Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

Reviewer #1 (Public Review):

In this manuscript, Kipfer et al describe a method for a fast and accurate SARS-CoV2 rescue and mutagenesis. This work is based on a published method termed ISA (infectious subgenomic amplicons), in which partially overlapping DNA fragments covering the entire viral genome and additional 5' and 3' sequences are transfected into mammalian cell lines. These DNA fragments recombine in the cells, express the full length viral genomic RNA and launch replication and rescue of infectious virus.

CLEVER, the method described here significantly improves on the ISA method to generate infectious SARS-CoV2, making it widely useful to the virology community.

Specifically, the strengths of this method are:

1. The successful use of various cell lines and transfection methods.
2. Generation of a four-fragment system, which significantly improves the method efficiency due to lower number of required recombination events.
3. Flexibility in choice of overlapping sequences, making this system more versatile.
4. The authors demonstrated how this system can be used to introduce point mutations as well as insertion of a tag and deletion of a viral gene.
5. Fast-tracking generation of infectious virus directly from RNA of clinical isolates by RT-PCR, without the need for cloning the fragments or using synthetic sequences.
   One weakness of the latter point, which is also pointed out by the authors, is that the direct rescue of clinical isolates was not tested for sequence fidelity.

The manuscript clearly presents the findings, and the proof-of-concept experiments are well designed.

Overall, this is a very useful method for SARS-CoV2 research. Importantly, it can be applicable to many other viruses, speeding up the response to newly emerging viruses than threaten the public health.

Reviewer #2 (Public Review):

The authors of the manuscript have developed and used cloning-free method. It is not entirely novel (rather it is based on previously described ISA method) but it is clearly efficient and useful complementation to the already existing methods. One of strong points of the approach use by authors is that it is very versatile, i.e. can be used in combination with already existing methods and tools. I find it important as many laboratories have already established their favorite methods to manipulate SARS-CoV-2 genome and are probably unwilling to change their approach entirely. Though authors highlight the benefits of their method these are probably not absolute - other methods may be as efficient or as fast. Still, I find myself thinking that for certain purposes I would like to complement my current approach with elements from authors CLEVER method.

The work does not contain much novel biological data - which is expected for a paper dedicated to development of new method (or for improving the existing one). It may be kind of shortcoming as it is commonly expected that authors who have developed new methods apply it for discovery of something novel. The work stops on step of rescue the viruses and confirming their biological properties. This part is done very well and represents a strength of the study. The properties of rescued viruses were also studied using NSG methods that revealed high accuracy of the used method, which is very important as the method relies on use of PCR that is known to generate random mistakes and therefore not always method of choice.

What I found missing is a real head-to-head comparison of the developed system with an existing alternatives, preferably some PCR-free standard methods such as use of BAC clones. There are a lot of comparisons but they are not direct, just data from different studies has been compared. Authors could also be more opened to discuss limitations of the method. One of these seems to be rather low rescue efficiency - 1 rescue event per 11,000 transfected cells. This is much lower compared to infectious plasmid (about 1 event per 100 cells or so) and infectious RNAs (often 1 event per 10 cells, for smaller genomes most of transfected cells become infected). This makes the CLEVER method poorly suitable for generation of large infectious virus libraries and excludes its usage for studies of mutant viruses that harbor strongly attenuating mutations. Many of such mutations may reduce virus genome infectivity by 3-4 orders of magnitude; with current efficiencies the use of CLEVER approach may result in false conclusions (mutant viruses will be classified as non-viable while in reality they are just strongly attenuated).