scholarly journals Assessment of Cas12a‐mediated gene editing efficiency in plants

2019 ◽  
Vol 17 (10) ◽  
pp. 1971-1984 ◽  
Author(s):  
Joan Miquel Bernabé‐Orts ◽  
Iván Casas‐Rodrigo ◽  
Eugenio G. Minguet ◽  
Viola Landolfi ◽  
Victor Garcia‐Carpintero ◽  
...  
Keyword(s):  
Gene Editing ◽  
Editing Efficiency

scholarly journals Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporationmediated delivery and embryo culture

MethodsX ◽  
2021 ◽  
pp. 101419
Author(s):  
Anil K Challa ◽  
Denise Stanford ◽  
Antonio Allen ◽  
Lawrence Rasmussen ◽  
Ferdinand K Amanor ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Gene Editing ◽  
Editing Efficiency

scholarly journals Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuki Yoshino ◽  
Shino Endo ◽  
Zhenghao Chen ◽  
Huicheng Qi ◽  
Gou Watanabe ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Recombination Activity ◽  
Site Specific ◽  
Editing Efficiency

scholarly journals CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Agronomy ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1033 ◽  
Author(s):  
Jake Adolf V. Montecillo ◽  
Luan Luong Chu ◽  
Hanhong Bae
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Gene Editing ◽  
Fine Tuning ◽  
Plant Genome ◽  
Detection Methods ◽  
Editing Efficiency ◽  
Targeted Gene Editing ◽  
Transgene Detection ◽  
Selection Of

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

A Highly Sensitive Assessment Tool for the Gene Editing Efficiency of Sickle Cell Disease Hemoglobin Beta Gene

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5557-5557
Author(s):  
Mandula Borjigin ◽  
Eric Brian Kmiec ◽  
Rigumula Wu
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Point Mutation ◽  
Sickle Cell ◽  
Gel Electrophoresis ◽  
Gene Editing ◽  
Cell Disease ◽  
Transcription Activator ◽  
Hematopoietic Stem ◽  
Editing Efficiency

Abstract In sickle cell disease, a single point mutation in hemoglobin β gene (HBB) results in the substitution of valine for glutamic acid at position 6 of the β globin protein sequence, causing the deformation of red blood cells into a sickle (or crescent) shape. With the development of powerful gene editing tools, scientists are initiating the correction of the point mutation of HBB gene in CD34+ hematopoietic stem cells and induced pluripotent stem cells. Although the results are very exciting, the evaluation method of the gene editing is primitive. Currently, the modification at the mutation site is identified and quantified using Restriction Fragment Length Polymorphism (RFLP), which involves PCR amplification, restriction enzyme digestion and gel electrophoresis. The accuracy of the gene editing efficiency depends heavily on the quantification of the DNA bands in the gel images, which is inherently imprecise. We have developed a novel technique to quantify the correction efficiency of HBB gene editing using a fluorescence tagging of the edited DNA sequence. This method provides excellent sensitivity and accuracy, and saves time and labor, eliminating a process of gel electrophoresis. We demonstrate the assessment of gene editing in HBB of K562 cells, in which the wild type HBB (βA gene) is converted to mutant βs using the gene editing tools (i.e. Transcription Activator-Like Effector Nucleases (TALENs) and single-stranded oligo deoxynucleotides (ssODNs)). We present limited information here due to the sensitivity of the intellectual property, but will discuss in detail the experimental procedures and data at the American Society of Hematology meeting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

2020 ◽  
Author(s):  
Xi Xiang ◽  
Kunli Qu ◽  
Xue Liang ◽  
Xiaoguang Pan ◽  
Jun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
High Efficiency ◽  
Outcome Data ◽  
Massively Parallel ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Sites ◽  
In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

scholarly journals Comparative Study Between the CRISPR/Cpf1 and CRISPR/Cas9 systems for Multiplex Gene Editing in Maize

2021 ◽  
Author(s):  
Chongzhi Gong ◽  
Shengchan Huang ◽  
Rentao Song ◽  
Weiwei Qi
Keyword(s):  
Gene Editing ◽  
Direct Repeat ◽  
Bzip Transcription Factor ◽  
Target Range ◽  
Editing Efficiency ◽  
Target Site Selection ◽  
Different Types ◽  
Selection For ◽  
Alternative Choice ◽  
New Mutations

Abstract Background: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully used for multiplex gene editing in crops. Although CRISPR/Cas9 system has been proved to be an efficient multiplex gene editing system in crops, it was still unclear how CRISPR/Cpf1, a natural direct repeat (DR)-based multiplex gene editing system, performed in crops. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. Results: The bZIP transcription factor Opaque2 (O2) was used as the target gene to evaluate the editing efficient of both systems. We found that in the T0 generation, the CRISPR/Cpf1 system showed low editing efficiency with only one mutation, while the CRISPR/Cas9 system generated many different types of on-target mutations. In the T1 generation, the CRISPR/Cpf1 system still showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. Conclusions: In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize. Thus, the CRISPR/Cpf1 system is a valuable alternative choice for gene editing in crops.

scholarly journals Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass

2021 ◽  
Author(s):  
Shaoting Weng ◽  
Yitian Zhao ◽  
Changhong Yu ◽  
Xiaofan Wang ◽  
Xuehan Xiao ◽  
...  
Keyword(s):  
Muscle Mass ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Specific Site ◽  
Target Tissue ◽  
Myostatin Gene ◽  
Editing Efficiency ◽  
Green Fluorescent

AbstractAn ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.

scholarly journals A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1108-1108
Author(s):  
Masoud Nasri ◽  
Perihan Mir ◽  
Benjamin Dannenmann ◽  
Diana Amend ◽  
Yun Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Gene Editing ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Editing Efficiency ◽  
Human Ipscs

Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficient Gene Editing Through an Intronic Selection Marker in Cells

2021 ◽  
Author(s):  
Shang Wang ◽  
Yuqing Li ◽  
Li Zhong ◽  
Kai Wu ◽  
Ruhua Zhang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Selection Marker ◽  
End Joining ◽  
Dna Variation ◽  
Editing Efficiency ◽  
Endogenous Gene ◽  
Positive Effects ◽  
Efficient Gene ◽  
Almost All ◽  
In Cells

Abstract Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. Despite the great importance and universality of gene editing, however, precision and efficiency are hard to achieve with the prevailing editing strategies, such as homology-directed DNA repair (HDR) and the use of base editors (BEs). Results & Discussion Our group has developed a novel gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves editing efficiency as high as 91% without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and editing efficiency, and our data suggest that GEIS has the potential to edit approximately 99% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

Block Polymer Micelles Enable CRISPR/Cas9 Ribonucleoprotein Delivery: Physicochemical Properties Affect Packaging Mechanisms and Gene Editing Efficiency

2019 ◽  
Vol 52 (21) ◽  
pp. 8197-8206 ◽  
Author(s):  
Zhe Tan ◽  
Yaming Jiang ◽  
Mitra S. Ganewatta ◽  
Ramya Kumar ◽  
Allison Keith ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Gene Editing ◽  
Polymer Micelles ◽  
Block Polymer ◽  
Editing Efficiency
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