Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology
Channabasavaiah B. Gurumurthy,
Surinder K Batra,
Kathleen A Becker,
James D Eudy,
Donald W Harms,
Kathryn E Hentges,
Catherine B Lawrence,
Moorthy P Ponnusamy,
Ronald J Redder,
Clifford J. Rosen,
Mark T Ruhe,
Ane M Salvador,
Brandon J Willis,
Christian S Wright,
Daniel J. Garry,
Antony D Adamson,
William R. Thompson,
KC. Kent Lloyd,
Joshua A Wood,
Joseph M Miano,
Posted 30 Aug 2018
bioRxiv DOI: 10.1101/393231 (published DOI: 10.1186/s13059-019-1776-2)
Posted 30 Aug 2018
CRISPR–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.
- Downloaded 2,467 times
- Download rankings, all-time:
- Site-wide: 2,572 out of 89,274
- In genetics: 200 out of 4,618
- Year to date:
- Site-wide: 15,000 out of 89,274
- Since beginning of last month:
- Site-wide: 35,845 out of 89,274
Downloads over time
Distribution of downloads per paper, site-wide
- 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!