59th Annual Meeting & ToxExpo March 15–19, 2020 • Anaheim, California SR01: Advances in CRISPR-Cas9 Tools and Applications for Toxicologists Continuing Education Course Sunday, March 15 | 7:00 AM to 7:45 AM Chair(s) Cheryl Rockwell, Michigan State University Elena Demireva, Michigan State University Primary Endorser Continuing Education Committee Other Endorser(s) Mechanisms Specialty Section Molecular and Systems Biology Specialty Section Presenters Elena Demireva, Michigan State University Christopher Vulpe, University of Florida
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59th Annual Meeting & ToxExpoMarch 15–19, 2020 • Anaheim, California
SR01: Advances in CRISPR-Cas9 Tools and Applications for Toxicologists
Continuing Education CourseSunday, March 15 | 7:00 AM to 7:45 AM
Chair(s) Cheryl Rockwell, Michigan State UniversityElena Demireva, Michigan State University
Primary EndorserContinuing Education Committee
Other Endorser(s)Mechanisms Specialty Section
Molecular and Systems Biology Specialty Section
Presenters Elena Demireva, Michigan State University
Christopher Vulpe, University of Florida
As a course participant, you agree that the content of this course book, in print or electronic format, may not, by any act or neglect on your part, in whole or in part, be
reproduced, copied, disseminated, or otherwise utilized, in any form or manner or by any means, except for the user’s individual, personal reference, or in compliance with the US
Government Copyright Law as it pertains to Fair Use, https://www.copyright.gov/fair-use/more-info.html.
The author(s) of each presentation appearing in this publication is/are solely responsible for the content thereof; the publication of a presentation shall not constitute or be
deemed to constitute any representation by the Society of Toxicology or its boards that the data presented therein are correct or are sufficient to support conclusions reached or
that the experiment design or methodology is adequate.
Course Participant Agreement
11190 Sunrise Valley Drive, Suite 300, Reston, VA 20191Tel: 703.438.3115 | Fax: 703.438.3113
7:05 AM–7:25 AM Latest Advances in CRISPR-Cas Technologies Elena Demireva, Michigan State University, East Lansing, MI 4
7:25 AM–7:45 AM Use of CRISPR/Cas9-Based Genome-Wide Screens in Toxicology from a User’s Perspective Christopher Vulpe, University of Florida, Gainesville, FL 28
Advances in CRISPR-Cas9 Tools and Applications for Toxicologists
The author declares no conflicts of interest including any financial interests or affiliation with any commercial organization that has a direct or indirect interest in the subject matter of this presentation.
In Bacteria: CRISPR-Cas Provides Adaptive Immunity
In nature, bacteria adapt to invading viruses by acquiring immunity to foreign genetic elements to protect against subsequent infections by the same pathogen
I. Adaptation II. Expression III. Interference and Immunity
Cas proteins CRISPR array
Bacterial cell
Viral DNA
Detection of foreign DNA
Cas1-Cas2
Transcription
Cas
Effector nuclease
Surveillance complex Immunity
Processing & assembly
Integration
Target interference
crRNAs
Knott and Doudna, 2018
In Eukaryotic Cells: Streptococcus pyogenes Cas9 First Adapted for DNA Editing
Jinek et al., 2012 ScienceJinek et al., 2013 ElifeCong et al., 2013 Science
CRISPR Genome Modifications Result from DNA Repair of DSBs
Ran et al., 2013 Nat Protoc
Error prone but high efficiency.Present in somatic and dividing cells.
Low efficiency, requires a DNA template. Limited in non-dividing cells.
