scholarly journals Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids

2021 ◽  
Vol 4 (10) ◽  
pp. e202000940
Author(s):  
Maarten H Geurts ◽  
Eyleen de Poel ◽  
Cayetano Pleguezuelos-Manzano ◽  
Rurika Oka ◽  
Léo Carrillo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Adult Stem Cell ◽  
Target Site ◽  
Whole Genome ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Oncogenic Mutations ◽  
Cancer Modeling ◽  
Adenine Base ◽  
Target Effects

Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell–derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9–mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing–repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

scholarly journals Evaluating CRISPR-based Prime Editing for cancer modeling and CFTR repair in intestinal organoids

2020 ◽  
Author(s):  
Maarten H. Geurts ◽  
Eyleen de Poel ◽  
Cayetano Pleguezuelos-Manzano ◽  
Léo Carrillo ◽  
Amanda Andersson-Rolf ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Application ◽  
Adult Stem Cell ◽  
Great Promise ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Intestinal Organoids ◽  
Oncogenic Mutations ◽  
Cancer Modeling ◽  
Adenine Base

AbstractPrime editing is a recently reported genome editing tool employing a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. The technique holds great promise for clinical application due to its versatility. Here, we explore the use of prime editing in human intestinal organoids. Common TP53 mutations were modeled in human adult stem cell with notable efficiency differences. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology directed repair and adenine base editing on the CFTR-R785* mutation. Despite encountering varying editing efficiencies and undesired mutations, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

scholarly journals Prime editing for functional repair in patient-derived disease models

2020 ◽  
Author(s):  
Imre F. Schene ◽  
Indi P. Joore ◽  
Rurika Oka ◽  
Michal Mokry ◽  
Anke H.M. van Vugt ◽  
...  
Keyword(s):  
Adult Stem Cells ◽  
Gene Editing ◽  
Therapeutic Potential ◽  
Gene Encoding ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Genome Wide ◽  
Culture Models ◽  
Liver Organoids ◽  
Target Effects

AbstractPrime editing is a novel genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. We develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers diseasecausing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

scholarly journals Prime editing for functional repair in patient-derived disease models

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Imre F. Schene ◽  
Indi P. Joore ◽  
Rurika Oka ◽  
Michal Mokry ◽  
Anke H. M. van Vugt ◽  
...  
Keyword(s):  
Adult Stem Cells ◽  
Gene Editing ◽  
Therapeutic Potential ◽  
Gene Encoding ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Genome Wide ◽  
Culture Models ◽  
Liver Organoids ◽  
Target Effects

Abstract Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

scholarly journals Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

2019 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Target Site ◽  
Gene Repression ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

scholarly journals Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Science ◽  
2019 ◽  
pp. eaav9973 ◽  
Author(s):  
Erwei Zuo ◽  
Yidi Sun ◽  
Wu Wei ◽  
Tanglong Yuan ◽  
Wenqin Ying ◽  
...  
Keyword(s):  
Spontaneous Mutation ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Spontaneous Mutation Rate ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Cell Embryo ◽  
Adenine Base ◽  
Target Effects

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

scholarly journals Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Syed A. K. Shifat Ahmed ◽  
Michelle Rudden ◽  
Sabrina M. Elias ◽  
Thomas J. Smyth ◽  
Roger Marchant ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Genomic Islands ◽  
Clinical Strain ◽  
Whole Genome ◽  
Synonymous Mutations ◽  
Wide Range

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

scholarly journals Mapping CRISPR-Cas9 public and commercial innovation using The Lens institutional toolkit

2021 ◽  
Author(s):  
Osmat Azzam Jefferson ◽  
Simon Lang ◽  
Kenny Williams ◽  
Deniz Koellhofer ◽  
Aaron Ballagh ◽  
...  
Keyword(s):  
Public Investment ◽  
Open Data ◽  
Regulatory Elements ◽  
Policy Makers ◽  
Base Editing ◽  
The Public ◽  
Homology Directed Repair ◽  
A Genome ◽  
Institutional Capability ◽  
Cellular Dna

AbstractCRISPR-Cas9 is a revolutionary technology because it is precise, fast and easy to implement, cheap and components are readily accessible. This versatility means that the technology can deliver a timely end product and can be used by many stakeholders. In plant cells, the technology can be applied to knockout genes by using CRISPR–Cas nucleases that can alter coding gene regions or regulatory elements, alter precisely a genome by base editing to delete or regulate gene expression, edit precisely a genome by homology-directed repair mechanism (cellular DNA), or regulate transcriptional machinery by using dead Cas proteins to recruit regulators to the promoter region of a gene. All these applications can be for: 1) Research use (Non commercial), 2) Uses related product components for the technology itself (reagents, equipment, toolkits, vectors etc), and 3) Uses related to the development and sale of derived end products based on this technology. In this contribution, we present a prototype report that can engage the community in open, inclusive and collaborative innovation mapping. Using the open data at the Lens.org platform and other relevant sources, we tracked, analyzed, organized, and assembled contextual and bridged patent and scholarly knowledge about CRISPR-Cas9 and with the assistance of a new Lens institutional capability, The Lens Report Builder, currently in beta release, mapped the public and commercial innovation pathways of the technology. When scaled, this capability will also enable coordinated editing and curation by credentialed experts to inform policy makers, businesses and private or public investment.

scholarly journals Effective and precise adenine base editing in mouse zygotes

2018 ◽  
Vol 9 (9) ◽  
pp. 808-813 ◽  
Author(s):  
Puping Liang ◽  
Hongwei Sun ◽  
Xiya Zhang ◽  
Xiaowei Xie ◽  
Jinran Zhang ◽  
...  
Keyword(s):  
Base Editing ◽  
Mouse Zygotes ◽  
Adenine Base

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

2021 ◽  
Vol 4 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Pin Lyu ◽  
Zuyan Lu ◽  
Sung-Ik Cho ◽  
Manish Yadav ◽  
Kyung Whan Yoo ◽  
...  
Keyword(s):  
Base Editing ◽  
Adenine Base

scholarly journals BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

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