Week 7: CRISPR Week

BioHack Academy: Fabio Ferreira - Digital DNA Design

This is the lecture about methodology of designing genes and how to get them.

Coding

（DNA配列に対して）Mutagenic event ->deletion -> insertion -> Substitution（置換）

CRISPR

Q. What is Linker loop?

細胞が壊れたとき（皮膚の怪我など）・・・DNAは自力で修復しようとする
→CRISPRはその仕組みを利用して、壊したDNAの配列の隙間に新たなDNA配列を挿入する

Design sgRNA: Tools and parakeets

・Target (coding and non-coding sequences)
・PAM(s)
・On-target efficiency
・Off-targets

There are online tools for the design.
・Species-specific (mice, zebra fishes, etc…)
・Coding vs non-coding sequences (Coding: place and write name of the genes.)
（既に用意されているものがあるということらしい。どっちがどっち？）
・integration of multiple parameters

Some online tools to select sgRNA

Fabio’s recommendation is “Benching”

Where to get the vectors (and sgRNAs)

A lot of companies sell the prasmids

• Addgene (Non-profit) 60€(?)
  https://www.addgene.org/guides/crispr/
  ↑There are also protocols for CRISPR.
  

How to use Benching

Pick your gene. For example, ENSG0000254647
↓
On Benching click “+”
-> DNA sequence
-> New DNA sequence
-> Search External Databases
-> Search for “ENSG0000254647”
-> Import

Homework

• Explain, draw/sketch and understand the CRISPR/Cas9 mechanism. Name the involved parts, expand and learn the acronym, explain why this technology is a break-through, explain how it can be programmed.
  
  
• Explain, draw/sketch and understand the DNA Repair mechanisms: HDR and NHEH
  
  
• Make an account at benchling.com and follow the CRISPR tutorial from Fabio Ferreira on Digital DNA Design.
  
  
• Extra Credit: Compare with other Genome Editing technologies like Restriction Enzymes, TALENs and ZFNs.
  
  

CRISPR Basic

What is CRISPR?

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats

Why this technology is a break-through?

Different methods of gene editing
———————————–
1st generation: ZFN
⇩
2nd generation: TALEN
⇩
3rd generation: CRISPR-Cas9
———————————–
The practices to lose certain DNA abilities or to insert other DNA sequences into another DNA by cutting & pasting DNA have been around for a while.However, with conventional methods, it is difficult to incorporate the gene in the targeted position.

CRISPR-Cas9 is an innovative method since it enables gene editing much more accurately than ever.
It is a technology that makes use of the DNA repair mechanism and has the advantage of being able to cut “only” one targeted spot in a long piece of DNA.

Since the accuracy and efficiency increased by CRISPR-Cas9, the extent of gene editing was highly expanded, it is applied to medicine, drug discovery, and agriculture studies.

CRISPR Cas9 - A Brief Introduction (YouTube)

Guiding, Cutting, Checking

Guiding

Making guide RNA to instruct CAS9 where to cut

1. setting the stage for sgRNA production
   
2. 60m Making sgRNA
   
3. 30m Destroying unnecessary DNA
   
4. cleaning sgRNA
   

Cutting

CAS9 precisely cuts DNA

1. Setting the stage for CAS9 cutting
   
2. Combining sgRNA + CAS9+targetDNA
   
3. 60-90m cutting targetDNA
   

Checking

Separate DNA fragment

1. Preparing the GEL
   
2. Running the GEL
   
3. Checking the GEL (UV light)
   

Heat shock? (PCR?)

Make sure you know definitions of:

• sgRNA
  sgRNA(segment RNA)=gRNA(guide RNA)
  “CRISPR/Cas9 gene targeting requires a custom single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a Cas9 nuclease-recruiting sequence (tracrRNA).”
  
  sgRNA is consists by 2 regions: crRNA sequences and tracrRNA.
  
  
  Citation above and image from How to design sgRNA sequences - Takara Bio
  
  
• PAM sequence
  PAM: “Protospacer Adjacent Motif” (プロトスペーサー隣接配列)
  Specific sequence of nucleotides, around 2-6 base pairs, that follow the protospacer sequence in a viral genome
  
  CRISPR cut target sequences only when PAM follows just behind that sequences. It is thought that PAM is the system that microbes distinguish themselves from viruses.
  
  
  Image from CRISPR Cas9 - A Brief Introduction(YouTube)
  
  
  
  
• Promoter
  Promoter is a region of DNA upstream of a gene where relevant proteins (ex. RNA polymerase and transcription factors) bind to initiate transcription of that gene.
  プロモーターとは遺伝子の一部で、RNAポリメラーゼが結合し、遺伝子の転写（DNA→RNA）を始める領域のこと。
  
  
• Plasmid
  
  
  Image from Science Learning Hub
  
  Plasmid is small circular DNA molecules which bacteria contains in addition to chromosome.
  Plasmids do not have genes necessary for normal life activities. However, genes in plasmids work independently when bacteria are exposed to special environments (high temperature, dryness, high salt content, etc.).
  Plasmids can copy themselves independently of the bacterial chromosome. Because of the independent replicability, aftificial plasmids are used as a vector for genetically modification in various scientific studies and industries.
  
  
• 5’ to 3’ Directionality (“5 prime to 3 prime)
  “5’ and “3’” indicate the carbon numbers in the DNA’s sugar backbone. DNA sequences always extend to 5’(five prime) -> 3’(3 prime) direction.
  
  
  Image from Wikipedia
  
  
• Enzyme
  “Enzymes are built of proteins folded into complicated shapes; they are present throughout the body.The chemical reactions that keep us alive – our metabolism – rely on the work that enzymes carry out.”
  Enzymes: How they work and what they do - Medical News Today
  
  Cas9 is also the enzyme which cut genome:a dual RNA-guided DNA endonuclease enzyme.
  
  
  

References

• CRISPR Cas9 - A Brief Introduction
  https://youtu.be/1aJxXWkE3Ek
  
  
• What is the PAM? - A CRISPR Whiteboard Lesson
  https://www.youtube.com/watch?v=iSEEw4Vs_B4
  
  
• CRISPR-Cas9 基本の「き」コスモ・バイオ株式会社
  https://www.cosmobio.co.jp/product/detail/crispr-cas9-introduction-apb.asp?entry_id=15520
  
  
• Wikipedia, “Directionality (molecular biology)”
  https://en.wikipedia.org/wiki/Directionality_(molecular_biology)
  
  
• Wikipedia, “Phosphodiester bond”
  https://en.wikipedia.org/wiki/Phosphodiester_bond
  
  
• CRISPR　そしてゲノム編集へ… RNA Japan
  https://www.rnaj.org/newsletters/item/844-nishimasu-1
  
  
• 国内初！ゲノム編集トマト 「シシリアンルージュハイギャバ」
  (Genetical modified tomato by CRISPR-Cas9 was developed in 2020)
  https://p-e-s.co.jp/higabatomato_column/genome-editing-for-sicilian-rouge/
  
  
• Benchiling
  https://www.benchling.com
  
  
• Bacterial DNA- the role of plasmids - Science Learning Hub
  https://www.sciencelearn.org.nz/resources/1900-bacterial-dna-the-role-of-plasmids
  
  
• 「ゲノム編集」とは？遺伝子治療への応用の可能性は？
  https://genetics.qlife.jp/interviews/dr-mashimo-20210215
  
  
• ゲノム編集の現状について（PDF）
  https://www8.cao.go.jp/cstp/tyousakai/life/haihu90/siryo4-1.pdf