DSB
Indel/random mutation
Precise modification
NHEJMMEJ
Rad51-dependentre-ligation of ends
HDRSSTRRad51-independent
Requires template
5’ -3’ - - 5’
- 3’
5’ -3’ - - 5’
- 3’Indel mutation PTC
Indel results in a frameshift and downstream PTC (premature termination codon)
DSB, bound by NHEJ repair machinery
Genomic DNA
DSB, bound by HDR repair machinery
Repairtemplate
Precise modification
5’-3’- -5’
-3’
5’-3’- -5’
-3’
5’-3’- -5’
-3’
Homologous recombination
Precise Genomic Modifications Directed by Different DNA Repair Templates
Template Repair Pathway Properties/LimitationsPlasmid, HA > 500bpdsDNA fragment
HDR • Low efficiency• Long homology arms—large constructs
dsDNA fragments with short HA 30–100bp
MMEJ/SSA • Indels at insertion sites• Off-target integration• May be promising with shorter HAs 24–48bp
ssODN (oligo)Max 200bp, HA 30–40bp
HDR/SSTR
• Limitations of insert size• Reduced off-target/random integration• Both NTS and TS templates can be used• Asymmetry of HA can be considered• Less toxicity than dsDNA templates
Long ss DNA (200bp–3kb)HA 40–200bp
Donor HDR Efficiency FactorsPosition of edit with respect to PAM and seed region of RNA/DNA hybridInsert length and sequence (secondary structures of long ssDNA can result in mutations)Locus dependency—open versus closed chromatin, structural topology, repetitive DNA, empirical testsDistance from DSB (insertion site) to start of homology armsgRNA cutting efficiency—important to validate gRNA efficiency prior to introduction of donor template
• Nicks are repaired with much higher fidelity than DSBs in mammalian cells via the BER pathway
• Mutating the catalytic residue of either nuclease domain of Cas9 converts it to a nickase- Cas9 H840A (HNH domain) nicks non-target strand (PAM strand)- Cas9 D10A (RuvC domain) nicks target strand Jinek et al., 2012 Science
Cong et al., 2013 Science
A Single Mutation Converts SpCas9 to a Nickase
Double nicking by Cas9n (D10A) with paired gRNAs
Ran et al., 2013 Cell
• Cas9n with paired gRNAs on opposite strands introduces DSBs with high specificity.
• Off-target activity reduced by 50- to 1,500-fold
• DSBs induced by double nicking are effectively repaired by both NHEJ and HDR
• Distance between nicks can be optimized
• HDR facilitated when gRNA orientation results in a 5’ overhang
Faster and cheaper (weeks to months to generate edited models)Easily accessible and adapted for different applicationsInherently programmable and easy to engineer, unlike ZFNs and TALENs editors (RNA-based versus protein-based recognition of target DNA)Functional in many species: mammalian, insects, nematodes, fish, and plants
Amenable for in vivo and ex vivo deliveryDirect editing of vertebrate and invertebrate embryos from multiple speciesDifferent formats for delivery—plasmid, RNP, virus, etc.Allows for multiplexing (generate multiple genomic modifications simultaneously)
No “scarring” at genome modification site
Applications beyond DSB generation, such as transcriptional regulation, genome imaging, epigenetic modifications, molecular recoding, chromatin looping
SpCas9 Research Has Paved the Way for Next-Gen Genome Engineering
• Extensively characterized for predicting on-target efficiency and off-target effects, tested and adopted for many different species and systems, new applications for whole genome and targeted screens, in vivo editing, lineage tracing, data recording using DNA, gene drives, and more.
• Major tool for genome editing in last seven years. Superior to prior methods such as ZFN/TALEN, ESCs HDR, transgenesis, siRNA.
• Optimization of repair template formats (ssODN, long ssDNA, dsDNA) for more efficient precise editing. Understanding of DSB DNA repair mechanisms.
• Vast natural diversity of CRISPR homologs and availability of engineered variants.• Structural studies of Cas9 complexes and extensive insertional mutagenesis studies—
inform how to engineer multi-protein modular Cas effector platforms for diverse applications.
