scholarly journals Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

2020 ◽  
Author(s):  
Benedetta Artegiani ◽  
Delilah Hendriks ◽  
Joep Beumer ◽  
Rutger Kok ◽  
Xuan Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Intestinal Cell ◽  
Precise Integration ◽  
Exogenous Dna ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Efficient Generation

AbstractCRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homology-independent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

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 Efficient Homology-directed Repair with Circular ssDNA Donors

2019 ◽  
Author(s):  
Sukanya Iyer ◽  
Aamir Mir ◽  
Joel Vega-Badillo ◽  
Benjamin P. Roscoe ◽  
Raed Ibraheim ◽  
...  
Keyword(s):  
Genome Editing ◽  
K562 Cells ◽  
Cost Effective ◽  
Cell Types ◽  
Traffic Light ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Cost Effective Method ◽  
Circular Ssdna ◽  
Fluorescent Tag

AbstractWhile genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

scholarly journals Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

2021 ◽  
Author(s):  
Jichen Bao ◽  
Silvan Scheller
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Value Added ◽  
Primary Metabolism ◽  
Methanococcus Maripaludis ◽  
Knock Out ◽  
Homology Directed Repair ◽  
A Genome ◽  
Product Scope ◽  
Positive Rate

Methanococcus maripaludis is a fast-growing and genetically tractable methanogen. To become a useful host organism for the biotechnological conversion of CO2 and renewable hydrogen to fuels and value-added products, its product scope needs to be extended. Metabolic engineering requires reliable and efficient genetic tools, in particular for genome editing related to the primary metabolism that may affect cell growth. We have constructed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery natively present in M. maripaludis. The toolbox enables gene knock-out with a positive rate typically above 89%, despite M. maripaludis being hyper-polyploid. We have replaced the flagellum operon (around 8.9kb) by a beta-glucuronidase gene to demonstrate a larger deletion, and to enable quantification of promotor strengths. The CRISPR/LbCas12a toolbox presented here is currently perhaps the most reliable and fastest method for genome editing in a methanogen.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

scholarly journals Rapid genome editing by CRISPR-Cas9-POLD3 fusion

2021 ◽  
Author(s):  
Ganna Reint ◽  
Zhuokun Li ◽  
Kornel Labun ◽  
Salla Keskitalo ◽  
Inkeri Soppa ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Cell Types ◽  
Cell Type ◽  
Repair Protein ◽  
Specific Manner ◽  
Dna Repair Protein ◽  
Cell Type Specific

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

scholarly journals Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

2020 ◽  
Vol 21 (19) ◽  
pp. 7353 ◽  
Author(s):  
Karim Shalaby ◽  
Mustapha Aouida ◽  
Omar El-Agnaf
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Viral Vectors ◽  
Scale Up ◽  
Cell Types ◽  
Delivery Systems ◽  
Different Cell Types ◽  
Immunogenic Response ◽  
Cas Gene

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.

scholarly journals Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

2020 ◽  
Vol 48 (7) ◽  
pp. e38-e38 ◽  
Author(s):  
Sara E DiNapoli ◽  
Raul Martinez-McFaline ◽  
Caitlin K Gribbin ◽  
Paul J Wrighton ◽  
Courtney A Balgobin ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Cell Types ◽  
Specific Cell ◽  
Loss Of Function ◽  
Genomic Deletions ◽  
Homology Directed Repair ◽  
Linear Dsdna ◽  
Rapid Generation

Abstract CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

scholarly journals Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does

2015 ◽  
Vol 20 (5) ◽  
Author(s):  
Silvia Petrezselyova ◽  
Slavomir Kinsky ◽  
Dominika Truban ◽  
Radislav Sedlacek ◽  
Ingo Burtscher ◽  
...  
Keyword(s):  
Quality Control ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Southern Hybridization ◽  
Embryonic Stem ◽  
Fluorescent Reporter ◽  
Exogenous Dna ◽  
Knock Out ◽  
Endogenous Genes ◽  
Pcr Method

AbstractClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

scholarly journals BMP signalling is critical for maintaining homeostasis of hair follicles and intestine in adult mice

2017 ◽  
Author(s):  
Aditi Nag ◽  
Pallavi Nigam ◽  
Abhishek L. Narayanan ◽  
Megha Kumar ◽  
Ritika Ghosal ◽  
...  
Keyword(s):  
Cell Types ◽  
Hair Follicles ◽  
Intestinal Cell ◽  
Knock Out ◽  
Adult Mice ◽  
Rapid Changes ◽  
Anagen Hair ◽  
Mouse Intestine ◽  
Bmp Signalling

AbstractBMP signalling play critical roles during embryonic development, however, its roles have not been extensively studied in the maintenance of adult tissue homeostasis. In this study we have used temporal knock out of Bmp2 and Bmp4 to uncover the role of BMP signalling in adult mice. We observed rapid changes in the adult hair follicles and the intestine within two days of depleting BMP signalling, which demonstrates the critical and acute requirement of BMP signalling in maintaining homeostasis in these tissues. In addition, our study demonstrates that while BMP signalling is required for maintenance of quiescence in telogen hair follicles, it is needed for survival and proliferation in late anagen hair follicles. Our study also reveals differential requirement of BMP signalling in differentiation of distinct intestinal cell types. In addition, Loss of BMP signalling rapidly promotes early neoplastic transformations in mouse intestine.

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.

Sign in / Sign up

Export Citation Format

Share Document