Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.
Abstract. The use of the split-middle line of progress, trend line, to measure teacher effectiveness in the literacy field is discussed. Three case studies are presented to illustrate the use of trend lines in evaluating best practices in providing effective literacy instruction for students in elementary classrooms. Initiating interactive dialogue and shared reading responses are the two strategies used to represent the probability of performance change in planning literacy lesson instruction. This discussion is an innovative use in the literacy field of observational data recording that graphically demonstrates inclining and declining confirmed effect and interpretive probability. The authors' intentions are to challenge educators to more closely examine graphic representation or reflect upon the frequency of use and effectiveness of reading strategies that may improve reading performance in literacy settings.
Writing is regarded as a particularly demanding process involving complex higher level thinking processes combined with the demands of certain meta-cognitive skills. Since academic success may be predicted by the student's level of reading comprehension and writing skills and with the recent adoption of the Common Core State Standards, it is an ideal time for conceptualizing how to improve writing instruction for elementary students. This mixed-methods study investigated the effects of process writing instruction on the development of second and third grade students' writing abilities focusing on wide reading and inquiry, writing frames, technology netbooks, and grammar/mechanics. Results indicate that all students involved advanced at least two stages on Gunning's scale of writing proficiency, increased in number of words written, addressed problems in writing mechanics, and improved selective language use. Findings are relevant to classroom teachers, specialists, and administrators alike.
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