SpCas9 DNA Editing Has Led to an Explosion of New Discoveries and Advances in the GE Field
• Natural diversity and evolution of CRISPR-Cas systems
• Engineering Cas Effectors beyond nuclease activity
• Restricting CRISPR-Cas activity with Anti-CRISPRs
Naturally Evolved Diversity of CRISPR SystemsClass 1 Class 2
Koonin et al., 2017 Curr Opin Microbiol
Mine Cas protein orthologs for new and distinct properties: PAM flexibility, smaller size, naturally high specificity, pre-crRNA processing activity, DNA shredders, RNA targeting
Exploring the Natural Diversity of CRISPR Systems
• Remarkable diversity across species of bacteria and archea: protein components, effector complex, cas operon locus architecture, and pre-crRNA processing.
• Class 1—multi-protein effector complexes
• Class 2—a single-component effector protein (e.g., Cas9 [Class 2, Type II subtype])
• All CRISPR-Cas systems rely on crRNA for guidance and targeting specificity
• The adaptation module, composed of Cas1 and Cas2 endonucleases, is shared by all known CRISPR-Cas systems
• Diversity observed at the level of processing of the pre-crRNA to mature crRNA guides, either via a Cas6-related ribonuclease or a housekeeping bacterial Rnase III
• More recently characterized Class 2 Type VI systems are the first variants to exclusively target RNA
Class I CRISPR Systems—Cas3 and Cascade• Most abundant in nature (~90% of CRISPR systems)• A multimeric DNA-targeting complex (Cascade) and a Cas3 helicase-nuclease • Cascade consists for 8-11proteins—multiple options for attachment of accessory modules• crRNA recognition spans >30bp providing potential for high specificity in larger genomes• Promiscuous PAM offers greater targeting flexibility • Cas3 nicks DNA upon recruitment and degrades target DNA upstream of PAM via 3’ -> 5’
exonuclease activity• CasE, a ribonuclease, mediates guide RNA processing—transcription of several guides from
the same promoter
Class I CRISPR Systems—Cas3 and Cascade• Cas3 exhibits great processivity of ssDNA degradation (DNA shredding)—allows for
long-range chromosomal modifications in human cells
• Many potential applications—antimicrobial, removal of transposons/integrated viral elements, exon-skipping, screening of non-coding regions
• Harness dCas9-specific DNA binding and processivity to recruit other functional domains/modules to precise genomic loci
• Avoid DSB toxicity and unwanted editing by-products• Cas-effector platforms offer limitless potential functionalities• Can be designed with both Class 1 and Class 2 CRISPR systems
1. Transcriptional regulators—CRISPRa and CRISPRi2. Epigenetic modifiers—alter DNA methylation and histone modification3. Base Editors—change single nucleotides without causing DSB4. Prime Editing—precision modification without DSB and need for a repair
template5. Other applications—chromosome imaging, identifying chromatin interactions
dCas9 linked to effector domains can potentially recruit any protein to any DNA sequence
Gilbert et al., 2013 Cell
Cas Effector Platforms
Genomic DNA target• Coding DNA• Non-coding DNA• Promoters• Regulatory elements• CpG island
• CRISPR activation—dCas9 is fused to transcriptional activator domains in a multi-modular cooperative manner to activate gene expression when guided to promoter regions
• Several different systems exist, including VPR, SAM, and SunTag activation systems
Transcriptional Effectors: CRISPRa
mRNA
SunTag
VP64
scFV-epitope recruitment
MS2p65HSF1
VP64
SAM
mRNA
VP64p65
RtamRNA
VPR
dCas9
Tanenbaum et al., 2014 CellKonermann et al., 2015 NatureChavez et al., 2015 Nat Methods
Tycko et al., 2017 Nat Methods
Transcriptional Effectors: CRISPRiCRISPR interference—repression of gene expression, two main approaches:1. Steric hinderance between dCas9 complex and transcriptional machinery,
depending on location can block transcription initiation or block elongation when RNAP collides with dCas9
