Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Transvenous embolization of vein of galen aneurysmal malformations with coils as a final procedure for cure: A single-institution experience of 18 years.

INTRODUCTION: Staged, transarterial embolization (TAE) is currently considered the gold standard for the treatment of vein of Galen aneurysmal malformation (VGAM); however, as transarterial access becomes restricted, further staged TAE may become ineffective or carry an increased risk of hemorrhagic or ischemic stroke when attempting complete obliteration. OBJECTIVE: To describe the first consecutive, retrospective series of VGAM treated with transvenous embolization (TVE) with coils alone, as the final treatment in staged endovascular therapy, at a single institution between January 2004 and September 2021. RESULTS: A total of 10 patients with a median age of 5.5 (IQR: 9.25) years were treated with coiling TVE. Patients were treated with a median number of 5 (IQR: 2.75) TAEs prior to the final TVE treatment. Complete or near-complete immediate angiographic obliteration was achieved in eight patients. Immediate post-procedural (within 48 h) hemorrhagic complications were noted in two patients (20%), one of whom passed away while the second suffered from hemiparesis. Stereotactic radiosurgery was performed in two patients with incomplete obliteration after TVE. The median follow-up time after TVE was 17 (IQR: 9) months. At long-term follow up (17 months) for the remaining nine patients, all VGAMs were completely obliterated. Long-term clinical deterioration compared to pre-TVE was noticed in one case. CONCLUSION: Transvenous coil embolization is a technically feasible but risky option, as a final-stage treatment for cure of VGAMs with restricted trans-arterial access. Although TVE with coils remains an effective therapeutic modality, we recommend continuing investigation of safer TVE techniques to achieve cure.

Transvenous embolization of vein of galen aneurysmal malformations using the "Chapot pressure cooker" technique.

METHODS: Two patients, one 5-year-old and one 7-year-old, both presented with congestive heart failure in the newborn period and were subsequently treated in the newborn period with multiple, staged TAEs with n-BCA for choroidal VGAMs. RESULTS: We achieved progressive reduction in shunting and flow but were unable to accomplish complete closure of the malformation: in both patients, a small residual with numerous perforators persisted. The decision was made to perform TVE using the CHPC. In this technique, a guiding catheter is placed transjugular into the straight sinus (SS). One or two detachable tip microcatheters are advanced to the origin of the SS. Another microcatheter is advanced and the tip placed between the distal marker and the detachment zone of the former. Coils and n-BCA are used to prevent reflux of Onyx. CONCLUSIONS: In this study, we recognized two important factors of traditional VGAM treatment that may cause interventionalists to consider the ChPC to treat VGAM: (1) without liquid embolic, deployed coils may not occlude the fistula entirely. (2) There is the concern of causing delayed bleeding should the arterial component of the fistula rupture. ChPC ameliorates these issues by offering complete closure of the fistula with liquid embolic material in TVE.