How to identify a new drug target? Cholera -Diphtheria Toxicity

1. Mobile-CRISPRi : Bringing CRISPRi to
Diverse Bacteria & Repurposing CRISPR
for Better Drug Use
Credit Seminar
NIKUNJ TYAGI
PhD Scholar
ICAR-NDRI 1

2. CRISPRi
• Clustered regularly interspaced short palindromic repeats interference
(CRISPRi), a versatile method of blocking gene expression using a catalytically
inactive Cas9 protein (dCas9).
• A nuclease-null Cas9 (dead Cas9, or dCas9) through single point mutations in
both the RuvC1 and HNH domains.(Qi et al,2013)
Cas9 uses two nuclease domains to cleave the two strands in the target DNA
2

3. Why Mobile CRISPRi?
Limitations with CRISPRi Knockdown:
CRISPRi has been transferred using species-specific or narrow host range
strategies
Every time Components need to be optimized for function in different species
• ‘Mobile-CRISPRi’, a suite of CRISPRi systems that combines modularity, stable
genomic integration and ease of transfer to diverse bacteria by conjugation.
Advantages:
• It Can be used in non-model bacteria species
• Its modular nature allows for the customization of gRNAs, strain specific
promoters, selection markers, and dCas9 proteins.
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4. Key Applications of CRISPRi
CRISPRi-Based Essential Gene Phenotyping
CRISPRi Knockdowns of Essential Genes Enable Discovery of Direct
Antibiotic Targets
Partial knockdown of the essential gene increased sensitivity to
existing Drug
CRISPRi Knockdowns Facilitate Genetic Analysis of Complex Pathways
Functional Analysis of the Essential Gene Network 4

5. 5

6. CRISPRi repression & Modularity
A sgRNA–dCas9 complex sterically blocks the
progression of RNAP, reducing gene expression.
Individual modules are flanked by unique
restriction sites.
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7. Strain construction using Mobile-CRISPRi
For Gammaproteobacteria,Tn7 Transposition system & RP4 transfer machinery
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8. Failure of Tn7 methodology
transfer
B. subtilis attTn7 site is not responsible for failure, other factors such
as transfer efficiency or transposon gene expression. 8

9. Strain construction via alternative approach
CRISPRi transfer using an integrative and conjugative element in Bacillus subtilis(ICEBs1)
9

10. Mobile-CRISPRi Stability
Mobile-CRISPRi stability after 50 generations of growth in the absence of
antibiotic selection, Stability was calculated as the plating efficiency on kanamycin versus no
antibiotic. 10

11. Mobile-CRISPRi Transfer Efficiency
Efficiency was calculated as:
transconjugants/total recipients.
Vary with increasing evolutionary distance
from E. coli 11

12. knockdown efficiency
Ranged from~8-fold in Pseudomonas aeruginosa to ~150-fold in S. aureus,
with a median knockdown of ~40-fold across all measured species. 12

13. dCas9 is degraded in P. aeruginosa, Myc tag fused to the C-terminus of dCas9
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14. Knockdown was titrable
Mobile-CRISPRi knockdown can be titrated with IPTG( units in μM).
Enables systematic study of essential genes.
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15. knockdown of native genes in P. aeruginosa
Target genes involved directly (phzA1 and phzM) or indirectly (pqsC) in pyocyanin
Biosynthesis. The loss of blue pigment indicates knockdown of the pyocyanin pathway
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16. Construction of CRISPRi Knockdown
library of Essential genes for E.
cloacae
a. sgRNAs were cloned as pool,
transformed and mated into
E.cloacae
b. sgRNAs were cloned
individually
Maximum twofold difference in representation,
Mobile-CRISPRi transfer and integration is highly
uniform, with essentially all variability derived from
the initial cloning step. 16

17. Fitness of strain library includes knockdown of amino acid
biosynthesis genes
Fitness of strains with sgRNAs targeting
amino acid biosynthesis and those
targeting some putative essential genes
decreased, whereas representation of
non-essential genes that are unrelated to
amino acid biosynthesis remained
constant
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18. Knockdown Library of Essential Genes For Bacillus subtilis
• Constructed an arrayed library of B. subtilis strains expressing
computationally optimized sgRNAs targeting the 289 known or proposed
essential genes
• The sgRNAs targeted unique DNA sequences at the 5’ends of genes, where
CRISPRi is most effective (Qi et al., 2013)
• Nearly all sgRNAs (94%) targeting bona fide essential genes (258 genes
total)decreased colony size on agar plates with xylose (>25% reduction in
area compared to the control)
• Use this essential knockdown library as a platform for drug target discovery
by screening against unique chemical compounds 18