2. dCas9 is fused to transcriptional repressor domains
Qi et al., 2013 Cell Larson et al., 2013 Nat Protoc
dCas9RNA Pol
Direction of transcriptionCollision with dCas9 complex
Epigenetic Effectors: Histone Modification• dCas9 epigenetic effectors can write or erase histone modifications, or directly alter DNA methylation
• Epigenetic effectors can be used to transiently or stably activate or repress specific genes or annotate non-coding genomic regions by changing heterochromatin/chromatin status
• Chromatin modifiers change acetylation or methylation of histones: - Acetylation modified by p300 HAT and HDAC3 effectors - Methylation modified by LSD1, KRAB, PRDM9
Hilton et al., 2015 Cano-Rodriguez et al., 2016Kearns et al., 2015 Kwon et al., 2017Gilbert et al., 2013Histone Modification
MeMe
target locus
dCas9
Histone modifying enzyme
gRNA AcAc
P300HDAC3
PRDM9 LSD1, KRAB
Epigenetic Effectors: DNA MethylationdCas9 epigenetic effectors that are coupled to DNA methylation enzymes include:
- Fusion of dCas9 to Tet1 for erasing methylation of CpGs- Fusion to Dnmta3 for de novo methylation of specific sequences
Improved Precision Editing Cas Effectors: Base EditorsTo achieve efficient editing of human pathogenic point mutations:• Increase precise editing efficiency over that of Cas9+HDR (typically 0.1%–5%), especially in somatic
cells• Reduce random editing products of NHEJ repair (indels); e.g., bi-allelic edits, mixture of genotypes• Eliminate potential DSB toxicity due to random editing by-products, unintended structural changes
such as chromosomal rearrangements, and activation of p53 pathway• Improve in vivo delivery of editing systems: eliminate co-delivery of DNA repair template
To address these goals, Dr. David Liu’s group has developed a series of base editors (BE):ABE—adenine base editorsCBE—cytosine base editors
• Allow for all four transition mutations (C→T, G→A, A→G, and T→C) • Expand the type of edits that can be achieved cleanly• Improve overall SNP repair efficiency• Expand the type of cells that can be targeted with BEs
CBE: Change a Single or a Window of NT from C>T (A>G)
• dCas9 or Cas9 nickase (nCas9) is fused to cytidine deaminases, which is a ssDNA deaminase targeting C residues in the Cas9 R-loop
• Deaminases combined with Cas9 nickase and uracil DNA glycosylase inhibitor modules increase the efficiency of changing cytidine to thymidine
• Repair T>C mutations or create KO or inactivate dominant negative alleles via the iSTOP approach
• iSTOP—convert any of four codons (CAA, CAG, CGA, and TGG) into a termination codon
Komor et al., 2016 Nature Kim et al., 2017 Nat BiotechnolBillon et al., 2017 Mol CellTycko et al., 2017 Nat Meth
CBE: Mechanism of Action• Deamination of cytosine (C) by a cytidine deaminase domain results in uracil (U) conversion• U can base-pair as a thymine (T), and upon DNA mismatch repair or replication replaced by a T• Selected a cytosine deaminase that uses ssDNA as a substrate—has access to the first 11bp of protospacer• First-generation BE can convert C to T within a short window of position 4–8 of protospacer (distal to PAM)
Further evolve variants to expand PAM range and reduce editing window Base editor PAM BE3 NGG VQR-BE3 NGAN EQR-BE3 NGAG VRER-BE3 NGCG SaBE3 NNGRRT SaKKH-BE3 NNNRRT
Approx. Editing Window (nt)Base editor Site A Site BBE3 4 6YE1-BE3 2 2YE2-BE3 2 2EE-BE3 2 2YEE-BE3 2 1
0%10%20%30%40%
Cas9 +ssODN
BE1 BE2 BE3
Relative BE efficiencies
% E
dite
d Ta
rget
Cs
Rees et al., 2017 Nat Comm
1. UGI (uracil glycosylse inhibitor) added to BE2 and BE32. Nickase (Cas9n) function restored in BE3
ABE: Engineering of A>G (T>C) Editors• Engineer a novel DNA deoxyadenosine deaminase from transfer RNA adenosine deaminase by
directed evolution mutagenesis, and fuse to a Cas9 nickase• Deaminate adenosine (A) to convert to inosine (I), which can base-pair with C and be replaced with G
