Precision RNAi using synthetic shRNAmir target sites

Reviewer #1 (Public Review):

In this manuscript the authors proposed a novel system by which they can suppress the expression of any gene of interest precisely and efficiently with a pre-validated, highly specific and efficient synthetic short-hairpin RNA. The idea of identifying potent artificial RNAi (ARTi) triggers is intriguing, and the authors successfully identify six ARTi with robust knockdown efficiency and limited to no off-target effects. As a proof-of-concept, the authors examined three oncology targets for validation, including EGFRdel19 (which already has a clinically approved drug for validation), KRASG12R (for which there are no in vivo compatible inhibitors yet) and STAG1 (which has a synthetic lethal interaction with recurrent loss-of-function mutations of STAG2). The authors demonstrated significant suppression of colony formation and in vivo tumor growth for all three oncology targets.

This novel system could serve as a powerful tool for loss-of-function experiments that are often used to validate a drug target. Not only this tool can be applied in exogenous systems (like EGFRdel19 and KRASG12R in this paper), the authors successfully demonstrated that ARTi can also be used in endogenous systems by CRISPR knocking in the ARTi target sites to the 3'UTR of the gene of interest (like STAG2 in this paper).

ARTi enables specific, efficient, and inducible suppression of these genes of interest, and can potentially improve therapeutic target validations. However, the system cannot be easily generalized as there are some limitations in this system:

• The authors claimed in the introduction sections that CRISPR/Cas9-based methods are associated with off-target effects, however, the author's system requires the use CRISPR/Cas9 to knock out a given endogenous genes or to knock-in ARTi target sites to the 3' UTR of the gene of interest. Though the authors used a transient CRISPR/Cas9 system to minimize the potential off-target effects, the advantages of ARTi over CRISPR are likely less than claimed.

• Instead of generating gene-specific loss-of-function triggers for every new candidate gene, the authors identified a universal and potent ARTi to ensure standardized and controllable knockdown efficiency. It seems this would save time and effort in validating each lost-of-function siRNAs/sgRNAs for each gene. However, users will still have to design and validate the best sgRNA to knock out endogenous genes or to knock in ARTi target sites by CRISPR/Cas9. The latter is by no-means trivial. Users will need to design and clone an expression construct for their cDNA replacement construct of interest, which will still be challenging for big proteins.

• The approach of knocking-out an endogenous gene followed by replacement of a regulatable gene can also be achieved using regulated degrons, and by tet-regulated promoters included in the gene replacement cassette. The authors should include a discussion of the merits of these approaches compared with ARTi.

Reviewer #2 (Public Review):

In this manuscript, Hoffmann et al. introduce a novel and innovative method to validate and study the mechanism of action of essential genes and novel putative drug targets. In the wake of many functional genomics approaches geared towards identifying novel drug targets or synthetic lethal interactions, there is a dire need for methods that allow scientists to ablate a gene of interest and study its immediate effect in culture or in xenograft models. In general, these genes are lethal, rendering conventional genetic tools such as CRISPR or RNAi inept.

The ARTi system is based on expression of a transgene with an artificial RNAi target site in the 3'-UTR as well as a TET-inducible miR-E-based shRNAi. Using this system, the authors convincingly show that they can target strong oncogenes such as EGFRdel19 or KRasG12 as well as synthetic lethal interactions (STAG1/2) in various human cancer cell lines in vivo and in vitro.

The system is very innovative, likely easy to be established and used by the scientific community and thus very meaningful.

Author Response:

Reviewer #1 (Public Review):

[…] This novel system could serve as a powerful tool for loss-of-function experiments that are often used to validate a drug target. Not only this tool can be applied in exogenous systems (like EGFRdel19 and KRASG12R in this paper), the authors successfully demonstrated that ARTi can also be used in endogenous systems by CRISPR knocking in the ARTi target sites to the 3'UTR of the gene of interest (like STAG2 in this paper).

We thank the referee for highlighting the novelty and potential of the ARTi system.

ARTi enables specific, efficient, and inducible suppression of these genes of interest, and can potentially improve therapeutic target validations. However, the system cannot be easily generalized as there are some limitations in this system:

• The authors claimed in the introduction sections that CRISPR/Cas9-based methods are associated with off-target effects, however, the author's system requires the use CRISPR/Cas9 to knock out a given endogenous genes or to knock-in ARTi target sites to the 3' UTR of the gene of interest. Though the authors used a transient CRISPR/Cas9 system to minimize the potential off-target effects, the advantages of ARTi over CRISPR are likely less than claimed.

We thank the reviewer for raising these very valid concerns about potential off-target effects related to the CRISPR/Cas9-based gene knockout or engineering of endogenous ARTi target sites. In contrast to conventional RNAi- and CRISPR-based approaches, such off-target effects can be investigated prior to loss-of-function experiments through comparison between parental and engineered cells, which in the absence of CRISPR-induced off-target events are expected to be identical. Subsequent ARTi experiments provide full control over RNAi-induced off-target activities through comparison of target-site engineered and parental cells. However, we agree that undetected CRISPR/Cas9-induced off-target events cannot be ruled out in a definitive way, which we will point out in our revised manuscript.

• Instead of generating gene-specific loss-of-function triggers for every new candidate gene, the authors identified a universal and potent ARTi to ensure standardized and controllable knockdown efficiency. It seems this would save time and effort in validating each lost-of-function siRNAs/sgRNAs for each gene. However, users will still have to design and validate the best sgRNA to knock out endogenous genes or to knock in ARTi target sites by CRISPR/Cas9. The latter is by no-means trivial. Users will need to design and clone an expression construct for their cDNA replacement construct of interest, which will still be challenging for big proteins.

We fully agree that the required design of gene-specific sgRNAs and subsequent CRISPR-engineering steps are by no means trivial. However, we believe that decisive advantages of the method, in particular the robustness of LOF perturbations and additional means for controlling off-target activities, can make ARTi an investment that pays off. In our experience, much time can be lost in the search for effective LOF reagents, and even when these are found, questions about off-target activity remain. While ARTi overcomes many of these challenges by providing a standardized experimental workflow, we do not propose to replace all other LOF approaches by this method. Instead, we would position ARTi as a unique orthogonal approach for the stringent validation and in-depth characterization of candidate target genes, as we will highlight in our revised discussion.

• The approach of knocking-out an endogenous gene followed by replacement of a regulatable gene can also be achieved using regulated degrons, and by tet-regulated promoters included in the gene replacement cassette. The authors should include a discussion of the merits of these approaches compared with ARTi.

We thank the reviewer for pointing out these alternative LOF methods. We had already included a brief discussion of chemical-genetic LOF methods based on degron tags. While we certainly share the current excitement about degron technologies, they inevitably require changes to the coding sequence of target proteins, which can alter their regulation and function in ways that are hard to control for. In our revised discussion, we will add a brief comparison to conventional tet-regulatable expression systems, which unlike ARTi require the use of ectopic tet-responsive promoters. Overall, we would position ARTi as an orthogonal tool that enables inducible and reversible LOF perturbations without changing the coding sequence and the endogenous transcriptional control of candidate target genes.

Reviewer #2 (Public Review):

[…] The system is very innovative, likely easy to be established and used by the scientific community and thus very meaningful.

We thank the reviewer for their enthusiasm about ARTi.