Peer review of: Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Barnes et. al. provide data supporting the implementation of a novel CRISPR based technology to 
the diagnostics of two high priority viral hemorrhagic fevers, LASV and EBOV, which have proven 
an incredible public health burden through out Western and Central Africa. The work is timely and 
attempts to address many issues that workers in these otherwise austere clinical settings are faced 
with in terms of utilizing cutting edge, or even basic, diagnostic services for high consequence 
emerging and endemic viral diseases. The study design and supporting statistical analysis appear 
adequate. A few issues that should be clarified or addressed prior to publication include. 
1. Line 94 and throughout text: References used, are these really the “gold standard” assays? Are 
there not commercial kits that target LASV? How does this statement fit in with WHO or US-CDC 
(or Africa CDC, or any CDC for that matter) guidance on LASV detection? Are these assays what 
MSF or other academic groups are using? I’m not sure there is a “gold standard” unless you are 
talking about a methodology ie RT-PCR. 
2. Hudson heating inactivation: This is a pretty big safety concern, I strongly recommend a 
statement about minimum biosafety conditions required to do this inactivation. I.E. use of a BSC, 
glove box, PPE requirements, screw cap or snap cap tube (the latter is a huge biosafety aerosol 
risk as snap cap tubes tend to pop when heated), any vortexing? Please provide more detail on the 
process as this manuscript will be looked at by folks attempting to replicate what you have done 
and providing a very firm and serious tone to biosafety here will be appreciated by all who read 
and evaluate your work. 
3. Line 133 and throughout text where any incidence of the word “isolate” is used. The authors are 
using this term way too loosely. An isolate, in the classical sense, refers to an “isolated virus”. A 
clinical sample could have a “swarm” of different “isolates” within it. So when you refer to a type 
of sample it is important to be mindful of what the implications are here. From this reviewer’s 
perspective, you were testing clinical samples, not isolates. Please review the rest of the 
manuscript for use of “isolates” and correct accordingly. 
4. Line 148-149, change sample storage to cold chain. 
5. I see there is use of full length viral sequences of EBOV that were used? Is this correct or did I 
misread something? The use of full length genomes of select agents where transcription or other 
biological processes is pretty tightly regulated by NIH-RAC. Can the authors confirm these 
experiments are exempt from NIH-RAC regulation? I understand that no attempts to produce 
infectious virus were made, but the use of full length EBOV genomes is creeping into a grey area 
and I would hate to have the authors get this published and not be in compliance with perhaps an 
otherwise not realized regulation. Please address this important issue. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
As the next-generation diagnostics, CRISPR-Dx has the merits of high sensitivity, much 
convenience, rapidness, etc. Among the CRISPR-Dx systems, Cas13-based and Cas12-based 
systems are the most promising. Through combing HUDSON, which provides a convenient solution 
for clinical sample preparation, and Cas13-based SHERLOCK system, the authors here developed 
deployable diagnostic tools for realtime detection of Ebola and Lassa viruses. As expected, the tool 
is of high specificity and sensitivity. Besides, with the assistance of HandLens, the results can be 
read out on mobile phones. Overall, the work is of importance, especially at the special moment. 
I only have several minor points. 
1. How to avoid aerosol contamination during the operations. 
2. During the whole processes, pipettes and incubators are still required, which is inconvenient and 
could restrict the technology from field application. The authors may discuss about this and find 
out a solution. 
3. Besides of SHERLOCK, there are also Cas12-based CRISPR-Dx. Can the authors explain the 

rationale of choosing SHERLOCK in detection of VHF viruses? 
4. As the ID NOW technology from Abbott has recently been applied in SARS-CoV-2 detection, 
which is extremely fast, the authors should compare their technology with ID now.