insertion. We cite previous work on the knox gene kn1, and discuss our results in the context of interactions between Mu-encoded products and the inherently negative regulation of neomorphic liguleless3 and rough sheath1 transcription.

scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kyoko Yoshizaki ◽  
Akihiro Hirata ◽  
Hiroyuki Matsushita ◽  
Naohito Nishii ◽  
Mifumi Kawabe ◽  
...  
Keyword(s):  
Mutant Allele ◽  
False Negative ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Buccal Swab ◽  
Wild Type ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
Rflp Assay

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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 Skeletal muscle PGC-1β signaling is sufficient to drive an endurance exercise phenotype and to counteract components of detraining in mice

2017 ◽  
Vol 312 (5) ◽  
pp. E394-E406 ◽  
Author(s):  
Samuel Lee ◽  
Teresa C. Leone ◽  
Lisa Rogosa ◽  
John Rumsey ◽  
Julio Ayala ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Exercise Training ◽  
Early Stage ◽  
Peroxisome Proliferator ◽  
Disuse Atrophy ◽  
Proof Of Concept ◽  
Peroxisome Proliferator Activated Receptor ◽  
Muscle Disuse ◽  
Training Response

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1β serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1β affects the detraining response following endurance training. First, we established and validated a mouse exercise-training-detraining protocol. Second, using multiple physiological and gene expression end points, we found that PGC-1β overexpression in skeletal muscle of sedentary mice fully recapitulated the training response. Lastly, PGC-1β overexpression during the detraining period resulted in partial prevention of the detraining response. Specifically, an increase in the plateau at which O2 uptake (V̇o2) did not change from baseline with increasing treadmill speed [peak V̇o2 (ΔV̇o2max)] was maintained in trained mice with PGC-1β overexpression in muscle 6 wk after cessation of training. However, other detraining responses, including changes in running performance and in situ half relaxation time (a measure of contractility), were not affected by PGC-1β overexpression. We conclude that while activation of muscle PGC-1β is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training, suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in preclinical models of muscle disuse atrophy.

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