19. Essential sgRNAs
Strain sgRNA Name Target Name Target Product Xylose Fitness
CAG74399 control control none 1
CAG74542 scr scr
small cytoplasmic RNA scRNA,
4.5S RNA, 0.1136
CAG74543 rnpB rnpB
RNA component of
ribonuclease P RNase P
catalytic subunit, ribozyme 0.1051
CAG74544 trnH trnH RNA-Asn 03026
CAG74545 dnaA dnaA
chromosomal replication
initiator protein DnaA 0.3038
CAG74546 dnaN dnaN
DNA polymerase III beta
subunit 0.2941
CAG74547 gyrB gyrB DNA gyrase subunit B 0.2046
CAG74548 gyrA gyrA DNA gyrase subunit A 0
CAG74549 guaB guaB
inosine-monophosphate
dehydrogenase 0.2667
CAG74550 serS serS seryl-tRNA synthetase 0.0838
CAG74551 dnaX dnaX
DNA polymerase III gamma and
tau subunits 0
CAG74552 holB holB
DNA polymerase III delta'
subunit 0.5933
CAG74553 metS metS methionyl-tRNA synthetase 0.1444
CAG74554 ipk ispE
4-cytidine 5'-diphospho-2-
methyl-D-erythritol kinase 0.0933
CAG74555 glmU gcaD
UDP-N-acetylglucosamine
pyrophosphorylase 0.1297
CAG74556 prs prs
phosphoribosylpyrophosphate
synthetase 0.092819

20. DRUG TARGET DISCOVERY
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21. • Emergence of multidrug-resistant pathogenic bacteria is an urgent
threat to human health.
• Knowledge of mode of action (MOA) is critical.
• Most pathogenic bacteria lack genetic tools to systematically perturb
the functions of essential genes that encode antibiotic targets.
• A low level of knockdown allowed sufficient growth to determine the
MOA of an uncharacterized antibiotic by virtue of its synergistic
effects on growth.
Antibiotic MOA studies
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22. sgRNA Name LB LB+DMSO MAC-0170636 LB+DMSO Ratio MAC-0170636 Ratio
control 1130.577971 1105.97793 1109.46056 0.9782 0.9813
aroE 1058.385563 1083.00509 1259.07685 1.0233 1.1896
birA 999.3125985 980.037564 964.86661 0.9807 0.9655
folE 1031.024828 1072.33913 885.025892 1.0401 0.8584
mgtE 846.021394 924.246392 861.997015 1.0925 1.0189
folK 985.5643417 1056.75042 896.657609 1.0722 0.9098
nifS 989.4788643 1045.67424 1038.19689 1.0568 1.0492
ftsL 1542.516643 1525.6424 397.473667 0.9891 0.2577
pdhA 995.8063676 1028.03438 974.433607 1.0324 0.9785
uppS 588.5087736 576.782258 0 0.9801 0.0000
hepS 843.0386722 899.222411 751.508393 1.0666 0.8914
dapF 1141.662111 1063.31409 980.540066 0.9314 0.8589
holA 1201.151362 1131.41214 1267.52132 0.9419 1.0553
racE 839.7644272 816.356112 579.61999 0.9721 0.6902
Test of Essential Gene Knockdown Library against MAC-0170636
LB -normalized colony size on LB platte,LB+DMSO-normalized colony size on LB+DMSO plates; tested because MAC-0170636 was dissolved in
DMSO, MAC-0170636 Ratio -colony size ratio of MAC-0170636 to LB ,(n=289), Undecaprenyl pyrophosphate synthetase (uppS) 22

23. Discovery of Direct Antibiotic Targets
Relative fitness of CRISPRi essential
gene Knockdown strains(n = 289)
Minimal inhibitory concentration (MIC) assay. The
MICs of these strains were (in mg/ml) sgRNAuppS +
0.05% xyl (0.012), sgRNAuppS (0.195), WT
(0.78), and uppS overexpression (3.125) 23

24. Purified B. subtilis UppS activity was inhibited
by MAC-0170636 with an IC50 of 0.79 mM indicating
that UppS is the direct target of MAC-0170636
UppS purified from Staphylococcus aureus,was
completely resistant to MAC-017063 as was.
S.aureus itself. 24

