4/20/2023 - Open Access

CRISPR-activated expression of collagen col-120 increases lifespan andheat tolerance
Anita Goyala1, Collin Y. Ewald1§
1Laboratory of Extracellular Matrix Regeneration, Institute of Translational Medicine, Department of Health Sciences andTechnology, ETH Zürich, Schwerzenbach CH-8603, Switzerland§To whom correspondence should be addressed: collin-ewald@ethz.ch

Abstract
Transgenic overexpression of collagen col-120 increases the lifespan of C. elegans. However, whether post-developmental
enhancement of collagen expression could also increase the lifespan is unknown. Recently, we described a method to induce
the expression of a target gene using catalytically dead Cas9 (dCas9)-engineered C. elegans via ingestion of bacteria
expressing a pair of promoter-specific single guide RNAs (sgRNA). Here, we cloned col-120 promoter-specific sgRNA oligo
pair into L4440-Biobrick-sgRNA and fed these bacteria to dCas9::VP64 transgenic C. elegans. We observed a similar
percentage of lifespan extension by post-developmentally dCas9-induced expression of col-120, as previously reported
through transgenic overexpression of col-120. Consistent with this result is that induction of another previously shown
longevity-promoting collagen, col-10, also increased lifespan. Furthermore, we found an enhanced resilience to heat stress andincreased expression of hsp-16.2 upon dCas9-activated col-120 expression. Together, these results provide an orthogonal
method to validate longevity by enhancing col-120 expression and point towards a potential role of collagen enhancement inthermotolerance.

4/20/2023 - Open AccessFigure 1. CRISPR-activation of collagens increases lifespan and heat stress resilience:4/20/2023 - Open AccessA) Schematic illustration of bacteria feeding expressing col-120 via promoter-specific sgRNA using the L4440-BiobricksgRNA plasmid to dCas9::VP64 transgenic C. elegans. B, D) Relative fold change in mRNA expression of col-120 gene inyoung adult (YA) and day three adults, respectively, when fed sgRNA col-120 or scr to eggs (B) or L4 (D) animals. qRT-PCR
experiment was performed in three or more biological replicates. Welch's two-tailed t-test was used for significance analysis.
C, E-J) Survival curves showing extension in lifespan in animals with a dCas9-activated expression of col-120 (C, E-G) andcol-10 (H-J) compared to scrambled (scr) control. C) eggs or (E-G) L4 dCas9::VP64 transgenic C. elegans were placed on
sgRNA-expressing bacteria. Lifespans were performed at 20 °C. Experiments were performed in four independent biologicaltrials for sgRNA col-120 and three independent biological trials for sgRNA col-10, as shown individually here. The MantelCox log-rank test was used for statistics. Data, details, and statistics of lifespan assays are in Extended Data. K) dCas9activated expression of col-120 enhanced the expression of hsp-16.2 promoter-driven GFP after a heat shock at 33 °C for 2
hours with 6 hours of recovery at 20 °C, as shown in the representative fluorescent images. The bars represent mean + SEM
and are a composite of three independent biological trials. The dots represent the quantified fluorescence intensity of Phsp16.2::GFP in arbitrary units of individual C. elegans. Welch’s t-test was used for significance analysis. L-O) Heat stresssurvival curves showed enhanced survival of animals with the dCas9-activated expression of col-120 to high temperature (35°C). Experiments were performed in four independent biological trials, as shown individually. The details of the heat
resistance assay, summary, and statistical analysis are in Extended Data. The Mantel-Cox log-rank test was used forsignificance analysis.

