scholarly journals Programming large target genomic deletion and concurrent insertion via a prime editing-based method: PEDAR

2021 ◽  
Author(s):  
Tingting Jiang ◽  
Xiao-Ou Zhang ◽  
Zhiping Weng ◽  
Wen Xue
Keyword(s):  
Transfection Efficiency ◽  
Exogenous Dna ◽  
Gene Therapies ◽  
Guide Rnas ◽  
Pathogenic Mutations ◽  
Cas9 Nuclease ◽  
Dna Strands ◽  
Dna Template ◽  
Genomic Insertions ◽  
Deletion Junction

Genomic insertions, duplications, and insertion/deletions (indels) account for ~14% of human pathogenic mutations. Current gene editing methods cannot accurately or efficiently correct these abnormal genomic rearrangements, especially larger alterations (>100 bp). Thus, developing a method to accurately delete insertions/duplications and repair the deletion junction could improve the scope of gene therapies. Here, we engineer a novel gene editor, PE-Cas9, by conjugating Cas9 nuclease to reverse transcriptase. Combined with two prime editing guide RNAs (pegRNAs) targeting complementary DNA strands, PE-Cas9 can direct the replacement of a genomic fragment, ranging from to ~1-kb to >10-kb, with a desired sequence at the target site without requiring an exogenous DNA template. In a reporter cell line, this PE-Cas9-based deletion and repair (PEDAR) method restored mCherry expression through in-frame deletion of a disrupted GFP sequence. We further show that PEDAR efficiency could be enhanced by using pegRNAs with high cleavage activity or increasing transfection efficiency. In tyrosinemia mice, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver. This study highlights PEDAR as a tool for correcting pathogenic mutations.

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 Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest,Drosophila suzukii

2021 ◽  
Author(s):  
Nikolay P. Kandul ◽  
Esther J. Belikoff ◽  
Junru Liu ◽  
Anna Buchman ◽  
Fang Li ◽  
...  
Keyword(s):  
Population Control ◽  
Invasive Pest ◽  
Drosophila Suzukii ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Efficient Gene ◽  
Transgenic Lines ◽  
Repeated Applications ◽  
Very High ◽  
Genetic Strategies

AbstractOriginally from Asia,Drosophila suzukii(Matsumura, 1931, Diptera:Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wingDrosophila(SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linkedyellowbody andwhiteeye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.

404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
T. S. Kim ◽  
Y. Cao ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
C. K. Park
Keyword(s):  
Gene Transfer ◽  
Transfection Efficiency ◽  
The Other ◽  
Control Group ◽  
Dna Uptake ◽  
Alkaline Lysis ◽  
Exogenous Dna ◽  
Other Hand ◽  
Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

scholarly journals CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthew A. Coelho ◽  
Etienne De Braekeleer ◽  
Mike Firth ◽  
Michal Bista ◽  
Sebastian Lukasiak ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Target Sites

scholarly journals Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

2021 ◽  
Author(s):  
Akihito Fukudome ◽  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Eswar Reddem ◽  
Francisco Martinez-Marquez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Rna Polymerases ◽  
Rna Recognition Motif ◽  
Rna Dependent Rna Polymerase ◽  
Guide Rna ◽  
Pol Iv ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Template

AbstractRNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3’ ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNA’s 3’ end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3’ end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3’ end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand.SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3’ end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

scholarly journals PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Chenlei Cai ◽  
Yuexia Xie ◽  
liangliang Wu ◽  
Xiaojing Chen ◽  
Hongmei Liu ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Target Genes ◽  
Transfection Efficiency ◽  
Tumor Xenograft ◽  
Migration And Invasion ◽  
High Morbidity ◽  
Hepatic Carcinoma ◽  
Gene Therapies

Abstract Hepatic carcinoma (HCC) is a lethal disease associated with high morbidity and poor prognosis. Recently years, gene therapies have offered novel modalities to improve the prognosis of HCC patients. MicroRNA-99a (miR-99a) is frequently down-regulated in HCC, where it acts as a tumor suppressor. Therefore, we constructed monomethoxy (polyethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine)-lactobionic acid- anti-vascular endothelial growth factor antibody (mPEG-PLGA-PLL-LA/VEGFab or PEAL-LA/VEGFab) nanoparticles (NPs) with highly specific targeting properties as carriers to restore the expression of miR-99a both in vitro and in vivo, to inhibit HCC progression. In vitro, PEAL-LA/VEGFab NPs showed more efficient delivery of miR-99a to HepG2 cells than the conventional transfection reagent LipofectamineTM2000 (Lip2000). The higher delivery efficiency associated with PEAL-LA/VEGFab NPs consequently resulted in down-regulation of target genes and suppression of the proliferation, migration and invasion of HepG2 cells. In vivo, miR-99a-PEAL-LA/VEGFab NPs inhibited tumor xenograft growth in HCC-bearing mice without causing obvious systemic toxicity. Our results demonstrate that PEAL-LA/VEGFab NPs selectively and effectively deliver miR-99a to HCC cells based on the double-targeting character of these nanoparticles, thereby offering potential for translation into effective clinical therapies for HCC.

205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 243
Author(s):  
S. N. Lanjewar ◽  
K. R. Bondioli
Keyword(s):  
Plasmid Dna ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Fibroblast Cells ◽  
Square Wave ◽  
Cas9 Nuclease ◽  
Swedish Mutation ◽  
Genomic Editing ◽  
App Gene ◽  
Lipofectamine 2000

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.

scholarly journals Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system

2020 ◽  
Vol 6 (8) ◽  
pp. eaay6812 ◽  
Author(s):  
Yu Zhang ◽  
Hui Li ◽  
Yi-Li Min ◽  
Efrain Sanchez-Ortiz ◽  
Jian Huang ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Dystrophin Gene ◽  
Adeno Associated Virus ◽  
Guide Rnas ◽  
Dystrophin Expression ◽  
Cas9 Nuclease ◽  
High Doses

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

scholarly journals Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

2020 ◽  
Author(s):  
Taylor L. Mighell ◽  
Andrew Nishida ◽  
Brendan L. O’Connell ◽  
Caitlin V. Miller ◽  
Sally Grindstaff ◽  
...  
Keyword(s):  
Low Cost ◽  
Targeted Sequencing ◽  
High Input ◽  
Selective Enrichment ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Fold Enrichment ◽  
Gc Bias ◽  
Dna Template

AbstractTargeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

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