Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9

Abstract

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.

Chromosome engineering in zygotes with CRISPR/Cas9

“…Although deletions of 65 kb have been generated by zygote injection previously (Zhang et al ., 2015), this is relatively small and the relationship between size and efficiency was unknown. We also wished to establish a reliable method for generating large duplications.…”

Customizing the genome as therapy for the β-hemoglobinopathies

“…47,48,58 Introduction of 2 engineered nucleases can result in targeted deletion or inversion, duplication, local indels at nuclease cleavage sites, or translocations/ chromosomal rearrangements. [60][61][62][63][64][65][66][67][68][69][70][71][72][73] Here, we review genome-editing approaches for genetic correction and disruption strategies for the b-hemoglobinopathies as summarized in Figure 1 and Table 1.…”

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

“…Therefore, this system can be used to generate genetically modified mice rapidly by microinjecting sgRNAs with Cas9 into fertilized eggs [4,5,6,7,8,9]. Several studies have reported the induction of an efficient CRISPR/Cas9-mediated genomic rearrangement such as deletion or inversion of ~100 kb [10,11,12]. However, the maximum size of indels that can be induced by the CRISPR/Cas9 system is still unclear.…”