One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas mediated genome engineering

By Hui Yang, Haoyi Wang, C. Shivalila, A. Cheng, Linyu Shi, R. Jaenisch. Cell, 2013

1,586 citations recorded by the source corpus.

SUMMARY The type II bacterial CRISPR/Cas system is a novel genome engineering technology with the ease of multiplexed gene targeting. Here we created reporter and conditional mutant mice by co-injection of zygotes with Cas9 mRNA, different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2 and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/454c35d7d3e31bcca5b97416e570dfbeb80bb3c4

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Citations in the corpus

A CRISPR/Cas9 toolkit for multiplex genome editing in plants (2014, background): “The CRISPR/Cas system has been harnessed to achieve efficient genome editing in a variety of organisms, including bacteria, yeast, plants, and animals, as well as human cell lines [12-27].”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, positive): “…animal models, Cas9 protein and transcribed sgRNA can be directly injected into fertilized zygotes to achieve heritable gene modification at one or multiple alleles in models such as rodents and monkeys (Wang et al., 2013; Li et al., 2013; Yang et al., 2013; Niu et al., 2014) (Figure 6D).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “Even circularized plasmid DNA can undergo this phenomenon (Yang et al., 2013).”
High-Content Screening for Quantitative Cell Biology. (2016, background): “Likewise, CRISPR/Cas9-mediated knock-in of a desired reporter cassette [63] (e.g., a gene encoding a ﬂ uorescent protein fused to a gene of choiceattheendogenouslocus)inhighereukaryoteswillenable invivo imagingandreducepotentialreagenttoxicityand the need for preparative procedures associated with…”