Description
Collagens form an extensive part of the extracellular matrix in all metazoans (Kramer, 1994). In C. elegans, there are 181
collagens, categorized as cuticular and basement membrane collagens (Teuscher et al. 2019). Upon longevity-promotinginterventions, such as reduced insulin/IGF-1 receptor signaling (rIIS), some key collagens, including col-120 and col-10,
showed prolonged expression during aging (Ewald et al. 2015). Both collagens, col-120 and col-10, are expressed during
larval development, with the highest expression at L4 but show a rapid decline in expression in young adults (Ewald et al.
2015; Teuscher et al. 2022). This progressive and age-associated decline of col-120 mRNA and protein levels is delayed byrIIS (Teuscher et al. 2022). Moreover, transgenic overexpression of collagen col-120 or col-10 is sufficient to increase thelifespan of C. elegans (Ewald et al. 2015). However, whether post-developmental enhancement of col-120 or col-10 issufficient to increase lifespan is unknown.
Several methods exist to force gene expression in C. elegans, such as using a heat-shock inducible promoter or a Tetracyclinecontrolled transcription system (Tet/Q) (Link et al. 1999; Bacaj and Shaham 2007; Mao et al. 2019). Yet, repeated heat shocks
or tetracycline-analog doxycycline used for induction of the Tet/Q hybrid system can increase the lifespan of C. elegans(Olsen et al. 2006; Houtkooper et al. 2013; Ye et al. 2014; Ewald et al. 2016; Vertti‐Quintero et al. 2021; Gao et al. 2022). To
overcome this with an orthogonal method, we employed a recently developed technique of feeding bacteria containing
promoter-specific sgRNA for inducing the transcriptional expression of the endogenous target genes in a transgenic
dCas9::VP64 C. elegans (Fischer et al. 2022) (Figure 1A). In brief, nuclease dead Cas9 (dCas9) is fused to the widely
accepted transcriptional activator domain VP64, and endogenous expression of dCas9::VP64 is driven by the ubiquitous sur-5
promoter in C. elegans (Fischer et al. 2022) (Figure 1A). The target gene promoter-specific sgRNAs are designed to be -50 to-400 bp upstream of the target gene's transcription start site (TTS) (Fischer et al. 2022). The pair of sgRNAs were cloned in
L4440_BioBrick-sgRNA plasmid to induce expression in E. coli. Similar to RNAi, sgRNA production is induced by ITPG
(Fischer et al. 2022). The simple cloning of specific sgRNAs and inducing gene transcription through feeding bacteria
expressing the sgRNAs makes this technique easy to adapt and feasible, similar to RNAi methods.
As described in Fischer et al. (2022), we cloned the col-120 (3+4 sgRNA pair) or col-10 (3+4, 5+6 sgRNA pairs) promoterspecific sgRNA oligo sequences in two consecutive sgRNA cassettes driven by T7 promoter with BioBrick flanking region in
the L4440_BioBrick-sgRNA vector. The construct was transformed into HT115 E. coli bacteria, and through the bacterialdelivery of the sgRNA to the modified dCas9::VP64 C. elegans strain, the expression of col-120 or col-10 was induced(Figure 1A). The advantage of this method is that the induction of expression of the target gene can be temporarily regulated
by controlling the timings of feeding to the animals.
We first placed eggs on sgRNA-expressing bacteria to dCas9-activate the expression of col-120 (Figure 1B) and found arobust lifespan increase (Figure 1C). Next, to post-developmentally enhance the expression of col-120, we placed L4 animals
on the sgRNA-expressing bacteria, which resulted in an increase in col-120 gene expression induction and increased lifespancompared to the control (scr = scrambled; Figure 1D-G). This validates the previously reported lifespan enhancement bytransgenic COL-120 overexpression (Ewald 2015). We also found an extension in lifespan upon feeding two different pairs ofcol-10 sgRNA post-developmentally (Figure 1H-J), further validating the longevity benefits of extracellular matrix
enhancement.

4/20/2023 - Open Access
One intriguing observation is that many collagen genes are highly upregulated upon short exposure to heat stress (Brunquell et
al. 2016). Therefore, we asked whether higher collagen expression levels could be protective against heat stress. To address
this, we fed col-120 sgRNA bacteria to dCas9 transgenic animals that drive GFP expression under the heat-shock promoter
hsp-16.2 (Phsp-16.2::GFP), placed them for two hours at 33 °C, and let them recover for six hours at 20 °C before quantifying
GFP fluorescence. We found increased levels of hsp-16.2 by dCas9-activated expression of col-120 (Figure 1K), suggesting anenhancement of the heat shock response. Furthermore, we found that dCas9-activated expression of col-120 enhanced survival
at 35 °C (Figure 1 L-O), suggesting that enhancing collagen expression improves heat resilience.
In summary, using dCas9-activation, we demonstrated that enhancing collagen expression promotes longevity and heat
resilience. This might provide a model to study the underlying mechanisms of extracellular matrix enhancement for healthy
aging.

