DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing

Xue, Chuang; Greene, Eric C.

doi:10.1016/j.tig.2021.02.008

Cited by 145 publications

(135 citation statements)

References 225 publications

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“…In fact, treated mice had an increase of about 1% of plasma hFIX while coagulation activity after the tail clip test was not statistically different from the one observed in untreated FIX KO mice. The lower efficacy observed in adult animals in the presence of nucleases could be related to the post-mitotic condition of the adult liver, in which DNA damage is mainly corrected by NHEJ and not by HDR ( Xue and Greene, 2021 ), limiting the overall gene targeting efficiency, as already observed in other models ( Yang et al, 2016 ). On the contrary, in the absence of nucleases we observed similar gene targeting rates in neonate and adult treatments ( Barzel et al, 2015 ).…”

Section: Discussionmentioning

confidence: 94%

“…In our previous work, we showed that the GeneRide strategy ( Barzel et al, 2015 ) coupled to the CRISPR/Cas9 platform efficiently targets the therapeutic transgene to the albumin locus in a Crigler-Najjar mouse model, leading to the complete rescue from neonatal lethality and phenotype abnormalities ( De Caneva et al, 2019 ). Importantly, this strategy is efficiently applicable to neonatal mice where hepatocytes are actively proliferating and the homologous directed repair (HDR) mechanism is active ( Xue and Greene, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

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Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Lisjak

Caneva²,

Marais³

et al. 2022

Front. Genome Ed.

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Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Lisjak

Caneva²,

Marais³

et al. 2022

Front. Genome Ed.

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“…Due to locus-dependent and physiological variables (e.g., gRNA efficiency, chromatin feature, sub-telomere/telomere location, and cell cycle), gene-editing efficiency can vary between loci and experiments. ( Ceccaldi et al., 2016 ; Gisler et al., 2019 ; Huang et al., 2021 ; Schep et al., 2021 ; Xue and Greene, 2021 ). The use of two separate RNPs, targeting DNA regions within proximity to the desired location of editing may increase editing efficiency if single RNP transformation does not generate the desired mutation.…”

Section: Limitationsmentioning

confidence: 99%

CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae

Huang

Cook

2022

STAR Protocols

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Summary Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae ), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .

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“…Here, we will use the term TMEJ to distinguish between the theta-mediated end joining pathway and alternative end joining in general, which may encompass other end joining activity. If a homologous sequence is present, TMEJ can act upon ssDNA revealed by short range end-resection (∼20 bp) ( Zhao B. et al, 2020 ; Xue and Greene, 2021 ). Whether TMEJ can also act upon long-range resected ends remains to be proven ( Truong et al, 2013 ).…”

Section: Double Strand Break Repair Pathways Contribution Is Influenced By Resection Rangementioning

confidence: 99%

“…The fourth form of DSB repair is SSA, which is also considered an alternative repair pathway and is less frequently activated. SSA acts upon long-range resected DNA ends (>1000 bp), utilizing homologies of more than 50 bp to anneal homologous sequences ( Zhao B. et al, 2020 ; Xue and Greene, 2021 ). The key player in SSA is Rad52 which interacts with the RPA coated ssDNA and aligns the complementary regions.…”

Section: Double Strand Break Repair Pathways Contribution Is Influenced By Resection Rangementioning

confidence: 99%

DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation

et al. 2021

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The superior dose distribution of particle radiation compared to photon radiation makes it a promising therapy for the treatment of tumors. However, the cellular responses to particle therapy and especially the DNA damage response (DDR) is not well characterized. Compared to photons, particles are thought to induce more closely spaced DNA lesions instead of isolated lesions. How this different spatial configuration of the DNA damage directs DNA repair pathway usage, is subject of current investigations. In this review, we describe recent insights into induction of DNA damage by particle radiation and how this shapes DNA end processing and subsequent DNA repair mechanisms. Additionally, we give an overview of promising DDR targets to improve particle therapy.

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DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing

Cited by 145 publications

References 225 publications

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae

DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation

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