scholarly journals 5' modifications improve potency and efficacy of DNA donors for precision genome editing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Krishna S Ghanta ◽  
Zexiang Chen ◽  
Aamir Mir ◽  
Gregoriy A Dokshin ◽  
Pranathi M Krishnamurthy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Triethylene Glycol ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Cultured Human Cells ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

scholarly journals 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

2018 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Gregoriy A. Dokshin ◽  
Aamir Mir ◽  
Pranathi Meda Krishnamurthy ◽  
Hassan Gneid ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genetic Basis ◽  
Cell Types ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.

scholarly journals Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

2021 ◽  
Vol 22 (16) ◽  
pp. 8571
Author(s):  
Christopher E. Denes ◽  
Alexander J. Cole ◽  
Yagiz Alp Aksoy ◽  
Geng Li ◽  
G. Gregory Neely ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Nuclear Dna ◽  
Great Promise ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Small Molecule Modulators

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

scholarly journals Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V

2021 ◽  
Author(s):  
Dimitra Synefiaridou ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genome Editing ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Human Pathogen ◽  
Gram Positive Bacteria ◽  
Homology Directed Repair ◽  
Dna Fragment ◽  
Opportunistic Human Pathogen ◽  
Template Dna

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells’ DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be potentially transported to other Gram-positive bacteria. Importance Streptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts in identifying new vaccine candidates. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

scholarly journals Precision genome editing in the CRISPR era

2017 ◽  
Vol 95 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Jayme Salsman ◽  
Graham Dellaire
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Point Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genetic Changes ◽  
New Era ◽  
Homology Directed Repair ◽  
Repair Template ◽  
Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

scholarly journals Author response: Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

2018 ◽  
Author(s):  
Natasa Savic ◽  
Femke CAS Ringnalda ◽  
Helen Lindsay ◽  
Christian Berk ◽  
Katja Bargsten ◽  
...  
Keyword(s):  
Dna Repair ◽  
Author Response ◽  
Covalent Linkage ◽  
Homology Directed Repair ◽  
Cas9 Nuclease ◽  
Repair Template

scholarly journals Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Dimitra Synefiaridou ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genome Editing ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Human Pathogen ◽  
Gram Positive Bacteria ◽  
Homology Directed Repair ◽  
Dna Fragment ◽  
Opportunistic Human Pathogen ◽  
Template Dna

AbstractCRISPR systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a novel, replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells’ DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain, can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches should be readily transportable to other Gram-positive bacteria.ImportanceStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and vaccine candidate efforts. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

scholarly journals Base and Prime Editing Technologies for Blood Disorders

2021 ◽  
Vol 3 ◽  
Author(s):  
Panagiotis Antoniou ◽  
Annarita Miccio ◽  
Mégane Brusson
Keyword(s):  
Genome Editing ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Great Promise ◽  
Blood Disorders ◽  
Major Drawback ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Advantages And Disadvantages

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches–including the delivery of base editors and the off-target events–and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

scholarly journals Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

2021 ◽  
Author(s):  
Irina Sizova ◽  
Simon Kelterborn ◽  
Valeriy Verbenko ◽  
Suneel Kateriya ◽  
Peter Hegemann
Keyword(s):  
Dna Repair ◽  
Chlamydomonas Reinhardtii ◽  
Genome Editing ◽  
Gene Targeting ◽  
Gene Inactivation ◽  
End Joining ◽  
Repair Gene ◽  
Algal Biotechnology ◽  
Homology Directed Repair ◽  
Random Integration

Abstract The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent non-homologous end-joining and polymerase theta (POLQ)-mediated end-joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect homology-directed repair of the inactivated paromomycin resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate homology-directed repair/gene inactivation. Our data suggests that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced homology-directed repair and random integration of transgenes, while KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in Chlamydomonas reinhardtii and can be used for future development of algal biotechnology.

scholarly journals Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

2018 ◽  
Author(s):  
Roswitha A. Aumann ◽  
Marc F. Schetelig ◽  
Irina Häecker
Keyword(s):  
Control Systems ◽  
Genome Editing ◽  
Pest Control ◽  
Genetically Modified Organisms ◽  
Ceratitis Capitata ◽  
Fluorescent Protein ◽  
Great Promise ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes

AbstractBackgroundThe Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic.ResultsHere we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring.ConclusionConsidering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

Sign in / Sign up

Export Citation Format

Share Document