Methods
Strains
C. elegans strains used in this study were maintained on E. coli OP50 seeded nematode growth medium (NGM) plates at 20°C. MIR249, risIs33 [K03A1.5p::3xFLAG::SV40-NLS::dCas9::SV40-NLS::VP64::HA + unc-119(+)] and MIR276, risIs33[K03A1.5p::3xFLAG::SV40-NLS::dCas9::SV40-NLS::VP64::HA + unc-119(+)]; gpIs1 [hsp-16.2p::GFP] (Fischer et al.
2022).
E. coli strains used in this study are OP50 (streptomycin resistance) for general maintenance of C. elegans, HT115(DE3)(ampicillin and tetracycline resistance) for inducing the target gene sgRNA, and DH5α (ampicillin resistance) for cloning thetarget gene sgRNA plasmid construct. OP50 seeded plates were prepared by spotting OP50 culture grown overnight at 37 °C
from a single colony in LB broth with streptomycin antibiotic (100 µg/mL). sgRNA constructs containing HT115 bacteria
primary culture were prepared by inoculating a single colony in 5 mL LB broth with ampicillin (50 µg/mL) and tetracycline(12.5 µg/mL) antibiotic and incubated at 37 °C overnight. The next day, 25 mL of LB broth with ampicillin (50 µg/mL) was
added to the primary culture and let it grow at 37 °C for 3 h. Cultures were then concentrated for 15 min at 3500 rpm at room
temperature. The pellet was resuspended in 5 mL LB broth with ampicillin (50 µg/mL) and Isopropyl-β-Dthiogalactopyranoside (IPTG) (3 mM), and the homogenous mix was spotted on the NGM plates. Plates were dried for one
day before use.
Cloning of col-10 and col-120 sgRNAs
Primers for the col-10 and col-120 promoter-specific sgRNA target sequences were designed according to the stringent rules
described in Fischer et al. (2022). Briefly, six best 20 nucleotide sequences flanked with NGG (sgRNA) were chosen from 50
to 400 bp region upstream of the TSS (Transcription start site) of the col-10 or col-120 gene, with >50 off-target scores andnumbered based on their relative distance from the respective TSS, with sgRNA 1 being closest and 6 being furthest awayfrom the TSS. Scr (scrambled) sgRNA sequences were randomly chosen 20 nucleotides with ≥50 % GC content that did notmatch to C. elegans genome upon BLAST. col-10 (3+4, 5+6) or col-120 (3+4) promoter-specific sgRNA oligo pairs weresuccessfully cloned into the L4440_BioBrick-sgRNA vector. Annealed oligos with appropriate overhangs of BbsI and BsaI
restriction sites were ligated to the vector with BbsI and BsaI sites. Ampicillin antibiotic resistance was the selection pressure
for positive ligated colonies. E. coli DH5α cells were used as the cloning host, and HT115(DE3) bacteria were used forselecting and propagating the positive clone.
Lifespan assay
After hypochlorite treatment, eggs obtained from gravid MIR249 adults were maintained on OP50-seeded NGM plates (except
for one biological replicate where eggs were grown on scr or sgRNA col-120 or col-10 HT115 bacteria-seeded NGM plates)
until the L4 stage. 30-40 L4 animals were then transferred to scr or sgRNA col-10 or col-120 HT115 bacteria seeded NGM
plates in replicates of five. 120 µL FUdR (final concentration of 50 µM) was top coated onto these plates. Survival of animals
was assessed by gently prodding their posterior end with platinum wire to score live or dead. Unhealthy animals with vulval
rupture and those crawling on the sides or under the agar were censored from the population throughout lifespan scoring.
Lifespan Scoring was started at day 7 of adulthood, L4 being taken as t=0 timepoint. Survival curves were plotted using
GraphPad Prism 9.2.0. Statistical analysis was performed using OASIS 2.0 software online. Lifespan summary for individual
experiments is provided in Extended Data.
Fluorescence Microscopy
Gravid MIR276 adults were treated with sodium hypochlorite solution to obtain eggs. The eggs were grown on scr or sgRNA
col-120 HT115 bacteria seeded NGM plate until young adult. Young adults were heat-shocked at 33 °C for 2 h. After recovery

