One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

Abstract

CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no ‘two-in-one’ KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.

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

“…The other is that we pre-selected fluorescence-positive founder (F 0 ) embryos after injection of the targeting system and before rearing for germline transmission screening. The importance of pre-selection for the enrichment of F 0 embryos bearing correct insertions has been demonstrated in our previous publication (Li et al, 2019) One of the major improvements of our Bi-FoRe strategy is the successful utilization of insertions occurring in both directions, due to the special design of our donor vector. Bidirectional KI enables concurrent generation of positive and negative alleles, allowing for in vivo monitoring of gene expression and conditional manipulation of gene function.…”

“…Based on these allele pairs, we successfully achieved conditional knockout and conditional rescue in parallel, both coupled with switch of fluorescent reporter genes, from positive and negative conditional alleles at the sox10 locus, respectively. Similar to other organisms, KI in zebrafish could be achieved by either homology-dependent or -independent approaches (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;Li et al, 2020). Inclusion of homology arms in the donor allows for precise integration of the donor through HR, while dramatically compromising the KI efficiency.…”

“…Complex genome modification techniques utilizing site-specific 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 particularly important in organisms lacking embryonic stem cell-based approaches, including zebrafish ( Danio rerio ).…”

“…Li et al reported a fluorescent gene-tagging method which introduced a fluorescent reporter into the last intron via nonhomologous end joining (NHEJ)-mediated targeted insertion in zebrafish using the CRISPR/Cas system (Li et al, 2015 ), taking advantage of its higher KI efficiency than homologous recombination (HR) (Auer et al, 2014 ). Recently, we and others further improved the technique and established “two-in-one” dual-function KI strategies to achieve conditional knockout (CKO) coupled with in-frame fusion of double fluorescent reporters to label two different alleles (positive/normal vs. negative/defective), facilitated by specially designs of dual-cassette donors (Li et al, 2019 , 2020 ). However, generation of conditional rescue alleles through these new approaches has not been reported, and a method for simultaneously generating CKO and conditional rescue allele pairs is also not available.…”

Scanning Various Nanomaterials and Variable Camouflages to Evaluate the Biocompatibility in Multi-organs of Embryos

“…[17] Since zebra sh has regeneration capability in its organs such as ns, central nervous system (CNS), heart, pancreas, liver, and kidney, it has been used for different models of injury for example in cardiovascular, neurological, and metabolic diseases. [18][19][20] The technology of transgenic and genome editing is mature and e cient in zebra sh, which made zebra sh as a common model for genetic screen and disease models. [21][22][23] Moreover, the zebra sh embryos are transparent and develop externally, a variety of imaging modalities were applicable to live image of embryogenesis and disease occurrence.…”