Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms To whom correspondence should be addressed alex.chavez@wyss.harvard.edu and gchurch@genetics.med.harvard.edu. Contributions: A.C. and M.T. conceived of the study. A.C., M.T., B.W.P., and R.C. designed and performed experiments. S.J.H., R.J.C., and J.B. performed experiments.B.E.C, B.E.H and N.P. designed and performed all experiments in Drosophila Melanogaster. D.T-O and E.J.K.K. performed RNA-seq experiments and analyzed data. J.J.C and G.M.C. supervised the study. A.C. and M.T. wrote the manuscript with support from all authors. Conflict of interest: G.M.C. has equity in Editas and Caribou Biosciences. Accession codes: Primary accessions: Gene Expression Omnibus GSE80611 HHS Public Access Author manuscript Nat Methods. Author manuscript; available in PMC 2016 November 23. Published in final edited form as: Nat Methods. 2016 July ; 13(7): 563–567. doi:10.1038/nmeth.3871. A uhor M anscript

Comparison of Cas9 activators in multiple species

A large number of computational tools have been documented in recent years for identification of target-specific valid single-guide (sg) RNAs (18–20 nucleotide long sequence) that is an important component for the efficient utilization of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats—CRISPR-associated Protein) system. Despite optimization of Cas9, other major concerns are on-target efficiency and off-target activity that depend upon the sequence(s) of target-specific sgRNA(s). However, a very little attention has been paid for identification of the best-hit sgRNA for precise targeting as well as minimizing the off-target effects. The aim of this present work is to offer comparative insight into existing CRISPR software tools with their unique features (including targeted genome) and utilities. These available web tools were found to be designed based upon only a few limited mathematical models. Among all these available web tools, three (Benchling, Desktop and CRISPR-P) have been curated as exclusively available for plant genome-editing purpose. These three software tools have been comprehensively described and analyzed with single same target enquiry from two randomly selected genes (IDM2 and IDM3 from Arabidopsis thaliana). Interestingly, all these selected tools generated different results (sgRNAs) even for the same query. In fact, the sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. Thus, there is an urgent requirement to pay attention for a validated sgRNA-designing tool for precise DNA editing in plants. In conclusion, this work will encourage building up a consensus for developing a universal valid sgRNA designing for different organisms including plants.

/pdf/an-overview-of-designing-and-selection-of-sgrnas-for-precise-4ya2scic9l.pdf

An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants

Die Pflanzenzucht nutzte bisher ungezielte und zeitintensive Verfahren, um Verbesserungen in den agronomischen Eigenschaften unserer Kulturpflanzen zu erreichen. Dies hat sich in den vergangenen Jahren grundlegend geändert, denn mit dem CRISPR/Cas‐System steht nun ein hochpräzises und zuverlässiges Werkzeug zur Verfügung, um diese Ziele schnell und effizient zu erreichen. Durch die programmierbare Induktion von Doppelstrangbrüchen können gezielt an fast jeder beliebigen Stelle im Genom die gewünschten Veränderungen vorgenommen werden, die sich nicht mehr von natürlichen Veränderungen unterscheiden lassen. So entstanden bereits eine Reihe von Kulturpflanzen mit agronomisch interessanten Eigenschaften. Die momentane Charakterisierung weiterer CRISPR/Cas‐Systeme erweitert nicht nur unseren molekularen Werkzeugkasten, sondern beginnt die gesamte Biotechnologie und Biologie grundlegend zu verändern.

Das CRISPR/Cas‐System

Snail-borne parasitic diseases represent an important challenge to human and animal health. Control strategies that target the intermediate snail host has proved very effective. Epigenetic mechanisms are involved in developmental processes and therefore play a fundamental role in developmental variation. DNA methylation is an important epigenetic information carrier in eukaryotes that plays a major role in the control of chromatin structure. Epigenome editing tools have been instrumental to demonstrate functional importance of this mark for gene expression in vertebrates. In invertebrates, such tools are missing, and the role of DNA methylation remains unknown. Here we demonstrate that methylome engineering can be used to modify in vivo the CpG methylation level of a target gene in the freshwater snail Biomphalaria glabrata, intermediate host of the human parasite Schistosoma mansoni. We used a dCas9-SunTag-DNMT3A complex and synthetic sgRNA to transfect B. glabrata embryos and observed an increase of CpG methylation at the target site in 50% of the hatching snails.

/pdf/hit-and-run-epigenetic-editing-for-vectors-of-snail-borne-2jnajipm.pdf

Hit-and-Run Epigenetic Editing for Vectors of Snail-Borne Parasitic Diseases

/pdf/development-of-tools-for-genetic-manipulation-of-5gbux4pqn7.pdf

Development of tools for genetic manipulation of Staphylococcus aureus

Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries. Since its inception as an exciting new genome-engineering platform, the CRISPR/Cas (CRISPR associated protein) system has evolved to be more than just a tool for targeted mutagenesis. Adapted from its natural role as part of the bacterial adaptive immune system, the CRISPR system has been simplified to consist of a single-guide RNA (sgRNA) and Cas nuclease (most commonly spCas9 derived from Streptococcus pyogenes). Comprised of nuclease binding stem-loop structures and a ∼20 bp 5'- overhang for sequence-specific DNA recognition, a Cas9-loaded sgRNA scans the DNA for its complementary target sequence. Novel applications for the CRISPR/Cas system have emerged with the disabling of the nuclease domains of the Cas9 protein, resulting in a catalytically-inactive Cas9 nuclease (dCas9). This was done by introducing two point mutations, H840A to the HNH domain and D10A to the RUVC domain, which eliminated its ability to cleave either strand of DNA while maintaining its sequence-specific DNA recognition capability (Jinek et al., 2012). Acting as a programmable DNA binding protein, the CRISPR gene-editing system may now be repurposed to carry out new functions such as manipulating gene expression and labeling loci for dynamic cell imaging. In this prospect article, we discuss a few of the new and exciting uses of the CRISPR system, beyond its most popular application as a designer nuclease. This article is protected by copyright. All rights reserved

A CRISPR reimagining: New twists and turns of CRISPR beyond the genome-engineering revolution.

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A CRISPR reimagining: New twists and turns of CRISPR beyond the genome-engineering revolution.