Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage: Techniques for Detecting, Predicting, and Mitigating the On- and off-target Effects of Cas9 Editing.

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.

The Expanding Class 2 CRISPR Toolbox: Diversity, Applicability, and Targeting Drawbacks.

The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.

Variability of ethics education in laboratory medicine training programs: results of an international survey.

BACKGROUND: Ethical considerations are increasingly important in medicine. We aimed to determine the mode and extent of teaching of ethics in training programs in clinical chemistry and laboratory medicine. METHODS: We developed an on-line survey of teaching in areas of ethics relevant to laboratory medicine. Reponses were invited from directors of training programs who were recruited via email to leaders of national organizations. RESULTS: The survey was completed by 80 directors from 24 countries who directed 113 programs. The largest numbers of respondents directed postdoctoral training of scientists (42%) or physicians (33%), post-masters degree programs (33%), and PhD programs (29%). Most programs (82%) were 2years or longer in duration. Formal training was offered in research ethics by 39%, medical ethics by 31%, professional ethics by 24% and business ethics by 9%. The number of reported hours of formal training varied widely, e.g., from 0 to >15h/year for research ethics and from 0 to >15h for medical ethics. Ethics training was required and/or tested in 75% of programs that offered training. A majority (54%) of respondents reported plans to add or enhance training in ethics; many indicated a desire for online resources related to ethics, especially resources with self-assessment tools. CONCLUSION: Formal teaching of ethics is absent from many training programs in clinical chemistry and laboratory medicine, with heterogeneity in the extent and methods of ethics training among the programs that provide the training. A perceived need exists for online training tools, especially tools with self-assessment components.

How to Write a Great Research Paper, and Get it Accepted by a Good Journal.

BACKGROUND: Knowing the best way of structuring your paper when writing it, and the most appropriate journal to send it to, really helps in getting your paper accepted. Also understanding how editors and publishers think and what they expect, and knowing how the peer review process works, is invaluable insight into the publishing process. RESULTS: After attending this two hour workshop, one in the Elsevier Publishing Connect Workshop series, participants will have a clear idea of the steps needed to be taken before starting to write a paper. They will also be able to plan writing manuscripts using the logical step sequence - not the sequence in which the paper will be read. Authors are also made aware of what aspects of their papers Editors and Publishers look at critically, and to ensure that in taking care of these areas, their papers are much more likely to be accepted. Dealing with referees' comments and the art of polite rebuttal are also described such that these can be used to improve the submitted paper suitably. Sensitive areas such as publishing ethics, plagiarism, duplicate publishing, etc are also clearly explained such that participants have a clear understanding of what is allowed, and what is not permitted. CONCLUSIONS: These insights into the publishing process will enable the participants to be more confident as an author in the world of science publishing, and will help them get their papers published more easily.

How to Write a Scientific Paper: Practical Guidelines.

Precise, accurate and clear writing is essential for communicating in health sciences, as publication is an important component in the university criteria for academic promotion and in obtaining funding to support research. In spite of this, the development of writing skills is a subject infrequently included in the curricula of faculties of medicine and allied health sciences. Therefore clinical investigators require tools to fill this gap. The present paper presents a brief historical background to medical publication and practical guidelines for writing scientific papers for acceptance in good journals.

Ketone enolization with lithium dialkylamides: the effects of structure, solvation, and mixed aggregates with excess butyllithium.

The effects of lithium dialkylamide structure, mixed aggregate formation, and solvation on the stereoselectivity of ketone enolization were examined. Of the lithium dialkylamides examined, lithium tetramethylpiperidide (LiTMP) in THF resulted in the best enolization selectivity. The stereoselectivity was further improved in the presence of a LiTMP-butyllithium mixed aggregate. The use of less polar solvents reduced the enolization stereoselectivity. Ab initio calculations predict LDA and LiTMP to form mixed cyclic dimers in ethereal solvents. The calculations also predict LiTMP-alkyllithium mixed aggregates to competitively inhibit the formation of less stereoselective LiTMP-lithium enolate mixed aggregates.