Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

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 realtime 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.

Zebrafish Cre/loxregulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

“…We recovered rb1-UFlip-On and rbbp4-UFlip-On precise integration alleles at a frequency of 4% (1/25) and 9% (1/11), and rb1-UFlip-Off and hdac1-UFlip-Off alleles at a frequency of 6% (2/33) and 10% (1/10), a reasonable number of founders to screen. Previously reported methods for recovery of intron targeted floxed conditional alleles varied widely in frequency, from 12% for homology directed integration from a linear template with long 1-1.5 kb homology arms (Hoshijima et al, 2016), 53% by homologous recombination from a circular plasmid with 1 kb long homology arms (Sugimoto et al, 2017), to 56% – 60% for integration by Non-Homologous End Joining after preselection of a primary reporter (Han et al, 2021; Li et al, 2019). Our results demonstrate efficient recovery of precision integration alleles without requiring preselection of embryos expressing a fluorescent primary reporter, expanding the possibilities for novel integration cassette design and gene modification.…”

“…We updated the UFlip construct by replacing the mRFP in the gene trap with 2A-RFP or 2A-KalTA4, which resulted in robust primary reporter expression after targeting in Tol2<14XUAS:RFP> embryos injected with hdac1-2AKalTA4-UFlip-Off or rb1-2AKalTA4-UFlip-Off , or WIK embryos injected with rbbp4-2AmRFP-UFlip-Off . Integration alleles generated with the updated UFlip construct will allow fluorescent labeling of cells with Cre/ lox conditional gene inactivation, as described in other recent studies (Han et al, 2021; Hans et al, 2021; Li et al, 2019), which is critical for lineage tracing and capturing mutant cells for downstream genomics applications.…”

“…Cre/ lox regulated conditional control of gene function in zebrafish was previously limited to generation of knock out alleles by random Tol2 transposon insertional mutagenesis with a floxed/FRT gene trap, which can be reverted by Cre or Flip mediated recombination (Clark et al, 2011; Ni et al, 2012; Trinh le et al, 2011). Only recently have gene editing methods been developed in zebrafish to simplify Cre driver isolation (Almeida et al, 2021; Kesavan et al, 2018) and generate genuine lox -regulated conditional alleles (Burg et al, 2018; Han et al, 2021; Hoshijima et al, 2016; Li et al, 2019; Sugimoto et al, 2017) by targeted integration. These advances in zebrafish Cre/ lox genetics are being driven by rapidly evolving methods for homology directed gene editing using CRISPR/Cas9 and TALEN endonucleases.…”

“…Stable inversion of the Zwitch cassette by Cre led to effective shha gene knockdown and defective shha signaling. More recently homology independent targeting strategies have been described to generate conditional knockouts with a linked reporter, or bidirectional knockin of a dual reporter gene trap cassette, providing a method for labeling conditional gene knockout cells (Han et al, 2021; Li et al, 2019). As an alternative to isolation of stable germline conditional alleles, rapid tissue specific gene knockdown and cell labeling can be achieved by somatic targeting with a floxed Cas9-2A-GFP; U6:gRNA transposon in a Cre-specific transgenic background, allowing for simultaneous bi-allelic inactivation with GFP cell labeling (Hans et al, 2021).…”

ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

“…Recently, Cas12a identified from Eubacterium rectale (ErCas12a, also known as MAD7 nuclease), was reported to induce efficient mutagenesis when injected as mRNA in zebrafish embryos, but also requires heatshock treatment [11]. Interestingly, the integration of large DNA fragments into the zebrafish genome has been frequently reported with the use of Cas9 in the form of mRNA [12,14,16,17,20,23,24], while, to date, no studies have reported successful NEHJ-or MMEJ-mediated knockin in zebrafish using the highly efficient RNP form of Cas9 [25][26][27]. Therefore, we hypothesized that the mRNA-active ErCas12a could be an ideal tool for large fragment knockin in zebrafish, particularly for homology-based approaches, which might be facilitated by the staggered DSBs.…”