scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

scholarly journals Plant-based biosensors for detecting CRISPR-mediated genome engineering

2021 ◽  
Author(s):  
Guoliang Yuan ◽  
Md Mahmudul Hassan ◽  
Tao Yao ◽  
Haiwei Lu ◽  
Michael Melesse Vergara ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Transient Expression ◽  
Gene Editing ◽  
Genome Engineering ◽  
Protoplast Transformation ◽  
Reliable System ◽  
Base Editing ◽  
Detection Systems ◽  
Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

scholarly journals CRISPR - CAS9 GENE EDITING: A REVIEW

2020 ◽  
Vol 8 (10) ◽  
pp. 1127-1132
Author(s):  
Devam Desai ◽  
◽  
Hiral Panchal ◽  
Shivani Patel ◽  
Ketul Nayak
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Sequence ◽  
Antiviral Defense ◽  
Defense System ◽  
Guide Rna ◽  
Therapeutic Applications ◽  
Current Review ◽  
Cas9 Nuclease ◽  
Cost Efficient

CRISPR is an RNA guided genome editing technique of genetic engineering which works like genetic scissors. Based on simplified version of bacterial CRISPR-Cas9 antiviral defense system. It is more accurate, faster and cost efficient than other genome editing methods. There are two components in this system: First component includes a single guide RNA (sgRNA) of system which will identify target sequence in genome and Second component will include Cas9 nuclease of system which will act as a pair of scissors to spilt the double strands of DNA. CRISPR has promising therapeutic applications. This current review focuses on mechanism, therapeutic applications, delivery systems, limitations and different approaches used for gene editing using CRISPR.

scholarly journals Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing inCryptococcus neoformansand Related Species

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Ping Wang
Keyword(s):  
Adverse Effects ◽  
Genome Editing ◽  
Genetic Markers ◽  
Related Species ◽  
Gene Editing ◽  
Low Cost ◽  
Constitutive Expression ◽  
Time Saving ◽  
Genetic Studies ◽  
Content Type

ABSTRACTCryptococcus neoformansand related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility. The technique has been successfully demonstrated inC. neoformansandCryptococcus deneoformansin which two DNA plasmids expressing either theStreptococcus pyogenesCAS9gene or the guide RNA (gRNA) were employed. However, potential adverse effects due to constitutive expression and the time-consuming process of constructing vectors to express each gRNA remain as a primary barrier for wide adaptation. This report describes the delivery of preassembled CRISPR-Cas9-gRNA ribonucleoproteins (RNPs) via electroporation that is able to generate edited mutant alleles. RNP-mediated CRISPR-Cas9 was used to replace the wild-typeGIB2gene encoding a Gβ-like/RACK1 Gib2 protein with agib2::NATallele via homologous recombination in bothC. neoformansandC. deneoformans. In addition, a DNA plasmid (pCnCas9:U6-gRNA) that expresses both Cas9 and gRNA, allowing for convenient yet low-cost DNA-mediated gene editing, is described. pCnCas9:U6-gRNA contains an endogenous U6 promoter for gRNA expression and restriction sites for one-step insertion of a gRNA. These approaches and resources provide new opportunities to accelerate genetic studies ofCryptococcusspecies.IMPORTANCEFor genetic studies of theCryptococcusgenus, generation of mutant strains is often hampered by a limited number of selectable genetic markers, the tedious process of vector construction, side effects, and other limitations, such as the high cost of acquiring a particle delivery system. CRISPR-Cas9 technology has been demonstrated inCryptococcusfor genome editing. However, it remains labor-intensive and time-consuming since it requires the identification of a suitable type III RNA polymerase promoter for gRNA expression. In addition, there may be potential adverse effects caused by constitutive expressions of Cas9 and gRNA. Here, I report the use of a ribonucleoprotein-mediated CRISPR-Cas9 technique for genome editing ofC. neoformansand related species. Together with the custom-constructed pCnCas9:U6-gRNA vector that allows low-cost and time-saving DNA-based CRISPR-Cas9, my approach adds to the molecular toolbox for dissecting the molecular mechanism of pathogenesis in this important group of fungal pathogens.

scholarly journals Evaluating the cleavage efficacy of CRISPR-Cas9 sgRNAs targeting ineffective regions of Arabidopsis thaliana genome

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11409
Author(s):  
Afsheen Malik ◽  
Alvina Gul ◽  
Faiza Munir ◽  
Rabia Amir ◽  
Hadi Alipour ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genome Editing ◽  
Statistical Tests ◽  
Gc Content ◽  
Proximal Region ◽  
Seed Region ◽  
Stem Loop ◽  
Guide Rnas ◽  
Sgrna Design ◽  
Almost All

The CRISPR-Cas9 system has recently evolved as a powerful mutagenic tool for targeted genome editing. The impeccable functioning of the system depends on the optimal design of single guide RNAs (sgRNAs) that mainly involves sgRNA specificity and on-target cleavage efficacy. Several research groups have designed algorithms and models, trained on mammalian genomes, for predicting sgRNAs cleavage efficacy. These models are also implemented in most plant sgRNA design tools due to the lack of on-target cleavage efficacy studies in plants. However, one of the major drawbacks is that almost all of these models are biased for considering only coding regions of the DNA while excluding ineffective regions, which are of immense importance in functional genomics studies especially for plants, thus making prediction less reliable. In the present study, we evaluate the on-target cleavage efficacy of experimentally validated sgRNAs designed against diverse ineffective regions of Arabidopsis thaliana genome using various statistical tests. We show that nucleotide preference in protospacer adjacent motif (PAM) proximal region, GC content in the PAM proximal seed region, intact RAR and 3rd stem loop structures, and free accessibility of nucleotides in seed and tracrRNA regions of sgRNAs are important determinants associated with their high on-target cleavage efficacy. Thus, our study describes the features important for plant sgRNAs high on-target cleavage efficacy against ineffective genomic regions previously shown to give rise to ineffective sgRNAs. Moreover, it suggests the need of developing an elaborative plant-specific sgRNA design model considering the entire genomic landscape including ineffective regions for enabling highly efficient genome editing without wasting time and experimental resources.

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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 Enhanced Cas12a multi-gene regulation using a CRISPR array separator

2021 ◽  
Author(s):  
Jens P. Magnusson ◽  
Antonio R. Rios ◽  
Lingling Wu ◽  
Lei S. Qi
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Gc Content ◽  
Gene Control ◽  
Naturally Occurring ◽  
Crispr Array ◽  
Endogenous Genes ◽  
Simultaneous Activation ◽  
Type V ◽  
Array Performance

AbstractThe type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

scholarly journals Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

2021 ◽  
Author(s):  
Xiaoen Huang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species ◽  
Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

scholarly journals Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3993-3993
Author(s):  
Linda Yingqi Lin ◽  
Samuele Cancellieri ◽  
Jing Zeng ◽  
Francesco Masillo ◽  
My Anh Nguyen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Clinical Trials ◽  
Genome Editing ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
African Ancestry ◽  
Great Promise ◽  
Personal Genome ◽  
Hematopoietic Stem ◽  
The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

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