4/20/2023 - Open Accessfrom shock for 6 h at 20 °C, approx. 30 animals were mounted onto a 2 % agarose pad and anesthetized with 20 mM
levamisole for imaging (Teuscher and Ewald, 2018). Animals were stacked together and captured at 10X with one or two
fields of view and then stitched together later (as shown in Figure 1K) using ImageJ (version 1.53e). Quantifying the total
fluorescence intensity by running a Python script, Green Intensity Calculator (available publicly in Github-Ewaldlab), in
ImageJ (Statzer et al. 2021). Statistical analysis of the quantified data was done using GraphPad Prism software (GraphPad
Prism 9.2.0). The experiment was performed in three biological batches, with n>80 total animals per condition.
Heat resistance survival assay
MIR249 (or MIR276 for Figure 1O) gravid adults were bleached to obtain eggs and grown on OP50-seeded NGM plates until
the L4 stage. L4 animals were fed with scr and sgRNA col-120 HT115 bacteria until adulthood day 3 at 20 °C. Day 3 animals
were shifted to 35 °C to initiate heat stress. Survival of the animals was scored every 2-3 h by gently prodding their tails with aplatinum wire. Survival curves were plotted using GraphPad Prism 9.2.0. Statistical analysis was performed using OASIS 2.0
software online. Survival summary for individual experiments is provided in Extended Data.
col-120 gene expression by qRT-PCR
Gravid MIR249 adults were treated with hypochlorite solution to obtain eggs. Eggs were either fed with col-120 sgRNA and
scr until young adult (YA) or grown on OP50 till L4 and then fed with col-120 sgRNA and scr until day 3 of adulthood.
Animals were then collected at YA and Day 3 for RNA isolation. RNA isolation was done using Trizol (Ambion #15596018)
method and purified by RNA clean and concentrator kit (Zymo Research #R1017) with in-column DNase (Ambion#AM2222) digestion. 1 µg of RNA was used for first strand cDNA synthesis (SuperScript III by Invitrogen #11752-250)
according to the manufacturer’s instructions. Gene expression levels were determined by quantitative real-time PCR (qRTPCR) using the PowerUp SYBR Green MasterMix (Applied biosystems by Thermo Fischer Scientific #A25742) and
QuantStudio PCR system (Applied Biosystems by Thermo Fischer Scientific) according to the manufacturer’s specifications.
Relative gene expression, normalized to the geometric mean of two housekeeping genes cdc-42 and pmp-3, was determined
between animals treated with col-120 sgRNA and scr. Statistical analysis was performed using GraphPad Prism 9.2.0. All theprimers used are listed in Extended Data.

Reagents
TRizol reagent (Ambion by Life Technologies) #15596018
RNA clean and concentrator kit (Zymo Research #R1017)
DNase I (RNase-Free) (Ambion #AM2222)
SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen #11752-250)
PowerUp SYBR Green MasterMix (Applied biosystems by Thermo Fischer Scientific #A25742)
Acknowledgements: We thank Michael Ristow Lab for providing dCas9::VP64 strain, Fabian Fischer for the help indesigning the col-10 and col-120 sgRNA primers for cloning, and CGC, which is funded by NIH Office of Research
Infrastructure Programs (P40 OD010440). Funding from the Swiss National Science Foundation Funding from the Swiss
National Science Foundation SNF P3 Project 190072 to CYE and AG. Figure 1A was created with biorender.org (license
number: CV24SPYHIS).

Extended Data
Description: Extended Data File. Resource Type: Dataset. File: col-120 figure data and analysis.xlsx. DOI: 10.22002/d1ffsa0b42

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Funding: Swiss National Science Foundation SNF P3 Project 190072
Author Contributions: Anita Goyala: investigation, conceptualization, data curation, formal analysis, methodology, writing original draft, writing - review editing. Collin Y. Ewald: conceptualization, funding acquisition, project administration,
supervision, writing - review editing.
Reviewed By: Anonymous
History: Received December 23, 2022 Revision Received April 5, 2023 Accepted April 11, 2023 Published Online April
12, 2023 Indexed April 26, 2023
Copyright: © 2023 by the authors. This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Citation: Goyala, A; Ewald, CY (2023). CRISPR-activated expression of collagen col-120 increases lifespan and heat
tolerance. microPublication Biology. 10.17912/micropub.biology.000730