after DNA repair or replication• Simultaneous nicking of non-edited strand favors removal of T instead from mismatch• Engineered seventh-generation ABEs have 50% editing efficiency in human cells, and <0.1% indel rate
• ABEmax and CBEmax with expanded targeting scope: e.g., xABEMax = ecTadA-ecTadA*(7.10)-nSpCas9 (xCas9)CP-CBEMax = rApobec1-nSpCas9 (CP#) -UGI-UGI
Huang et al., 2019 Nat Biotechnol 37(6):626
• Continuous directed evolution of base editors to improve editing efficiency and target sequence compatibility
Thuronyi et al., 2019 Nat Biotechnol 37(9):1070
Newest Base Editor Variants
• BE4Max, evoAPOBEC1-BE4max, evoFERNY -BE4max, evoCDA1-BE4max, and more . . .
Prime Editing• A new method for introduction of small targeted insertions or deletions, and
base conversions without DSB, or donor template • Possible edits with PE:
- All four transition point mutations (C>T, G>A, A>G, T>C), - All eight transversion point mutations (C>A, C>G, G>C, G>T, A>C, A>T, T>A, T>G) - Insertions (1 bp to ≥ 44 bp) - Deletions (1 bp to ≥ 80 bp)
• Higher or similar efficiency and fewer by-products than DSB/HDR-based CRISPR• Additional DNA-RNA hybridization points in PE greatly decrease off-target effects
Overview of Prime Editing
Primer editor and pegRNA
Cas9 nickase
pegRNA3’-
5’-
Reverse transcriptase (RT) domain
PAM
Protospacer
pegRNA nick site Primer Editing rangefor Cas9 PE
5’ ···
3’ ···
··· 3’
··· 5’
Target DNA • Cas9 nickase fused to an engineered M-MLV reverse transcriptase (RT)
• A 3’ extended prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit
• pegRNA binds to primer binding site (PBS) on the edited strand
• Hybridization of pegRNA to primer binding site primes RT to direct synthesis of the edited DNA
• 5′ flap excision and 3′ flap ligation from a branched intermediate drive incorporation of the edited DNA strand creating heteroduplex DNA
• DNA repair resolves the mismatched heteroduplex to copy the edit on the complementary strand
• Both BE and PE allow for precise, targeted mutations without inducing DSBs, requiring DNA donor templates, or relying on low-efficiency HDR pathways
• Both can edit human cells and work in dividing and somatic cells
• Both have a reduced number of components that need to be delivered for in vivo and therapeutic applications
• BEs are useful for single nucleotide transition mutations, with newer BE versions performing with higher efficiency and almost undetectable indel rates
• PEs are less dependent on PAM location or local sequence constraints
• PEs can generate targeted deletions, insertions, and all possible 12 single-base conversions
• PE can theoretically correct the majority (~89%) of all known human pathogenic genetic variants
• PEs have higher indel rates than BE; therefore, for transition mutations PE or BE should be used depending on the desired edit and the target sequence context
Summary of Base Editing (BE) and Prime Editing (PE) Technologies
Restricting CRISPR-Cas Activity with Anti-CRISPRs• Anti-CRISPRs (Acr) are small protein inhibitors of CRISPR systems (~90 a.a)• Acrs evolved in bacteriophage as defenses against CRISPR immune
systems of their bacterial host • Function as “off switches” of Cas9 activity in mammalian cells• Many applications for effective control of Cas9 activity:
• Restrict Cas9 expression temporally or spatially• Prolonged Cas9 expression increases off-target effects and risk of
chromosomal abnormalities—especially problematic for therapeutic applications
• In development of GE models, prolonged Cas9 activity can lead to mosaicism of edited alleles and increase frequency of random indels
• Potential fail-safe measure to prevent gene-drive propagation • Robust, versatile, and genetically encodable approach to limit Cas9
activity compared with other methods, such as drug-inducible or light-inducible Cas9 variants
Pawluk et al., 2016 Cell
