Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation

Abstract

Body pigmentation is a limitation for in vivo imaging and thus for the performance of longitudinal studies in biomedicine. A possibility to circumvent this obstacle is the employment of pigmentation mutants, which are used in fish species like zebrafish and medaka. To address the basis of aging, the short-lived African killifish Nothobranchius furzeri has recently been established as a model organism. Despite its short lifespan, N. furzeri shows typical signs of mammalian aging including telomere shortening, accumulation of senescent cells and loss of regenerative capacity. Here, we report the generation of a transparent N. furzeri line by simultaneous inactivation of three key loci responsible for pigmentation. We demonstrate that this stable line, named klara, can serve as a tool for different applications including behavioral experiments and the establishment of a senescence reporter by integration of a fluorophore into the cdkn1a (p21) locus and in vivo microscopy of the resulting line.

Rapid and precise genome engineering in a naturally short-lived vertebrate

“…More recently, longer CRISPR/Cas9-mediated knock-in insertions (~1 kb) were achieved in killifish (in F0 individuals) with 18–30% efficiency, though germline transmission was not shown ( Oginuma et al, 2022 ). Moreover, knock-in of a long (1.5 kb) insertion was achieved for a specific locus with ~11% efficiency and successful germline transmission (germline transmission rate not shown) ( Krug et al, 2023 ). Our approach for CRISPR/Cas9-mediated knock-in in killifish allows long insertions (up to 2 kb), has >40% efficiency, and high germline transmission rates.…”

“…Previous studies have developed different CRISPR/Cas9-mediated long insertion knock-in approaches not only in killifish but also in other teleost species, such as zebrafish and medaka ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Oginuma et al, 2022 ; Seleit et al, 2021 ; Wierson et al, 2020 ). Key points of difference between these approaches include (1) type of donor repair template – plasmid donor (with in vivo linearization) ( Oginuma et al, 2022 ; Wierson et al, 2020 ), dsDNA PCR amplification product ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Seleit et al, 2021 ), or cloning-free synthetic dsDNA (as reported here), (2) use of chemical modification on linear dsDNA HDR repair template to prevent unwanted integration events (e.g.…”

“…Previous studies have developed different CRISPR/Cas9-mediated long insertion knock-in approaches not only in killifish but also in other teleost species, such as zebrafish and medaka ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Oginuma et al, 2022 ; Seleit et al, 2021 ; Wierson et al, 2020 ). Key points of difference between these approaches include (1) type of donor repair template – plasmid donor (with in vivo linearization) ( Oginuma et al, 2022 ; Wierson et al, 2020 ), dsDNA PCR amplification product ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Seleit et al, 2021 ), or cloning-free synthetic dsDNA (as reported here), (2) use of chemical modification on linear dsDNA HDR repair template to prevent unwanted integration events (e.g. concatemerization) and boost HDR efficiency – biotin ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Seleit et al, 2021 ), or IDT’s proprietary modification available with Alt-R HDR Donor Block (as reported here), and (3) length of the homology arms – short (24–40 bp; Seleit et al, 2021 ; Wierson et al, 2020 ), middle-range (150–200 bp; as reported here), or long (300–900 bp; Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ).…”

“…Key points of difference between these approaches include (1) type of donor repair template – plasmid donor (with in vivo linearization) ( Oginuma et al, 2022 ; Wierson et al, 2020 ), dsDNA PCR amplification product ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Seleit et al, 2021 ), or cloning-free synthetic dsDNA (as reported here), (2) use of chemical modification on linear dsDNA HDR repair template to prevent unwanted integration events (e.g. concatemerization) and boost HDR efficiency – biotin ( Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ; Seleit et al, 2021 ), or IDT’s proprietary modification available with Alt-R HDR Donor Block (as reported here), and (3) length of the homology arms – short (24–40 bp; Seleit et al, 2021 ; Wierson et al, 2020 ), middle-range (150–200 bp; as reported here), or long (300–900 bp; Gutierrez-Triana et al, 2018 ; Krug et al, 2023 ). While it is difficult without side-by-side experiments to compare each knock-in approach, high insertion efficiencies (>40%) and germline transmission have been achieved with different methods, for example, in zebrafish using plasmid donors with in vivo linearization and short homology arms ( Wierson et al, 2020 ), in medaka using dsDNA PCR product donors, biotin modification and short homology arms ( Seleit et al, 2021 ), and (reported here) in killifish using synthetic dsDNA donors, IDT’s proprietary modification (Alt-R HDR Donor Block) and middle-range length (150–200 bp) homology arms.…”

A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

“…The copyright holder for this preprint this version posted May 1, 2023. ; https://doi.org/10.1101/2023.05.01.538839 doi: bioRxiv preprint heterogeneity of cancer and metastasis. Together, we add to the continently expanding toolkit for the killifish model system [64][65][66][67] , by providing a transformative resource for advanced colorbased anatomical and lineage analyses during vertebrate aging and disease.…”