Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

Daichi G. Suzuki,Hiroshi Wada,Shin-ichi Higashijima

doi:10.1038/s41598-021-99338-1

Daichi G. Suzuki, Hiroshi Wada + Show 1 more

Open Access

https://doi.org/10.1038/s41598-021-99338-1

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Abstract

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

Highlights

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience
We found two homologs of Hsp70like genes in the P. marinus and L. camtschaticum genomes, one of which we named LcHsp70A
Knock-in transgenic zebrafish and medaka can be efficiently generated via non-homologous end joining (NHEJ) by co-injection of two short guide RNA (sgRNA), donor plasmids, and Cas[9] mRNA

Summary

Introduction

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. The lamprey belongs to the basal-most group of vertebrates, the cyclostomes, and retains ancestral characteristics, such as lack of a jaw and paired fins This animal has attracted attention from a wide range of researchers, including evolutionary developmental biologists and n euroscientists[1,2,3,4,5,6]. We succeeded in generating F 0 knock-in lampreys (for the Arctic/Japanese lamprey L. camtschaticum) expressing reporter genes, the genomic integration of which was determined by PCR and sequencing This method is simple and versatile for various experimental aims. The efficiency of obtaining transgenic lampreys is very high (20–35% of survivors) Based on these results, we believe that this CRISPR-Cas9-mediated versatile knock-in method opens up new research horizons using lamprey and possibly other “non-model” organisms in the sense of molecular genetics

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