Gene Editing Using ssODNs with Engineered Endonucleases

Chen, Fuqiang; Pruett-Miller, Shondra M.; Davis, Gregory D.

doi:10.1007/978-1-4939-1862-1_14

Cited by 29 publications

(23 citation statements)

References 22 publications

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“…Although HR is typically less efficient, it is often more desirable because it can lead to a precise genome modification, including the knockin of fluorescent markers or loxP sites to make conditional alleles. The donor DNA for HR can be introduced from plasmids or as a single-stranded oligonucleotide [159]; the latter approach has recently been improved by taking advantage of the asymmetric release of one DNA end from the strand not bound to the RNA [160] and by using modified (phosphorothioate) single-stranded oligonucleotide which are more stable in cells [161]. Cas9 and the gRNA can be expressed in cells from expression vectors or the Cas9 mRNA and guide RNA can be directly transfected.…”

Section: Democratization Of Genome Editing: Crispr-cas9mentioning

confidence: 99%

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair

2016

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DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

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Section: Democratization Of Genome Editing: Crispr-cas9mentioning

confidence: 99%

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair

2016

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“…Although oligodeoxynucleotides (ODNs) probably do not serve as natural HDR donors, many laboratories have reported that PGE can be routinely obtained by exogenously transfecting ODN donors into cells (Chen et al 2015;Richardson et al 2016). Since ODN donors are likely unable to physically form Holliday junctions and therefore cannot engage DSBR, they must perforce utilize some form of the SDSA or ssDI pathways during PGE (Storici et al 2006;Davis and Maizels 2014).…”

mentioning

confidence: 99%

Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussianlike distributions. Interestingly, single-nick-induced PGE using ODN donors produced conversion tracts biased either mostly uni-or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.

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“…First, a ribonucleoprotein complex (RNP) comprised of sgRNA mixed with recombinant Cas9 endonuclease was delivered into schistosome eggs isolated from livers of experimentally-infected mice (eggs termed ‘LE’, ‘liver eggs’) by electroporation. In addition, Homology Directed Repair (HDR) of CRISPR/Cas9-induced DSBs at the ω1 locus in the presence of a donor DNA template was investigated [39–41]. A single stranded oligodeoxynucleotide (ssODN) of 124 nt in length was delivered to some LE as a template for HDR of chromosomal double stranded breaks (DSBs) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Ittiprasert

Mann

Karinshak

et al. 2018

Preprint

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34 35 CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested 36 this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This 37 secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative 38 immuno-pathological readouts for programmed genome editing. Schistosome eggs were either 39 exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary 40 to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and 41 the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor 42 transgene that encoded six stop codons, flanked by 50 nt-long 5'-and 3'-microhomology arms 43 matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso 44 analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, 45deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion 46 of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ω1-edited 47 eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the 48 ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including 49 IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines 50followed the exposure to those of ω1-mutated schistosome eggs. Following injection of 51 schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas 52surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome 53 editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by 54 homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the 55 capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of 56 pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype 57showcase the novel application of programmed gene editing in and functional genomics for 58 schistosomes. 59 60 61 Introduction 62 63Schistosomiasis is considered the most problematic of the human helminth diseases in terms of 64 morbidity and mortality [1][2][3][4]. The past decade has seen major advances in knowledge and 65 understanding of the pathophysiology, developmental biology, evolutionary relationships and 66 genome annotation of the human schistosomes [5][6][7][8][9][10][11][12][13][14][15][16]. Establishing CRISPR/Cas9 genome 67 editing in schistosomiasis would greatly enable effective functional genomics approaches. The 68 stable CRISPR/Cas9-based site-specific gene mutation and phenotyping will drive innovation 69 and a deeper understanding of schistosome pathogenesis, biology and evolution [17]. 70 71The schistosome egg plays a central role both in disease transmission and pathogenesis [1]. The 72 appearance of S. mansoni eggs in host tissues by six to seven week...

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Gene Editing Using ssODNs with Engineered Endonucleases

Cited by 29 publications

References 22 publications

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair

Mechanisms of precise genome editing using oligonucleotide donors

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

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