Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.

Objectives: To determine if the timing of excess fluid accumulation (fluid overload) is associated with adverse patient outcomes. Design: Secondary analysis of a prospectively collected dataset. Setting: PICU of a tertiary care hospital. Patients: Children 3 months to 25 years old admitted to the PICU with expected length of stay greater than or equal to 48 hours. Interventions: Patients were dichotomized by time of peak overload: peak fluid overload from ICU admission (Day0) to 48 hours (Day3–7) and peak fluid overload value after 48 hours of ICU admission, as well as time of first-time negative daily fluid balance: net fluid out greater than net fluid in for that 24-hour period. Measurements and Main Results: There were 177 patients who met inclusion criteria, 92 (52%) male, with an overall mortality rate of 7% (n = 12). There were no differences in severity of illness scores or fluid overload on Day0 between peak fluid overload from ICU admission (Day0) to 48 hours (Day3–7) (n = 97; 55%) and peak fluid overload value after 48 hours of ICU admission (n = 80; 45%) groups. Peak fluid overload value after 48 hours of ICU admission was associated with a longer median ICU course (8 [4–15] vs 4 d [3–8 d]; p ≤ 0.001], hospital length of stay (18 [10–38) vs 12 [8–24]; p = 0.01], and increased risk of mortality (n = 10 [13%] vs 2 [2%]; χ2 = 7.6; p = 0.006]. ICU length of stay was also longer in the peak fluid overload value after 48 hours of ICU admission group when only patients with at least 7 days of ICU stay were analyzed (p = 0.02). Timing of negative fluid balance was also correlated with outcome. Compared with Day0–2, a negative daily fluid balance on Day3–7 was associated with increased length of mechanical ventilation (3 [1–7] vs 1 d [2–10 d]; p ≤ 0.001) and increased hospital (17 [10–35] vs 11 d [7–26 d]; p = 0.006) and ICU (7 [4–13] vs 4 d [3–7 d]; p ≤ 0.001) length of stay compared with a negative fluid balance between Day0–2. Conclusions: Our results show timing of fluid accumulation not just peak percentage accumulated is associated with patient outcome. Further exploration of the association between time and fluid accumulation is warranted.

BACKGROUND:The current study was conducted to compare simultaneously obtained bone marrow (BM) cytogenetics (CTG), peripheral blood (PB) and BM fluorescence in situ hybridization (FISH), and quantitative real-time polymerase chain reaction (Q-PCR) for BCR-ABL1 in monitoring response to treatment with tyrosine kinase inhibitors and homoharringtonine (HHT) in patients with chronic myeloid leukemia (CML). METHODS: PB and BM FISH (n ¼ 112 samples) and/or Q-PCR (n ¼ 132 samples) for BCR-ABL1 were simultaneously obtained in 70 patients with Philadelphia chromosome-positive (Phþ) CML in chronic (68%), accelerated (16%), and blast phase (16%) before the initiation of therapy and during the course of treatment with imatinib (IM) (n ¼ 40 patients), dasatinib (n ¼ 20 patients), nilotinib (n ¼ 4 patients), bosutinib (n ¼ 18 patients), or HHT (n ¼ 4 patients) for patients with newly diagnosed (n ¼ 13 patients), IM-sensitive (n ¼ 34 patients), IM-resistant (n ¼ 30 patients), or IM-intolerant (n ¼ 9 patients) disease. Eighteen patients were found to have Phþ variants or karyotypic abnormalities in addition to the Phþ. RESULTS: Excellent correlations (r) were observed between PB and BM FISH (r ¼ 0.95) and PB and BM Q-PCR (r ¼ 0.87), as well as BM CTG and PB FISH (r ¼ 0.89) and PB Q-PCR (r ¼ 0.82). This correlation was not affected by the presence of the Phþ variant or additional chromosomal abnormalities, the presence of ABL1 kinase domain mutations, phase of the disease, or treatment. CONCLUSIONS: PB FISH and Q-PCR appear to be reliable methods with which to monitor response to modern therapy in patients with all phases of CML.

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