scholarly journals Enhance genome editing efficiency and specificity by a quick CRISPR/Cas9 system

2019 ◽  
Author(s):  
Yingying Hu ◽  
Zhou Luo ◽  
Jing Li ◽  
Dan Wang ◽  
Hai-Xi Sun ◽  
...  
Keyword(s):  
Genome Editing ◽  
Transmission Rate ◽  
Hek293t Cell ◽  
Knock Out ◽  
Editing Efficiency ◽  
Mammalian Cell Lines ◽  
Cas9 Protein ◽  
Underlying Causes ◽  
Target Effect ◽  
Hek293t Cell Line

AbstractCRISPR/Cas9 is a powerful genome editing tool that has been successfully applied to a variety of species, including zebrafish. However, targeting efficiencies vary greatly at different genomic loci, the underlying causes of which were still elusive. Here we report a quick CRISPR/Cas9 system, designated as qCas9, which exhibits accelerated turnover of Cas9 protein in zebrafish. Our data showed that qCas9 significantly improved targeting efficiency, including both knock-out and knock-in in F0 embryos, and yielded higher germline transmission rate in founder screen. Importantly, qCas9 showed little to no off-target editing in zebrafish and profoundly reduced off-target effect in HEK293T cell line. In summary, our findings demonstrate that qCas9 is a simple, economic and highly effective method to improve genome editing efficiency in zebrafish embryos and also holds great potential in reducing off-target effect in mammalian cell lines.

scholarly journals No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)

2016 ◽  
Author(s):  
Nay Chi Khin ◽  
Jenna L. Lowe ◽  
Lora M. Jensen ◽  
Gaetan Burgio
Keyword(s):  
Cell Line ◽  
Genome Editing ◽  
Sanger Sequencing ◽  
Hek293t Cell ◽  
Human Cell Line ◽  
Blastocyst Stage ◽  
Editing Efficiency ◽  
Stage Of Development ◽  
Mouse Zygotes ◽  
Hek293t Cell Line

AbstractA recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’- phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

scholarly journals One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

2021 ◽  
Vol 22 (5) ◽  
pp. 2249
Author(s):  
Fuminori Tanihara ◽  
Maki Hirata ◽  
Nhien Thi Nguyen ◽  
Osamu Sawamoto ◽  
Takeshi Kikuchi ◽  
...  
Keyword(s):  
Gene Editing ◽  
Multiple Gene ◽  
Knock Out ◽  
Editing Efficiency ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
One Step ◽  
Step Generation ◽  
The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

scholarly journals Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wen Xu ◽  
Wei Song ◽  
Yongxing Yang ◽  
Ying Wu ◽  
Xinxin Lv ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
High Fidelity ◽  
Knock Out ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Modification ◽  
Targeted Genome Modification ◽  
Target Effect

Abstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. Conclusions We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

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 AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liyang Zhang ◽  
John A. Zuris ◽  
Ramya Viswanathan ◽  
Jasmine N. Edelstein ◽  
Rolf Turk ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Genome Editing ◽  
Nk Cell ◽  
Basic Research ◽  
Cell Recognition ◽  
Site Specific ◽  
Editing Efficiency ◽  
Rapid Generation ◽  
Therapeutic Cell

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

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 Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hek293t Cell ◽  
Good Manufacturing Practice ◽  
Clinical Grade ◽  
Cell Bank ◽  
Advanced Therapy ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed.Methods: Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Genome editing's potential target diseases in the cardiovascular field

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Cardiovascular Diseases ◽  
Genome Editing ◽  
Target Cells ◽  
Clinical Use ◽  
Editing Efficiency ◽  
Potential Target ◽  
Genome Wide ◽  
Significant Barrier

Two types of cardiovascular diseases can be cured or prevented using genome editing. The liver is the organ that has received the most attention in terms of clinical genome editing for cardiovascular diseases. Off-target mutagenesis is a concern of any form of genome editing. Off-target mutations in target cells or tissues may lead to undesirable functional phenotypes, including cancer. For therapeutic editing of the heart, the authors claim it's critical to achieve high editing efficiency at a chosen genomic site in a desired tissue. Off-target editing can be tested genome-wide unbiasedly using newer cell-based methods. For low off-target impact, well-designed gRNAs are important. The delivery of genome editors to target tissues and cells is a significant barrier to clinical use.

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.

A G-quadruplex motif at the 3′ end of sgRNAs improves CRISPR–Cas9 based genome editing efficiency

2018 ◽  
Vol 54 (19) ◽  
pp. 2377-2380 ◽  
Author(s):  
Smita Nahar ◽  
Paras Sehgal ◽  
Mohd Azhar ◽  
Manish Rai ◽  
Amrita Singh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Editing Efficiency ◽  
G Quadruplex

Incorporating biologically stable G-quadruplex modification at 3′ end of sgRNAs increases efficiency in zebrafish without compromising specificity.

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