Targeted truncation of the ADAM17 cytoplasmic domain in mice results in protein destabilization and a hypomorphic phenotype.

A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17-/- mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.

Generation of Mouse Model (KI and CKO) via Easi-CRISPR.

Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).

New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing.

CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5 kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.

Inhibition of mTORC1 kinase activates Smads 1 and 5 but not Smad8 in human prostate cancer cells, mediating cytostatic response to rapamycin.

Although hyperactivated mTOR is well recognized as being pivotal to prostate cancer growth and progression, the underlying mechanisms by which it promotes such responses remain incompletely understood. Here, we show that rapamycin activates Smads 1 and 5 in human prostate cancer cells and tissues through blocking mTORC1 kinase. Small hairpin RNA-based gene silencing and gene overexpression approaches reveal that Smads 1 and 5 mediate, whereas Smad8 represses, rapamycin-induced cell death and expression of the bone morphogenetic protein (BMP) transcriptional target Id1 in human prostate cancer cell lines. Moreover, such phospho-Smad1/5-mediated rapamycin responses were blocked by LDN-193189 (a BMPRI kinase inhibitor) or Noggin (a BMP antagonist) in LNCaP prostate cancer cells. Likewise, the mTOR kinase inhibitors Ku-0063794 and WYE-354 each enhanced phosphorylation of Smad1/5. Intriguingly, silencing raptor alone enhanced, whereas silencing rictor repressed, the phosphorylation of Smad1/5, indicating that mTORC1 represses, whereas mTORC2 activates, BMP signaling. Immunohistochemical analysis showed increased levels of phospho-Smad1/5 concomitant with suppression of phospho-S6 and survivin levels in PC3 human prostate cancer xenografts in athymic mice administered rapamycin (intraperitoneally, 5 mg/kg/d, 2-6 days). Moreover, we show that compared with prostate tumor tissue from untreated patients, levels of phospho-Smad1/5 were significantly elevated in the prostate tumor tissue of patients with high-risk prostate cancer who received 8 weeks of the rapalog everolimus as part of a neoadjuvant clinical trial before undergoing local definitive therapy by radical prostatectomy. Taken together, our data implicate Smads 1, 5 and 8 as potential prognostic markers and therapeutic targets for mTOR inhibition therapy of prostate cancer.

Insulin-like growth factor I suppresses bone morphogenetic protein signaling in prostate cancer cells by activating mTOR signaling.

Insulin-like growth factor (IGF) I and bone morphogenetic proteins (BMP) are critical regulators of prostate tumor cell growth. In this report, we offer evidence that a critical support of IGF-I in prostate cancer is mediated by its ability to suppress BMP4-induced apoptosis and Smad-mediated gene expression. Suppression of BMP4 signaling by IGF-I was reversed by chemical inhibitors of phosphoinositide 3-kinase (PI3K), Akt, or mTOR; by enforced expression of wild-type PTEN or dominant-negative PI3K; or by small hairpin RNA-mediated silencing of mTORC1/2 subunits Raptor or Rictor. Similarly, IGF-I suppressed BMP4-induced transcription of the Id1, Id2, and Id3 genes that are crucially involved in prostate tumor progression through PI3K-dependent and mTORC1/2-dependent mechanisms. Immunohistochemical analysis of non-malignant and malignant prostate tissues offered in vivo support for our model that IGF-I-mediated activation of mTOR suppresses phosphorylation of the BMP-activated Smad transcription factors. Our results offer the first evidence that IGF-I signaling through mTORC1/2 is a key homeostatic regulator of BMP4 function in prostate epithelial cells, acting at two levels to repress both the proapoptotic and pro-oncogenic signals of BMP-activated Smads. We suggest that deregulation of this homeostatic control may be pivotal to the development and progression of prostate cancer, providing important implications and new potential targets for the therapeutic intervention of this malignancy.