Abstract
Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Highlights
Complex genome modification techniques utilizing sitespecific knockin (KI) to achieve gene expression labeling by protein tags or gene inactivation or reactivation via Cre/loxP system have advanced remarkably since the emergence of engineered endonucleases such as TALENs and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system (Zu et al, 2013; Auer et al, 2014; Shin et al, 2014; Li et al, 2015; Hoshijima et al, 2016; Sugimoto et al, 2017; Burg et al, 2018; Luo et al, 2018; Li et al, 2019, 2020)
This donor vector could be introduced into the CRISPR/Cas9 site in the target gene through targeted insertion in either orientation via the nonhomologous end joining (NHEJ) pathway activated by the CRISPR/Cas9 system (Fig. S1) (Lin et al, 2014)
We developed an efficient flipping donor strategy based on dual-functional forward and reverse (FoRe) and bidirectional FoRe (Bi-FoRe) donors to simultaneously generate positive and negative conditional alleles, coupled with fluorescent allele tagging/geno-tagging, through NHEJ-mediated targeted insertion facilitated by the CRISPR/Cas system
Summary
Complex genome modification techniques utilizing sitespecific knockin (KI) to achieve gene expression labeling by protein tags or gene inactivation or reactivation via Cre/loxP system have advanced remarkably since the emergence of engineered endonucleases such as TALENs and the CRISPR/Cas system (Zu et al, 2013; Auer et al, 2014; Shin et al, 2014; Li et al, 2015; Hoshijima et al, 2016; Sugimoto et al, 2017; Burg et al, 2018; Luo et al, 2018; Li et al, 2019, 2020) These technical developments are important in organisms lacking embryonic stem cell-based approaches, including zebrafish (Danio rerio). A method which could simultaneously generate fluorescent labeling of both positive and negative conditional alleles in the same target gene through a single experiment has not been established
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
