This document discusses CRISPR-Cas9 gene editing. It begins by defining gene/genome editing and describing what CRISPR is and how it functions in bacteria. CRISPR uses Cas9 proteins and guide RNA to create double-strand breaks in DNA at targeted locations. There are two main pathways for repairing double-strand breaks - nonhomologous end joining and homology-directed repair. The document then outlines the general workflow for CRISPR gene editing, including designing guide RNAs, selecting expression systems, transfecting cells, selecting clones, and characterizing gene editing through assays like sequencing and western blot.

1. 1
CRISPR-Cas9

2. 2
What is Gene/Genome Editing?
• A process whereby researchers can introduce a modification into
an endogenous gene
• Disruption, Insertion, Replacement at a locus in the genome
– Control gene expression
– Create SNP
– Create Reporter fusions while maintaining endogenous gene
regulation

3. 3
What is CRISPR (CRISPR-Cas; CRISPR-Cas9)?
 Mechanism of adaptive immunity
in bacteria and archaea
 Evolved to adapt and defend
against foreign genetic material
(e.g. phage)
 Several different types of
CRISPR pathways in bacteria
and archaea
 Type II: CRISPR-Cas9. Creates a
double-strand break in the
targeted DNA
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
Cas proteins: CRISPR-Associated proteins

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How does CRISPR-Cas9 edit genome?
 Natural bacterial system (Type II):
crRNA + tracrRNA + Cas9 protein
 Two components: single chimeric
guide RNA + Cas9 protein
 Design crRNA to target any sequence
next to a PAM (NGG/NCC) in the
genome.
 Cas9 creates a double strand break
(DSB) in the genome .
 DSB occurs on both strands, 3 base
pairs upstream of the PAM.
 DSB is repaired by either NHEJ or HR
tracrRNA: trans-activating CRISPR RNA (tracrRNA)
PAM: Protospacer Adjacent Motif

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Two major repair pathways of DSBs
NHEJ HR

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General workflow

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Current CRISPR workflow-1
1. Design and selection of targeting sequences (by algorithm)
2. Synthesis of DNA insert oligos
3. Clone into CRISPR/Cas9 expression vector (from several sources)
4. Sequencing
5. Plasmids purification

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Current CRISPR workflow-2
6. Transfect cells
7. Selection e.g. antibiotic
8. Clonal Isolation
9. Clonal characterization with
further analysis and Phenotypic
assay
transfection antibiotic
Clonal
Isolation
Mismatch detection assay
Sanger sequencing
Western blot
Phenotypic assay
Clonal
characterization
antibiotic

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To knockout a gene using
CRISPR/Cas9 through NHEJ
Ex.1

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Design tools/algorithm
Step1: Design
 Selection criteria
 Nearly all gRNAs can create DSBs
 Not all DSB cause functional knockout of the protein
 Avoid off-targets
 Optimize Function and Specificity

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E-CRISPR Design Tool
http://www.e-crisp.org/
Nat. Methods. 11(2), 122-123 (2014)

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Edit-R algorithm
http://dharmacon.gelifesciences.com/gene-editing/edit-r/custom-crrna/
1. Gene or DNA Seq
2. Protein coding gene/miRNA/lncRNA
3. Species

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Expression system for gRNA and Cas9
Step2
 All in one vectors (selection markers/ lenti-backbone)
 Two vectors (gRNA and Cas9)
 gRNAs + cas9 mRNA
 gRNA + Cas9 protein

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transfection antibiotic
Clonal
Isolation
antibiotic
How can gene editing be detected and
characterized?
Step3
Clonal selection, isolation and expansion

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transfection antibiotic
Clonal
Isolation
Mismatch detection assay
Sanger sequencing
Western blot
Phenotypic assay
Clonal
characterization
antibiotic
How can gene editing be detected and
characterized?
Different analysis methods will provide
varying degrees of information
Step4

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Protein knockout confirmed by Western blot
UN = untreated HEK293T cells
wt = wild type
ht = heterozygous (both edited)
hm = homozygous (both edited)
wt ht hm
clone #:
16
Residual/truncated protein
may not be detected