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 Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

2021 ◽  
Author(s):  
Eleonora I. Ioannidi ◽  
Matthew T. N. Yarnall ◽  
Cian Schmitt-Ulms ◽  
Rohan N. Krajeski ◽  
Justin Lim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
High Efficiency ◽  
End Joining ◽  
Differentiated Cells ◽  
Dividing Cells ◽  
Gene Integration ◽  
Fluorescent Tagging ◽  
Non Homologous End Joining ◽  
Drag And Drop

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

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 Cas9-mediated genome editing in the methanogenic archaeonMethanosarcina acetivorans

2017 ◽  
Vol 114 (11) ◽  
pp. 2976-2981 ◽  
Author(s):  
Dipti D. Nayak ◽  
William W. Metcalf
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Genetic System ◽  
Nonhomologous End Joining ◽  
Methanosarcina Acetivorans ◽  
End Joining ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Insertion And Deletion

Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeonMethanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon,Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.

scholarly journals Improved methods and optimized design for CRISPR Cas9 and Cas12a homology-directed repair

2021 ◽  
Author(s):  
Mollie S Schubert ◽  
Bernice Thommandru ◽  
Jessica Woodley ◽  
Rolf Turk ◽  
Shuqi Yan ◽  
...  
Keyword(s):  
Optimized Design ◽  
End Joining ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Mammalian Cell Lines ◽  
Non Homologous End Joining ◽  
Dna Template ◽  
Donor Template

CRISPR-Cas proteins are used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a DNA template during repair allows for precise introduction of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein complexes with synthetic guide RNAs. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. Additionally, we employ chemically modified HDR donor templates in combination with a small molecule to boost HDR efficiency up to 10-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: www.idtdna.com/HDR

scholarly journals Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pan P. Li ◽  
Russell L. Margolis
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Neurodegenerative Disorder ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
End Joining ◽  
Human Ipscs ◽  
Non Homologous End Joining ◽  
Transient Overexpression ◽  
Donor Template

AbstractCas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

CRISPR/Cas9 mediated knockout of the DES gene alleles with desminopathy-related heterozygous gain-of-function mutations

2021 ◽  
pp. 37-44
Author(s):  
К.С. Кочергин-Никитский ◽  
А.В. Лавров ◽  
Е.В. Заклязьминская ◽  
С.А. Смирнихина
Keyword(s):  
Genome Editing ◽  
Genetic Defect ◽  
Gene Mutations ◽  
Respiratory Muscles ◽  
Surgical Approaches ◽  
Nonsense Mutations ◽  
Guide Rna ◽  
Guide Rnas ◽  
Patient State ◽  
Targeted Nucleases

Наследственные кардиомиопатии характеризуются неблагоприятным прогнозом и низкой пятилетней выживаемостью пациентов с выраженной клиникой. При этом лечение, за исключением хирургического, в основном паллиативное, во многих случаях лишь трансплантация сердца может улучшить состояние пациента и прогноз. Часть наследственных кардиомиопатий ассоциирована с аутосомно-доминантными мутациями в гене DES, кодирующем белок промежуточных филаментов десмин, дефекты в котором ведут к развитию десминопатий с вовлечением наиболее активно работающих мышц - скелетных, миокарда, мышц дыхательной системы. Новые терапевтические подходы, основанные на методах геномного редактирования, могут позволить устранить каузативный генетический дефект. Так как имеются данные об отсутствии клинических симптомов у людей с гетерозиготными нонсенс мутациями в гене DES, по-видимому, имеется возможность снизить тяжесть протекания десминопатий путем нокаута мутантного аллеля в случае гетерозиготной мутации. Целью работы являлась проверка возможности специфического нокаута аллелей гена DES, несущих гетерозиготные мутации, ассоциированные с десминопатиями, методами геномного редактирования. Нами был получен генетический материал трех пациентов с десминопатиями, связанными с мутациями в гене DES (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Направляющие РНК, совместимые с нуклеазами SaCas9 и eSpCas9(1.1), были подобраны, используя онлайн сервис Benchling, и клонированы в плазмиды, несущие соответствующие эндонуклеазы Cas9. Редактирующие плазмиды котрансфицировали в клетки HEK293T вместе с «таргетными» плазмидами, содержащими участки гена DES с мутациями. Анализ характерных для негомологичного соединения концов инделов в выделенной из клеток спустя 48 часов после трансфекции тотальной ДНК проводился посредством TIDE-анализа полученных сиквенсов целевых участков, либо методом Т7Е1 анализа. Наибольшая средняя эффективность 2,22% (до 8,06%) показана при использовании sgRNA на мутацию c.330_338del в комбинации с eSpCas9(1.1). Эффективность других комбинаций направляющих РНК и Cas9 не превышала 3%. Достигнутая эффективность нокаута очевидно недостаточна для коррекции десминопатии на уровне организма. Необходимость специфического нокаутирования мутантных аллелей не позволяет использовать другие направляющие РНК для CRISPR/Cas9, поэтому необходимо совершенствование разработанных систем для повышения их эффективности либо использование новых, более эффективных, направляемых нуклеаз. Hereditary cardiomyopathies are characterized by the generally poor prognosis and low 5-year survival of patients with severe symptoms. Besides surgical approaches, cardiomyopathy therapy mainly palliative and often heart transplantation is the only option to improve patient state and prognosis. Some of these pathologies are associated with the autosomal-dominant DES gene mutations. DES encodes intermediate filaments protein desmin, which defects causes desminopathies involving most active muscles such as skeletal muscles, myocardium and respiratory muscles. New therapeutic based on genome editing approaches could be used to correct causative genetic defect. There are data that heterozygous nonsense mutations in DES gene may be asymptomatic. Thus there is, apparently, a possibility to decrease severity of desminopathy using mutant allele knockout. Purpose. The aim of this work was to test the possibility of specific knockout of the DES gene alleles with heterozygous desminopathy-associated mutations by means of genome editing methods. Materials. We received genetic materials of three patients with desminopathy caused by DES gene mutations (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Guide RNA, compatible with nucleases SaCas9 and eSpCas9(1.1) were designed using online service Benchling and cloned into plasmids with corresponding Cas9 nucleases. Editing plasmids were cotransfected into HEK293T cells with “target” plasmids, containing DES gene sites with mutations. NHEJ-produced indels were assessed using TIDE-analysis with amplified and sequenced sites or using T7E1 analysis. Results. Combination sgRNA for c.330_338del with eSpCas9(1.1) demonstrated most mean efficiency of 2,22% (up to 8,06%). Others combinations of sgRNAs and Cas9 efficiency did not overcome 3%. Conclusions. Achieved knockout efficiency is evidently not enough for organism-level desminopathy correction. The need for specific knockout of mutated alleles does not allow usage of different guide RNAs for CRISPR/Cas9, so it is necessary to improve the developed systems to increase their efficiency or to use new, more efficient, targeted nucleases.

scholarly journals Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Tao Guo ◽  
Yi-Li Feng ◽  
Jing-Jing Xiao ◽  
Qian Liu ◽  
Xiu-Na Sun ◽  
...  
Keyword(s):  
Genome Editing ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals A modular cloning toolkit for genome editing in plants

2019 ◽  
Author(s):  
Florian Hahn ◽  
Andrey Korolev ◽  
Laura Sanjurjo Loures ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Higher Order ◽  
Plant Science ◽  
Golden Gate ◽  
Coding Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Science Community ◽  
Modular Cloning ◽  
Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Sign in / Sign up

Export Citation Format

Share Document