scholarly journals A Pipeline for Precise and Efficient Genome Editing by sgRNA-Cas9 RNPs in Drosophila

2020 ◽  
Author(s):  
Kevin G. Nyberg ◽  
Joseph Q. Nguyen ◽  
Yong-Jae Kwon ◽  
Shelby Blythe ◽  
Greg J. Beitel ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Background ◽  
Target Genes ◽  
Region Of Interest ◽  
Cutting Efficiency ◽  
Eyes Absent ◽  
Homology Directed Repair ◽  
Eye Morphology ◽  
Cas9 Protein ◽  
Balancer Chromosomes

ABSTRACTGenome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

scholarly journals A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Daisuke Matsumoto ◽  
Hirokazu Tamamura ◽  
Wataru Nomura
Keyword(s):  
Cell Cycle ◽  
Genome Editing ◽  
Target Genes ◽  
Genetic Alterations ◽  
Homology Directed Repair ◽  
Activation System ◽  
A Cell ◽  
Cycle Dependent ◽  
Natural Inhibitors ◽  
Target Effects

Abstract The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

scholarly journals Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

2021 ◽  
Vol 2 ◽  
Author(s):  
Daniel Allen ◽  
Michael Rosenberg ◽  
Ayal Hendel
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Nuclear Staining ◽  
Two Component System ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Therapeutic Benefits ◽  
A Genome ◽  
Cas9 Protein

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair

2016 ◽  
Author(s):  
Tony Gutschner ◽  
Monika Hämmerle ◽  
Giannicola Genovese ◽  
Giulio F. Draetta ◽  
Lynda Chin
Keyword(s):  
Cell Cycle ◽  
Protein Engineering ◽  
Genome Editing ◽  
Homology Directed Repair ◽  
Cas9 Protein

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

scholarly journals Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz0051 ◽  
Author(s):  
Xinyu Ling ◽  
Bingteng Xie ◽  
Xiaoqin Gao ◽  
Liying Chang ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Specific Chemical ◽  
Site Specific ◽  
Additional Tool ◽  
Homology Directed Repair ◽  
Chemically Modified ◽  
Dna Template ◽  
Oligonucleotide Conjugates

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

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 Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

2019 ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A.M. Martin ◽  
Pierre Boudinot ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Cell Line ◽  
Genome Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Genetic Resistance ◽  
Model Organisms ◽  
Fish Cell ◽  
Genome Wide ◽  
Lentivirus Transduction

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

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