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CRISPR Technologies�and�gRNA Design Tutorial

How To Grow (Almost) Anything

Asia & Oceania Chapter Review

Presenter: Cholpisit (Ice) Kiattisewee

April 15th 18th, 2024

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MolES, University of Washington

Seattle, Washington

UW

aka UDUB

MolES

DUBS!!!

GO DAWGS

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Overview

  • CRISPR in Nature:�battles with phage
  • Repurposing CRISPR �for Genome Editing
  • Precise Gene Editing�with next-gen CRISPR

Gene Editing

  • CRISPR interference:�tuning down expression
  • CRISPR activation:�switch-on expression
  • Expanded CRISPRa/i toolkit

Gene Regulations

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Adapted from CRISPRpedia by Innovative Genomics Institute (IGI)

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Central Dogma Recap

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

Storage

Message

Function

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CRISPR systems for Biological Programming

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

Alba Burbano & Kiattisewee et al. Annu. Rev. Chem. Biomol. Eng. (2024)

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Prefer the video version? Check this out!

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

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Naturally occurring CRISPR systems

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

Clustered Regularly Interspaced Short Palindromic Repeats

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CRISPR-Cas immunity mechanism

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

Is this a suicide attempt?

No! PAM is out there!

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Protospacer Adjacent Motifs (PAM)

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

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Diversity of CRISPR Systems

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CRISPR Gene Editing

Precise Gene Editing

CRISPR in Nature

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Repurpose Bacterial Immune System for Gene Editing

CRISPR in Nature

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Precise Gene Editing

CRISPR Gene Editing

Key Challenges

​

  • Delivery Method
    • As DNA for expression
    • As RNP Complex

​

  • Component Optimization
    • Cas protein
    • guided RNA
    • DNA targets

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Components of CRISPR Machinery

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

Cas protein

Figures adapted from CRISPRpedia | Innovative Genomics Institute

guide RNA

DNA context

CRISPR complex

How to engineer a better CRISPR symtem?

Protein Engineering

RNA Engineering

DNA Target Screening

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Repurpose Bacterial Immune System for Gene Editing

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

Figures adapted from CRISPRpedia | Innovative Genomics Institute

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Repurpose Bacterial Immune System for Gene Editing

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

Figures adapted from CRISPRpedia | Innovative Genomics Institute

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Repurpose Bacterial Immune System for Gene Editing

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

Figures adapted from CRISPRpedia | Innovative Genomics Institute

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CRISPR Editing to Cure Genetic Diseases

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

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Frangoul et al. N. Engl. J. Med. (2021)

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CRISPR Editing for Food Security

CRISPR in Nature

Precise Gene Editing

CRISPR Gene Editing

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Brandt et al. Annu. Rev. Food Sci. Technol. (2019)

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Engineered CRISPR systems Beyond HDR

CRISPR Gene Editing

CRISPR in Nature

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Precise Gene Editing

Nickase – Cut only one strand, more precise if cut twice

Base-Editing – Coupled with Base-modifying enzyme

Prime-Editing – Specific editing with RNA as a template

Transposase – Locus-specific insertion like transposon

Pool Screening – Massively-parallel gRNA targeting

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Beyond CRISPR Editing: nCas9 – Nickase

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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Wang et al. Annu. Rev. Biochem. (2016)

Increased Accuracy!

Enables other applications: Precise Editing

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Beyond CRISPR Editing: Base-Editing

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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Rees et al. Nat. Rev. Genetics (2018)

Edit can still occur at multiple bases

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Beyond CRISPR Editing: Prime-Editing

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

21

Chen et al. Nat. Rev. Genetics (2022)

Template included in pegRNA sequence

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Beyond CRISPR Editing: Transposase

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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Wang et al. Nat. Rev. Microbiol. (2022)

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Beyond CRISPR Editing: Transposase in Microbiome

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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Vo et al. Nat. Biotechnol. (2020)

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Beyond CRISPR Editing: Pool Screening

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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gRNA can be constructed in a big library

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Beyond CRISPR Editing: Pool Screening

CRISPR Gene Editing

CRISPR in Nature

Precise Gene Editing

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Selected References

  • CRISPRpedia, Innovative Genomics Institute, UCB & UCSF
    • a free, textbook-style resource that explains and illustrates all things CRISPR
  • Bacterial CRISPR Technologies: Alba Burbano & Kiattisewee et al. Annu. Rev. Chem. Biomol. Eng. (2024)
  • Beyond CRISPR Editing: Wang et al. Annu. Rev. Biochem. (2016)
  • Base Editing: Rees et al. Nat. Rev. Genetics (2016)
  • Prime Editing: Chen et al. Nat. Rev. Genetics (2022)
  • Food Supply: Brandt et al. Annu. Rev. Food Sci. Technol. (2019)
  • Sickel Cell Disease Cure: Frangoul et al. N. Engl. J. Med. (2021)
  • Structural Biology: Wang et al. Nat. Rev. Microbiol. (2022)