Liu et al. 2019 Mol Cell 73(3):611
In vitro enzymatic assay of WT AcrIIA2 and AcrIIA2 with Ala substitutions of residues that interact with sgRNA-bound SpyCas9
Anti-CRISPRs• Encoded by acr genes, which parallel the diversity of CRISPR
systems• Acrs evolved against both class 1 (AcrI) and class 2 (AcrII)
systems • Distinct Acr families inhibit CRISRP-Cas of different subtypes
and species—providing options for narrow and broad inhibition
• Known mechanisms of how Acrs block DNA cleavage by Cas9: - Disabling nuclease domains without effect on DNA binding- Blockade of catalytic DNA cleavage site- Dimerization of Cas9 to prevent active conformation- Destabilization dCas9 binding to DNA by obstructing PAM access
• Versatility of Acr mechanisms offers flexibility in designing Cas9 inhibition strategies for different applications
Pawluk et al., 2016 Cell Liu et al., 2019 Mol Cell
A. Domain organization of SpyCas9.
B. Ribbon and surface representations of the AcrIIA2-SpyCas9-sgRNA complex. The color-coding used for Cas9 is identical to that used in (A). AcrIIA2 is shown in magenta.
Harrington et al., 2017 Cell
A
B
(4) RNA-Targeting CRISPR-Cas Systems• Cas13 (Class 2, Type VI) systems are programmable RNA-guided RNases • Cas13 family contains at least four subtypes (Cas13a, Cas13b, Cas13c, and Cas13d)• Evolved as defense against RNA bacteriophages or to degrade transcripts of DNA viruses• Collateral RNase activity after cleavage of target transcript leads to nonspecific RNA degradation of
nearby transcripts (undetectable in mammalian or plant cells) • Expression of Cas13 proteins with crRNA and target RNA leads to cellular toxicity, suggestive of
induction of dormancy or programmed cell death in bacteria• Main advantage—changes to gene expression or function can be transient and reversible and
engineered without a permanent disruption of genome• RNA-targeting is not reliant on cellular DNA repair pathways• Applications:
• Transcript knockdown therapeutics, RNA base editing, antimicrobial, alternative splicing• CRISPR-Dx—rapid, cheap, portable, and ultrasensitive detection of nucleic acids
Abudayyeh et al., 2016 Science Pickar-Oliver and Gersbach, 2019 Nat Rev Mol Cell Biol
Target specificity encoded by a 28–30nt spacer of crRNA
Cas13 complexes with crRNA via hairpin recognition
Diagnostic Applications of Cas13 (SHERLOCK)• Co-opt nonspecific RNase activity to cleave fluorescent RNA reporters upon target recognition
allowing real-time detection of the target and combine with isothermal amplification (RPA)• Method achieves ultrasensitive (aM) detection of nucleic acids with 1bp sensitivity
- Single molecule detection confirmed by ddPCR• Many applications—pathogen detection, rapid genotyping, identification of tumor mutations,
Summary and ConclusionsRapid adoption and success of SpCas9 editing in eukaryotic systems led to “CRISPR revolution”• Propelled discovery of multiple new systems to target DNA and RNA, manipulate epigenome and transcriptome• Led to development of new genome engineering applications—research, diagnostic, translational, environmental• Development of Cas effector platforms with potential to recruit any protein unit to any DNA/RNA sequence
Diversity of CRISPR Cas systems• Addresses PAM flexibility, Cas protein size, specificity, component requirements, distinct nuclease, Cascade + Cas3
new tools for mammalian cell GE • Diversity of CRISPR and anti-CRISPRs offers vast expansion of CRISPR tools for existing and new applications• Directed evolution and protein engineering—key to creating tailored CRISPR systems with novel functionality
Next-gen precise GE • New and rapidly evolving Base Editing and Prime Editing• Precise changes in DNA or RNA, without DSB, need for donor template, or reliance on HDR repair pathways • Important advance for clinical applications, in vivo delivery, and editing of somatic cells
Thank You for Your Attention!
