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

AlJanahi, Aisha A.; Lazzarotto, Cícera R.; Chen, Shirley; Shin, Tae–Hoon; Cordes, Stefan; Fan, Xing; Jabara, Isabel; Zhou, Yifan; Young, David J.; Lee, Byung‐Chul; Yu, Kyung–Rok; Li, Yuesheng; Toms, Bradley; Tunc, Ilker; Hong, So Gun; Truitt, Lauren L.; Klermund, Julia; Andrieux, Geoffroy; Kim, Miriam; Cathomen, Toni; Gill, Saar; Tsai, Shengdar Q.; Dunbar, Cynthia E.

doi:10.1016/j.ymthe.2021.06.016

Cited by 18 publications

(8 citation statements)

References 62 publications

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“…CAST-Seq is a highly sensitive assay to nominate off-target (OT) sites by identifying gross chromosomal aberrations, such as large deletions and inversions at the on-target sites of designer nucleases. It was applied successfully to samples treated with various CRISPR-Cas9 nucleases ( Turchiano et al, 2021 ; AlJanahi et al, 2022 ) but, to date, only to a single TALEN pair targeting the HBB locus ( Turchiano et al, 2021 ). The fact that the bioinformatic nomination of an off-target site is fundamentally different for a dimeric TALEN pair as compared to a monomeric CRISPR-Cas nuclease, prompted us to revisit OT-activity of previously published TALEN targeting the CCR5 gene ( Mussolino et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

et al. 2023

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Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.

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Section: Resultsmentioning

confidence: 99%

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

et al. 2023

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“… 10 Similarly, we did not appreciate any negative impacts of our editing strategy on cell viability or engraftment potential, despite the previously reported Cas9-induced p53 induction or the known occurrence of large scale genomic rearrangements caused by double-strand break formation. 13 , 14 …”

Section: Discussionmentioning

confidence: 99%

Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates

Petty,

Radtke,

Fields

et al. 2023

Molecular Therapy - Methods & Clinical Development

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“…Off-target sites were selected as previously described ( 65 ), using an in-house Python script modified for the rhesus macaque genome from the published prediction algorithm (https://zlab.bio/guide-design-resources). Each potential off-target site that has 3 or less mismatches to the gRNA were scored and among them, the top 11 predicted sites were selected for design of flanking PCR primers (Table S2).…”

Section: Methodsmentioning

confidence: 99%

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

Lee,

Gin,

et al. 2023

Preprint

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For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively.CD33knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction inCD33KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

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

Cited by 18 publications

References 62 publications

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

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