Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish

Gintautas Tamulaitis,Cecilia Winata,Thomas Fricke,Matthias Bochtler,Charles Weige,Maciej Lapinski,Dalia Smalakyte,Abhishek Pateria,Agnieszka Kolano,Virginijus Siksnys,Michal Pastor

doi:10.1089/crispr.2020.0032

Abstract

RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.

Highlights

Chronic knockout and acute knockdown of genes often lead to drastically different phenotypes, in ways that are only partly explained by technical limitations.[1,2]knockdown experiments remain interesting even in the era of facile DNA knockout generation using CRISPR-Cas[9]
The Cas/Csm proteins coding genes were co-expressed with a synthetic CRISPR array (Fig. 1B), which contained either four identical copies of a spacer or four different spacers
As our method targets mRNAs in the cytoplasm that are expected to be spliced already, targeting regions had to be placed in exons

Summary

Introduction

Chronic knockout and acute knockdown of genes often lead to drastically different phenotypes, in ways that are only partly explained by technical limitations.[1,2]knockdown experiments remain interesting even in the era of facile DNA knockout generation using CRISPR-Cas[9]. Endoribonuclease-prepared short interfering RNA reportedly leads to nonspecific developmental defects,[5] presumably due to overload of the endogenous interference pathways. Morpholino-mediated knockdown is the leading technology for RNA silencing in zebrafish.[7,8,9] The method is well established, but some limitations exist. At lower concentrations, silencing is frequently incomplete,[10] while at higher concentrations, off-target effects are common and have to be carefully controlled.[1,11] Recently, dCas9-mediated transcriptional inhibition[12] has been tested in zebrafish as an alternative to morpholinos, but success has only been demonstrated in a single case using relatively early embryos.[13] For maternal transcripts, morpholinobased knockdown is problematic, and transcription suppression can altogether not be used

Methods

Results

Conclusion