This CRISPR Cas9 knockout cell line protocol is summerizing the stadard processes of projects from Vitro Biotech.

CRISPR Cas9 is a powerful tool for gene editing, allowing precise editing of genes at specific locations. With the participation of guide RNA (gRNA) and Cas9 protein, the genomic DNA of the cell to be edited will be treated as viral or exogenous DNA and accurately cut. Using this principle, the most commonly used and efficient method for gene knockout cells is to use gRNA vectors to transfect into Cas9 stable cell lines, and obtain gene knockout cell lines through monoclonal isolation and screening.

This gene knockout protocol provides the complete procedures for CRISPR knockout cell line generation. It includs:
Part 1: gRNA Design and Vector Cloning
CRISPR-cas9 knockout protocol (Part 2): Cell Transfection 
CRISPR gene knockout protocol (Part 3): Single Cell Isolation and Positive Clones Validation

Here is the part 1 of this CRISPR knockout cell line protocol--gRNA Design and Vector Cloning.

KO cells generation protocol

▍  Design gRNAs targeting gene of interest

Using an online gRNA design tool CRISPRdirect to design gRNAs:
1.Enter the target sequence
2.Choose species
3.Click “Design”
4.Click “Show highly specific target only”

▍  Key Points for gRNA Selection

1. Gene knockout targets should be designed downstream of the start codon or within the exon range downstream of the start codon.
2. Different gRNAs have significant differences in gene knockout efficiency. Therefore, it is recommended to design 2 to 3 targets at the same time to perform the experience and then select the target with better CRISPR knockout effect.
3. In a gRNA(N1-N20 NGG), the base close to PAM are important for the specificity of the target site, and the mismatch of the first 7 to 12 bases has a small impact on the cutting efficiency of Cas9. The designed target sequence should be BLAST tested in the gene library.
4. Using Cas9Nicknase needs to select a pair of targets. Generally, target pairs with a distance of 20-30 bp are selected on the sense chain and the antisense chain respectively. The CRISPR knockout efficiency of multiple target pairs often varies greatly. Due to the long turnaround of gene knockout experiments, it is necessary to test and select the targets before formally performing CRISPR knockout on the target cell line.

▍  Cloning sequence design

Design insertion oligonucleotide sequence (oligonucleotides must be purified by PAGE):

Forward sequence: 5’ACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT3’

Reverse sequence: 3’TGTGGCNNNNNNNNNNNNNNNNNNNCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAA5’

Synthesis of cloning fragments:

1. Dilute the oligonucleotide with water to 100μM. The annealing reaction solution is prepared according to the following formula:
● Forward oligonucleotide (100μM) 5μl
● Reverse oligonucleotide (100μM) 5μl
● NaCl 100 mM (final concentration)
● Tris‐Cl pH7.4 50 mM (final concentration)
● Add water until the whole volume reach to 50μl

2. Mix the prepared annealing reaction buffer solution, briefly centrifuge, and place it on a PCR instrument. Run the following procedures: 90℃ 4 min, 70℃ 10 min, 55℃ 10 min, 40℃ 10 min, 25℃ 10 min. The annealed oligonucleotides can be used immediately or stored at 20℃ for  long time storage.

▍  gRNA vector cloning

The 2 μg gRNA backbone vector was digested with EcoRV. Generally, the enzyme digestion can be completed with about 20 to 30 units of enzyme for about 3 hours. After digestion, we suggest using agarose gel to collect the linearized vector. Quantify the linearized vector. Typically, the working concentration of the linearized carrier is 50 to 100 ng/μl. Dilute 100x annealed double stranded oligonucleotides (10 μM) to 1x with water for ligation.

Ligation reaction system:
T4 DNA ligase 5U
EcoRV 5U
Linearized vector 2 μl
1x annealed double stranded oligonucleotides 1 μl
10× ligase Buffer 1 μl
50% PEG4000 1 μl
Add water to 10 μl

Reaction condition:
22℃ 30 min, 37℃ 15 min

Note:
The efficiency of Blunt-ended ligation is low, and adding PEG4000 to the ligation system can improve the ligation efficiency.
Adding EcoRV enzyme to the ligation system can significantly increase the positive rate.

▍  E.coli competent cells transformation and positive clones identification by PCR and sequencing

Select the positive clones for sequencing verification. After verification, extract the plasmid for transfection test to determine whether the sgRNA is valid. After gRNA vectors transfect to Cas9 stable cell lines, DNA can be extracted directly. Through sequencing verification, if there are overlapping peaks appear near the target site, it indicates that the sgRNA is effective, and if not, the sgRNA is ineffective.