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

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.

Precise Transcript Targeting by CRISPR-Csm Complexes

“…We chose the Type III-A Csm complex from Streptococcus thermophilus for several reasons: 1. it has been extensively characterized biochemically, structurally, and in bacteria (Staals et al 2014; Zhu et al 2018; You et al 2019; Tamulaitis et al 2014; Jia et al 2019; Guo et al 2019; T. Y. Liu, Iavarone, and Doudna 2017; Mogila et al 2019), 2. functions optimally at 37C, 3. has been demonstrated to work in zebrafish upon ribonucleoprotein (RNP) microinjection (Fricke et al 2020), and 4. has fewer components than the analogous Type III-B Cmr complex (Staals et al 2013). We began by verifying proper expression of each individual protein component (Csm1-5 and Cas6) in immortalized human embryonic kidney (HEK293T) cells.…”

“…Recently, St Csm was shown to be effective at depleting GFP mRNA upon microinjection of bacterially purified RNP into transgenic zebrafish embryos (Fricke et al 2020). While demonstrating proof of principle, RNP delivery of multi-subunit CRISPR-Cas effectors is not ideal for several reasons: 1. delivery is often difficult and short-lived compared to DNA-delivery methods, 2. the RNP may be unstable and prone to disassembly, and 3. for every new crRNA, the entire RNP must be re-purified from bacteria or reconstituted from individually purified subunits in the proper ratio.…”

CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research

“…To date, several Cas enzymes have been used for programmable RNA knockdown. The RNA-targeting type III CRISPR-Cas systems enabled targeted cleavage of desired RNAs in prokaryotes and zebrafish. , However, they have not yet been characterized for RNA-targeting in mammalian cells. In human cells, protein knockdown has been achieved via RNA-targeting using Cas9 enzymes derived from Streptococcus pyogenes (SpCas9), Streptococcus thermophilus (SthCas9), and Francisella novicida (FnCas9) .…”

Advances in Reproductive Endocrinology and Neuroendocrine Research Using Catfish Models

“…However, in recent years, the field of reverse genetics has been evolving widely with the development of novel genome editing technologies, such as RNA interference (RNAi), zinc finger nucleases (ZFN) and plasmids, morpholinos, TALEN and CRISPR/Cas9 for functional analysis including targeted gene knockdown and knockout in various species including zebrafish, tilapia, and catfish [ 206 , 207 , 208 , 209 , 210 , 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 ]. Morpholinos, on the other hand, provide better specificity than RNAi (siRNA/shRNA/esiRNA) by decreasing the possibility of catastrophic off-target antisense effects [ 219 ], and has been widely used for studies in zebrafish and goldfish [ 217 , 218 ].…”