Acknowledgements:
Support:Michigan State UniversityOffice of the SVP for Research and InnovationInstitute for Quantitative Health Science and Engineering
Michigan State University:Dr. Huirong XieBana AbolibdehDr. Richard Neubig
SOT OrganizersDr. Cheryl RockwellDr. Christopher VulpeKevin Merritt
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• Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R, Ciccia A. (2017). CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. Mol Cell 67(6):1068–1079.
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The presenter does not have any financial interest or affiliation with a commercial organization that has a direct or indirect interest in the subject matter of my presentation.
1. CRISPR as tool in functional toxicology2. Targeted CRISPR versus genome-wide CRISPR in
toxicology3. Applications of targeted CRISPR in toxicology4. Applications of genome-wide CRISPR in toxicology5. Future applications of CRISPR in toxicology6. CRISPR conclusions and caveats
Abbreviations
CRISPR: clustered regularly interspaced short palindromic repeats
Functional profiling—systematically testing multiple genes for their functional role, if any, in toxicity, by perturbing their function
The study of the requirement for the biological activities of genes and corresponding proteins in the response to, and effect on, an organism by a toxicant
OR if you muck it up (the gene) and bad (or good) things happen, then it’s probably important
Gene Function ToxicityAssess function
in cell or organism
As related to role, if any, in
toxicity
Usually mutate
gene
Targeted CRISPR versus Genome-Wide CRISPR
Gene 1KO
Xone
oneGene of interestone
one
Targeting sgRNA togene of interest CAS9
Introduce intocell/organism
Xone
Screen for KO
Isolate KO cell/organism Assess function
in cell/organism
two three n
Multiple genes of interest
Targeting sgRNA to genes of interest
Xone
1X
two
2X
three
3Xn
n
Introduce into cell One per cell
Gene 1KO
Gene 2KO
Gene 3KO
Gene nKO
Generate pool of mutants
Screen for sensitivity to toxicantto identify the important genes
Single Gene ModificationsGenome-Wide Screening Approaches
Gene 1KO
Assess function/phenotype
in cell/organism
Gene 1KO
Gene 2KO
Gene 3KO
Gene nKO
Pool of mutants
Gene nKO
Identifykey
genes
Toxicant Toxicant
or
Sensitivity
Toxicant and ToxinsAcetaldehydeArsenic Trioxide- Cytotoxicity APAPC. difficile Toxin A & BTriclosanParaquat
Regulators and Response PathwaysAHR InductionNRF2 inductionArsenic—unfolded protein responseUnfolded protein responseKarlgren et al., Drug Metab Dispos 2018;46:1776–1786
Karlgren et al., Drug Metab Dispos 2018;46:1776–1786 Karlgren et al., Journal of Pharmaceutical Sciences 106 (2017) 2909–2913
4 bp deletion in exon 4—leads to FS
Express Human MDR1
KO MDCKCanine MDR1
Characterize multiple individual clones
CRISPR “KO” of Toxicant Transporter in a Cell Line
• Single gene KO studies are already widespread in toxicology •More common in whole organism studies• Likely to be utilized extensively in cell lines/iPSCs• KOs must be subject to same scrutiny as other mutants
•Genome-wide screens emerging in toxicology • Limited to cell lines currently with all caveats• Computational analysis is evolving• Beginning to help define MOA/novel players in toxicology
If I only had legs, I could get out of
this dish
Future Applications of CRISPR in Toxicology • CRISPR variants •CRISPR a/i, multiple Cas enzymes, base editors, epigenetic
Reviews• Shen H, McHale CM, Smith MT, Zhang L. Functional Genomic Screening Approaches in Mechanistic Toxicology and Potential
Future Applications of CRISPR-Cas9. Mutation Research Reviews in Mutation Research. 2015;764:31–42. • Sobh A, Vulpe C. CRISPR Genomic Screening Informs Gene–Environment Interactions. Current Opinion in Toxicology. 2019;18:46–
53. • Karlgren M, Simoff I, Keiser M, Oswald S, Artursson P. CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition
Toolbox. Drug Metab Dispos. 2018;46(11):1776–86.