25. Generation of sensitized strains for MOA studies
knockdown of folA increases sensitivity to trimethoprim in multiple species, shifting the
MIC by 2–4-fold, E. aerogenes (left),K. pneumoniae (middle) and P. aeruginosa (right)
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26. Fully induced CRISPRi targeting folA was lethal in E. aerogenes, Klebsiella pneumoniae and P. aeruginosa 26

27. Genome-scale screens elucidate
pathways controlling cholera/diphtheria
toxicity
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28. CRISPRi screen for genes that
modulate sensitivity to a
chimeric toxin composed of the
diphtheria toxin catalytic A
subunit covalently linked to
cholera toxin (CTx-DTA)
Model for CTx-DTA
binding, retrograde
trafficking,
retrotranslocation, and
cellular toxicity
CRISPRi Reveals Pathways and Complexes that Govern Response to
Cholera and Diphtheria Toxin
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29. Experimental Procedure
• K562 cells expressing the CRISPRi sgRNA library and
dCas9 treated with several pulses of CTx-DTA over the
course of 10 days.
• Enrichment and depletion of many sgRNAs indicating
genes that modulate both resistance and sensitivity to
a selective pressure
• GSEA revealed that KEGG pathways enriched for top
protective hit genes were ‘‘Infection with Vibrio
cholera’’ and ‘‘Glycosphingolipid biosynthesis, ganglio-
series’’ while gene sets for top sensitizing genes
included ‘‘ribosome’’ and ‘‘proteasome.
• GSEA analysis provides support for the high specificity
in hit gene identification by CRISPRi approach
Vibrio chlorerae infection
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30. Dissection of biological pathways
CRISPRi screen identified protective
effect of knockdown.
Two top pathways identified are:
 Diphthamide biosynthetic
pathway
 Ganglioside biosynthetic pathway
 knockdown of members
of the ERAD E3 ubiquitin ligase
complex, SYVN1 and SEL1L
rendered cells resistant to CTx-DTA.
 Factors that mediate cytosolic
degradation of ERAD substrates
(UBXD2 or erasin, and the
proteasome) appeared as sensitizing
hits.
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31. Role of ERAD factors in Toxin Retrotranslocation
B4GALNT1 repression blocks toxin uptake, whereas SEL1L repression prevents
toxin retrotranslocation from the membrane fraction to the cytosol.
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32. Effective Knockdown of Noncoding RNAs
• K562 cells expressing dCas9 were transduced with sgRNAs targeting lncRNAs and
quantified the amount of transcript knockdown by qPCR.
XIST is undetectable in cells transduced with sgXIST-1. 32

33. Conclusion
• Mobile-CRISPRi provides a streamlined suite of tools for using CRISPRi in many
types of bacteria: lab-adapted, environmental isolated, and disease-associated
• CRISPRi knockdown of essential genes enables discovery of direct targets of
uncharacterized compounds. CRISPRi portability suggests its utility in future
organism-specific drug-discovery efforts
• Other complexes and pathways identified here including several involved in
RNA processing, had not previously been linked to cholera toxin biology,
highlighting the potential of CRISPRi as a discovery platform
• CRISPRi can effectively repress lncRNA expression, enabling future systematic
studies of noncoding gene function
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34. References
1. Peters, J.M. et al. Enabling genetic analysis of diverse bacteria with Mobile-
CRISPRi. Nature Microbiology volume 4, pages244–250 (2019)
2. Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of
essential genes in bacteria. Cell 165, 1493–1506 (2016).
3. Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene
repression and activation. Cell 159, 647–661 (2014).
4. Choi, K.-H. et al. A Tn7-based broad-range bacterial cloning and expression
system. Nat. Methods 2, 443–448 (2005).
5. Johnson, C. M. & Grossman, A. D. Integrative and conjugative elements
(ICEs):what they do and how they work. Annu. Rev. Genet. 49, 577–601
(2015).
6. Tan, S.Z. et al. A Robust CRISPR Interference Gene Repression System
in Pseudomonas. J Bacteriol 200:e00575-17. (2017).
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36. Mobile-CRISPRi overview
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38. • Gene set enrichment analysis (GSEA) (also functional enrichment analysis) is
a method to identify classes of genes or proteins that are over-represented in
a large set of genes or proteins, and may have an association with
disease phenotypes. The method uses statistical approaches to identify
significantly enriched or depleted groups of genes. Transcriptomics
technologies and proteomics results often identify thousands of genes which
are used for the analysis
• Knockdown of other Ganglioside like GM1b was sensitizing, confirmed that
GM1a is the relevant cell-surface receptor.
• CRISPRi Reveals Pathways and Complexes that Govern Response to Cholera
and Diphtheria Toxin
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