Targeted chromosomal deletions and inversions in zebrafish

Abstract

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%–15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

“…Importantly, engineered chromosomally stable clones could be obtained using a standard subcloning and PCR screening procedure. Large deletions up to 24 megabases have been reported using zinc finger nucleases (Lee et al 2010(Lee et al , 2012 or TALENs (Gupta et al 2013;Kim et al 2013;Xiao et al 2013), but the efficiency cannot always be inferred because few studies reported the isolation of single clones carrying these deletions. In cases when single clones were isolated, they were retrieved at a surprisingly high frequency (;0.5%) , given the fact that TALENs are generally believed to be less efficient than the CRISPR/Cas9 system.…”

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.…”

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

“…Several studies indicate that simultaneous targeting of two loci using two pairs of TALENs can induce large genomic deletions [34][35][36][37], inversions as well as chromosomal translocations [38,39]. Using this dual TALEN approach, we and other groups have demonstrated that large deletions of 1-80 kb can be efficiently created in zebrafish [33,[40][41][42]. These large deletions have been used to knock out non-coding RNA genes, gene clusters and gene regulatory elements.…”