Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Abstract

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

The yin and yang of kidney development and Wilms’ tumors

“…Mice might very well be one of the most difficult model organisms to model Wilms' tumors. However, the recent advances in gene editor technologies, such as zinc finger nucleases, TAL effector nucleases, and CRISPR/Cas9, have opened up genomic engineering possibilities in these species to comparable levels that so far have only been possible in mice (Li et al 2013a,b;Honda et al 2014;Park et al 2014;Yang et al 2014). Maybe the time is ripe to reconsider the best possible experimental model for Wilms' tumor research.…”

CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening

“…plasmodium [12], Cryptosporidium [13], tunicates [14], wheat [15], rice [16], tomato [17], silk worms [18], C. elegans [19], beetles [20], sea lampreys [21], zebrafish [22], salmons [23], pigs [24], rats [25], goats [26], rabbits [27], and many more).…”

The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding

“…In these studies, only a single construct for expression of Cas9 nuclease and a specifically designed sgRNA are needed for transformation. Since then, due to its ease of implementation and robustness the CRISPR-Cas9 system has been utilized widely for genome engineering in various organisms, including plants [13–15], insects [16], fish [17], rabbits [18], pigs [19], mice [20], monkeys [21], and human cells [22, 23]. A large number of publications using the CRISPR-Cas9 technology came up rapidly since the first reports and promoted our understanding and applications of the system.…”