Dataset Information

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

ABSTRACT: The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

SUBMITTER: AlJanahi AA

PROVIDER: S-EPMC8753565 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

AlJanahi Aisha A AA Lazzarotto Cicera R CR Chen Shirley S Shin Tae-Hoon TH Cordes Stefan S Fan Xing X Jabara Isabel I Zhou Yifan Y Young David J DJ Lee Byung-Chul BC Yu Kyung-Rok KR Li Yuesheng Y Toms Bradley B Tunc Ilker I Hong So Gun SG Truitt Lauren L LL Klermund Julia J Andrieux Geoffroy G Kim Miriam Y MY Cathomen Toni T Gill Saar S Tsai Shengdar Q SQ Dunbar Cynthia E CE

Molecular therapy : the journal of the American Society of Gene Therapy 20210624 1

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corr ...[more]

PMID: 34174439

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Project description:BackgroundUnhealthy consumption of alcohol is a major public health crisis with strong associations between immunological dysfunctions, high vulnerability to infectious disease, anemia, and an increase in the risk of hematological malignancies. However, there is a lack of studies addressing alcohol-induced changes in bone marrow (BM) and hematopoiesis as fundamental aspects of immune system function.MethodsTo address the effect of chronic alcohol consumption on hematopoietic stem and progenitor cells (HSPCs) and the BM niche, we used an established rhesus macaque model of voluntary alcohol drinking. A cohort of young adult male rhesus macaques underwent a standard ethanol self-administration protocol that allowed a choice of drinking alcohol or water 22 hours/day with periods of forced abstinence that elevated subsequent intakes when alcohol availability resumed. Following the last month of forced abstinence, the monkeys were euthanized. HSPCs and bone samples were collected and analyzed in functional assays and by confocal microscopy.ResultsHSPCs from alcohol animals exhibited reduced ability to form granulocyte-monocyte and erythroid colonies in vitro. HSPCs also displayed a decrease in mitochondrial oxygen consumption linked to ATP production and basal respiratory capacity. Chronic alcohol use led to vascular remodeling of the BM niche, a reduction in the number of primitive HSPCs, and a shift in localization of HSPCs from an adipose to a perivascular niche.ConclusionsOur study demonstrates, for the first time, that chronic voluntary alcohol drinking in rhesus macaque monkeys leads to the long-term impairment of HSPC function, a reduction in mitochondrial respiratory activity, and alterations in the BM microenvironment. Further studies are needed to determine whether these changes in hematopoiesis are persistent or adaptive during the abstinent period and whether an initial imprinting to alcohol primes BM to become more vulnerable to future exposure to alcohol.

Project description:Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94-99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40-75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental in vivo platform to test innovative interventions aimed at curing HIV infection in humans.

Dataset Information

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

Publications

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

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