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Overview

  • CRISPR in Nature:�battles with phage
  • Repurposing CRISPR �for Genome Editing
  • Precise Gene Editing�with next-gen CRISPR

Gene Editing

  • CRISPR interference:�tuning down expression
  • CRISPR activation:�switch-on expression
  • Expanded CRISPRa/i toolkit

Gene Regulations

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Gene Regulation by deactivated Cas proteins

CRISPRa/i Tools Development

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPR/Cas9 🡪 Tuning knob

CRISPRi (interference)

CRISPRa (activation)

  • Recruit by dCas9
  • Recruit by gRNA

CRISPR tools are less developed in bacteria

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Initial Development of CRISPRi in prokaryote

CRISPRa/i Tools Development

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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Qi et al. Cell. (2013)

CRISPRi works by physically blocking RNAP

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Initial Development of CRISPRi in prokaryote

CRISPRa/i Tools Development

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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Qi et al. Cell. (2013)

CRISPRi can be tuned by gRNA structure

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Multiplexing CRISPRa in eukaryote

CRISPRa/i Tools Development

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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Zalatan et al. Cell. (2015)

RNA scaffold enable extra programming layer

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Fishing Expedition of Bacterial Activator

CRISPRa/i Tools Development

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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Dong et al. Nat. Commun. (2018)

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Let’s try design CRISPR sgRNA!!!

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Alba Burbano & Kiattisewee et al. Annu. Rev. Chem. Biomol. Eng. (2024)

Cas protein

gRNA

DNA target

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Let’s try design CRISPR sgRNA!!!

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Cas protein

gRNA

DNA target

Kiattisewee & Karanjia et al. ACS. Synth. Biol. 2022

PAM-flexible dSpRY

+ activator

Addgene: pCK341

Reporter gene: Addgene: pCK760

E. coli genome: GenBank: U00096.3

Spacer

gRNA Hairpin

Addgene: pCK411.RR1

Addgene: pCK364.306

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gRNA expression cassette in bacteria

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Spacer

gRNA Hairpin

CRISPRi - pCK411.RR1

CRISPRa - pCK364.306

Ones can also install cut-site between 1) and 2) so that spacer can be changed by PCR with only one oligonucleotide order

Oligo order is ~$10-20 per 60 bases

20 nt is a regular spacer size

40 nt can be attributed to priming and overhang site

Go to

Benchling

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sgRNAs can be used to program phenotypes

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Green

Reporter genes Addgene: pCK760

Pmed

Pweak

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sgRNAs can be used to program phenotypes

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CRISPR interference

Colorless

Reporter genes Addgene: pCK760

Pmed

Pweak

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sgRNAs can be used to program phenotypes

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CRISPR interference and activation

Red

Reporter genes Addgene: pCK760

Pmed

Pweak

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sgRNAs can be used to program phenotypes

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Try knocking out

ampC gene

---ampicillin resistance

E. coli genome: GenBank: U00096.3

Pre-downloaded on Benchling here

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What about Prime-Editing?

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Let’s get started on the Addgene page

Tong et al. Nat. Commun. (2021)

PBS: Primer-Binding-Site

RTT: Reverse-Transcription-Template

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What about Prime-Editing?

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Tong et al. Nat. Commun. (2021)

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Benchling Sequences Shown in the talk

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CRISPRa/i gRNA design

  • pCK411.RR1 for expression gRNA
  • Reporter: pCK760 medium-sfGFP_weak-mRFP
  • Other plasmids - dCas9+Activator: pCK341

Prime-Editing gRNA design

  • pPEgRNA with primers added
    • PEgRNA_PCR for TAA insertion to GFP coding sequence
    • pPEgRNA_SpeI-HindIII as a backbone for adding PEgRNA insert

Tong et al. Nat. Commun. (2021)

Kiattisewee & Karanjia et al. ACS. Synth. Biol. 2022

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Editing Windows for different CRISPR systems

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Alba Burbano & Kiattisewee et al. Annu. Rev. Chem. Biomol. Eng. (2024)

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Tackling Challenges for Effective CRISPRa

CRISPRa/i Tools Development

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

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Fontana & Sparkman-Yager et al. Curr. Opin. Biotechnol. (2020)

  1. Protein Engineering
  2. RNA Engineering
  3. DNA Engineering

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Optimization of CRISPRa complex positioning

CRISPRa/i Tools Development

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

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Fontana & Dong et al. Nat. Commun. (2020)