Selected Individual KO Studies• Zagorski JW, Maser TP, Liby KT, Rockwell CE. Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and
H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing. J Pharmacol Exp Ther. 2017;361(2):259–67.• Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL (2018) AHR2 Required for Normal Behavioral Responses and Proper
Development of the Skeletal and Reproductive Systems in Zebrafish. PLOS ONE 13(3): e0193484. • Simoff I, Karlgren M, Backlund M, Lindstrom AC, Gaugaz FZ, Matsson P, Artursson P. Complete Knockout of Endogenous Mdr1
AK. CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism. Drug Metab Dispos. 2017;45(8):957–65.
• Xia P, Zhang X, Xie Y, Guan M, Villeneuve DL, Yu H. Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening. Environ Sci Technol. 2016;50(19):10682–92.
• Sundberg CD, Hankinson O. A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1. Toxicol Sci. 2019;170(2):310–9.
• Shortt K, Heruth DP, Zhang N, Wu W, Singh S, Li DY, Zhang LQ, Wyckoff GJ, Qi LS, Friesen CA, Ye SQ. Identification of Novel Regulatory Genes in APAP-Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen. Sci Rep. 2019;9(1):1396.
• Tao L, Tian S, Zhang J, Liu Z, Robinson-McCarthy L, Miyashita SI, Breault DT, Gerhard R, Oottamasathien S, Whelan SPJ, Dong M. Sulfated Glycosaminoglycans and Low-Density Lipoprotein Receptor Contribute to Clostridium Difficile Toxin A Entry into Cells. Nat Microbiol. 2019.
• Tao L, Zhang J, Meraner P, Tovaglieri A, Wu X, Gerhard R, Zhang X, Stallcup WB, Miao J, He X, Hurdle JG, Breault DT, Brass AL, Dong M. Frizzled Proteins Are Colonic Epithelial Receptors for C. difficile Toxin B. Nature. 2016;538(7625):350–5.
• Kerins MJ, Liu P, Tian W, Mannheim W, Zhang DD, Ooi A. Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor. Mol Cell Biol. 2019;39(13).
• Panganiban RA, Park HR, Sun M, Shumyatcher M, Himes BE, Lu Q. Genome-Wide CRISPR Screen Identifies Suppressors of Endoplasmic Reticulum Stress-Induced Apoptosis. Proc Natl Acad Sci USA. 2019;116(27):13384–93.
Genome-Wide Screens
Key Concepts/Confusions in Genome-Wide CRISPR Screening
- “In vitro”—using cell lines with all the accompanying issues and caveats- e.g., metabolism, immortalized cells, toxicokinetics
- Any or every gene can be targeted in your library BUT- Only a single gene is inactivated (KO) in each cell- A pool (library) of individual mutant cells each containing a KO of single gene
represents all genes
- The gene on each chromosome are KO’d, but the mutations are different on each chromosome
- Each cell with a KO is TAGGED/FLAGGED with unique DNA barcode (sgRNA) so you can see it in a crowd (pool)
- Generally measuring growth advantage or disadvantage of mutant cells in response to environmental exposure such as toxicant