CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.
AimsUK guidance was recently developed for the treatment of anal cancer using intensity-modulated radiotherapy (IMRT). We audited the current use of radiotherapy in UK cancer centres for the treatment of anal cancer against such guidance. We describe the acute toxicity of IMRT in comparison with patient population in the audit treated with two-phase conformal radiotherapy and the previous published data from two-phase conformal radiotherapy, in the UK ACT2 trial.Materials and methodsA Royal College of Radiologists' prospective national audit of patients treated with radiotherapy in UK cancer centres was carried out over a 6 month period between February and July 2015.ResultsTwo hundred and forty-two cases were received from 40/56 cancer centres (71%). In total, 231 (95%) underwent full dose radiotherapy with prophylactic nodal irradiation. Of these, 180 (78%) received IMRT or equivalent, 52 (22%) two-phase conformal (ACT2) technique. The number of interruptions in radiotherapy treatment in the ACT2 trial was 15%. Interruptions were noted in 7% (95% confidence interval 0–14%) of courses receiving two-phase conformal and 4% (95% confidence interval 1–7%) of those receiving IMRT. The percentage of patients completing the planned radiotherapy dose, irrelevant of gaps, was 90% (95% confidence interval 82–98%) and 96% (95% confidence interval 93–99%), in two-phase conformal and IMRT respectively. The toxicity reported in the ACT2 trial, in patients receiving two-phase conformal in the audit and in patients receiving IMRT in the audit was: any toxic effect 71%, 54%, 48%, non-haematological 62%, 49%, 40% and haematological 26%, 13%, 18%, respectively.ConclusionsIMRT implementation for anal cancer is well underway in the UK with most patients receiving IMRT delivery, although its usage is not yet universal. This audit confirms that IMRT results in reduced acute toxicity and minimised treatment interruptions in comparison with previous two-phase conformal techniques.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.