Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology, bioRxiv, 2018-08-31

AbstractCRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

biorxiv genetics 200-500-users 2018

SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues, bioRxiv, 2018-08-28

SUMMARYFluorescent in situ hybridization (FISH) reveals the abun-dance and positioning of nucleic acid sequences in fixed sam-ples and can be combined with cell segmentation to produce a powerful single cell gene expression assay. However, it re-mains difficult to label more than a few targets and to visu-alize nucleic acids in environments such as thick tissue sam-ples using conventional FISH technologies. Recently, meth-ods have been developed for multiplexed amplification of FISH signals, yet it remains challenging to achieve high lev-els of simultaneous multiplexing combined with high sam-pling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that serve as targets for sensitive fluores-cent detection. We establish that SABER effectively ampli-fies the signal of probes targeting nucleic acids in fixed cells and tissues, can be deployed against at least 17 targets si-multaneously, and detects mRNAs with high efficiency. As a demonstration of the utility of SABER in assays involv-ing genetic manipulations, we apply multiplexed FISH of reporters and cell type markers to the identification of en-hancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit to allow rapid and cost effective multiplexed imaging.

biorxiv genetics 200-500-users 2018

 

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