CRISPRa is limited by strict distance requirements

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Effect of guide RNA folding

CRISPRa/i Tools Development

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

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Fontana & Dong et al. Nat. Commun. (2020)

Fontana & Sparkman-Yager et al. Curr. Opin. Biotechnol. (2020)

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PAM-Flexible dCas9s expanded targetability

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

Kiattisewee & Karanjia et al. ACS. Synth. Biol. 2022

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PAM-Flexible dCas9s expanded targetability

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

Kiattisewee & Karanjia et al. ACS. Synth. Biol. 2022

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CRISPRa/i could be implemented in diverse systems

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

SoxS interacts with the C-terminal domain

of the RNA polymerase α subunit

Traits of chassis for industrial biotechnology

Dong et al. 2018 Nat. Commun.

Nikel et al. 2016 Curr. Opin. Chem. Biol.

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Porting CRISPRa into Pseudomonas putida

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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Kiattisewee et al. Metab. Eng. (2021)

CRISPRa/i Tools Development

P. putida genetic tool is less characterized

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Porting CRISPRa into Pseudomonas putida

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

Kiattisewee et al. Metab. Eng. (2021)

Integrated machinery performs better

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Rules for effective CRISPRa are also ported

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

Correlation of CRISPRa regulation in E. coli and P. putida

Under investigation

RNA folding

Weak 🡪 high FA

60-120 upstream

Kiattisewee et al. Metab. Eng. (2021)

CRISPRa rules are likely portable to other organisms

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The rules are also portable to cell-free system (CFS)

Design Rules for CRISPRa/i

CRISPRa/i

Gene Regulation

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CRISPRa/i Tools Development

Tickman & Alba-Burbano et al. Cell Systems (2022)

CRISPRa rules are also conserved in CFS

Allowed rapid prototyping of genetic circuitry

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Further Applications of CRISPRa/i Programs

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

CRISPRa/i Tools Development

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Fontana & Sparkman-Yager & Faulkner et al. bioRxiv (2023)

CRISPR/Cas9 🡪 Tuning knob

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Further Applications of CRISPRa/i Programs

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

CRISPRa/i Tools Development

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Fontana & Sparkman-Yager & Faulkner et al. bioRxiv (2023)

CRISPR/Cas9 🡪 Tuning knob

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Further Applications of CRISPRa/i Programs

CRISPRa/i

Gene Regulation

Design Rules for CRISPRa/i

CRISPRa/i Tools Development

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Fontana & Sparkman-Yager et al. Curr. Opin. Biotechnol. (2020)

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Selected References

  • Dong, C.; Fontana, J.; Patel, A.; Carothers, J. M.; Zalatan, J. G. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria. Nat. Commun. 2018, 2489.
  • Fontana, J.+; Dong, C.+; Kiattisewee, C.; Chavali, V.P.; Tickman, B.I., Carothers, J.M., Zalatan, J.G. Effective CRISPRa-mediated control of gene expression in bacteria must overcome�strict target site requirements. Nat. Commun. 2020, 11, 1618.
  • Fontana, J.; Sparkman-Yager, D.; Zalatan, J.G.; Carothers, J.M. Challenges and opportunities with CRISPR activation in bacteria for data-driven metabolic engineering. Curr. Opin. Biotechnol. 2020
  • Kiattisewee, C.; Dong, C.; Fontana, J.; Sugianto, W.; Peralta-Yahya, P.; Carothers, J. M.; Zalatan, J. G. Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida. Metab. Eng. 2021, 66, 283-295.
  • Tickman, B. I.+; Alba Burbano, D.+; Chavali, V. P.; Kiattisewee, C.; Fontana, J.; Khakimzhan, A.; Noireaux, V.; Zalatan, J. Z.; Carothers, J. M. Multi-Layer CRISPRa/i Circuits for Dynamic Genetic Programs in Cell-Free and Bacterial Systems. Cell Systems. 2021, 13, 1-15.
  • Kiattisewee, C.+; Karanjia, A.V.+; Legut, M.; Daniloski, Z.; Koplik, S. E.; Nelson, J.; Kleinstiver, B. P.; Sanjana, N. E.; Carothers, J. M.; Zalatan, J. G. Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants. ACS. Synth. Biol. 2022, 11, 4103-4112.
  • Fontana, J.+; Sparkman-Yager, D.+; Faulkner, I. D. +; Cardiff, R.; Kiattisewee, C.; Walls, A.; Primo, T. G.; Kinnunen, P. C.; Garcia Martin, H.; Zalatan, J. G.*; Carothers, J. M.* Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling. bioRxiv. DOI: https://doi.org/10.1101/2023.11.17.567465

All questions are welcome!!!

E-mail me at cholpisit@gmail.com