Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system

Abstract

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms.

CRISPR/Cas9, a universal tool for genomic engineering

“…Причем выявление этой зависимо сти представляет предмет активных научных дискуссий (Xiao et al, 2013;Canver et al, 2014;He et al, 2015;Kraft et al, 2015;Song et al, 2016). Согласно данным из работы Kraft с коллегами (2015), взаимосвязь между размером модификации и частотой ее возникновения под действием CRISPR/Cas9-системы в эмбриональных стволовых клет ках мыши отмечена не была.…”

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

“…Taking the advantage of highly efficient and specific DNA cleavage, CRISPR/Cas9 technology was used to successfully modify human cells at target sites precisely [6]. Compared with ZFNs and TALENs, CRISPR/Cas9 system revealed its accessible, versatile and precise merits for targeted genome editing in a large variety of species, such as mouse [7], rat [8], pig [9], goat [10], rabbit [11], zebrafish [12] and even plants [13,14]. …”

CRISPR/Cas9–Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits

“…The protocol for microinjection of pronuclear-stage zygotes has been described previously by Song 11 and Yuan et al 12 A mixture of Cas9 mRNA (200 ng/lL) and CRYAA-sgRNA (50 ng/ lL) was coinjected into the cytoplasms of pronuclear-stage zygotes. These zygotes were then immediately transferred into the oviducts of surrogate rabbits.…”

“…Vector construction and in vitro transcription were carried out as described previously by Song et al 11 Two complementary DNA oligonucleotides were annealed at 958C for 5 minutes to generate double-strand DNA, which was then cloned into a BbsIdigested pUC57-simple vector expressing Cas9 (Addgene ID 51307). In vitro transcription was performed using commercial kits (mMessagem Machine SP6 and MAXI script T7; Ambion, Austin, TX, USA).…”