Precise transcript targeting by CRISPR-Csm complexes
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Abstract
Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans -cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90–99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.
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We also compared KD efficiency of crRNAs targeting intronic versus exonic regions for the same two RNAs (Fig. 2D).
Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?
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Thus, fluorescently-tagged Csm can be used for easy visualization of RNA in living cells.
Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?
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A more large-scale analysis must be performed to determine optimal spacer design criteria, and to test how different factors (melting temperature, GC-content, target site availability) influence KD efficiency
Agree! Seems like these are critical steps for establishing Csm-mediated RNA KD as robust and specific technique. Do you have plans for further characterization?
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We varied the GFP-targeting spacer length from 24-48 nt in increments of 4 and assayed GFP KD
You measured GFP fluorescence, but did you also measure RNA levels directly? I'm curious how KD efficiency of targets like MALAT1, NEAT1 or TARDBP would change with spacer length.
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We also compared KD efficiency of crRNAs targeting intronic versus exonic regions for the same two RNAs (Fig. 2D).
Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?
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Thus, fluorescently-tagged Csm can be used for easy visualization of RNA in living cells.
Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?
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We varied the GFP-targeting spacer length from 24-48 nt in increments of 4 and assayed GFP KD
You measured GFP fluorescence, but did you also measure RNA levels directly? I'm curious how KD efficiency of targets like MALAT1, NEAT1 or TARDBP would change with spacer length.
-
A more large-scale analysis must be performed to determine optimal spacer design criteria, and to test how different factors (melting temperature, GC-content, target site availability) influence KD efficiency
Agree! Seems like these are critical steps for establishing Csm-mediated RNA KD as robust and specific technique. Do you have plans for further characterization?
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