CRISPR KEY POINTS

What is NHEJ?

Nonhomologous end joining (NHEJ) is an endogenous repair pathway that repairs double strand DNA break (DSB).

Is NHEJ error prone?

Yes. It is an error prone repair, often creates small deletions or insertions at the repair joint. These small mutations are referred to as indels.

How can NHEJ be used for genome editing?

When indels create a frameshift at the coding region, the result can be the loss of expression of the gene (knock-out). NHEJ can also be used to achieve deletion of the DNA sequence between 2 DSBs.

Which DNA repair pathway is needed for CRISPR knock-in? What can be used as a repair template?

Homology directed repair pathway is needed for knocking in a precise sequence, it can be either small sequence substitution or insertion.

A repair template is needed, it can be either a double strand or single strand DNA. The repair template has homology arms on each side of the intended mutation.

What are the 2 components of CRISPR/Cas9?

1. Cas9 protein

2. Guide RNA

Function of Cas9 protein?

Endonuclease that can be activated by gRNA complementing with the target sequence and create a double strand DNA break

Function of guide RNA?

RNA component including the 20nt sequence at the 5’ responsible for targeting specificity

What is PAM?

PAM stands for protospacer adjacent motif, it is a short DNA sequence adjacent to the target sequence on the target DNA.

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For Cas9, PAM is the 3-nucleotide sequence NGG on the target DNA 3’ to the gRNA sequence. PAM is not on the CRISPR gRNA, it’s on the target DNA.

Is PAM  necessary for CRISPR/Cas9 to work?

• PAM is essential, DNA sequence without PAM won’t be cleaved by CRISPR/Cas9. This restricts the choice of gRNAs to only DNA regions have the PAM.

• Cas12a recognizes a different PAM (NTTT), it can be used to target sequences that have TTT. There are ongoing efforts of searching for nucleases with relaxed PAM requirements.

How to reduce off target effect of CRISPR/Cas9?

• Find unique gRNA sequence with low similarity to other sequences in the genome.

• Use transient transfection to deliver CRISPR, avoid Lentiviral system that integrates CRISPR into the genome.

How to deliver CRISPR/Cas9 into the cells?

They can be transfected into the cells as plasmid, RNA, or in any combination if Cas9 protein and gRNA are in the same cell at some point.

• You can use plasmids that express Cas9 or gRNA or all in one, you can use Cas9 protein and synthesized gRNA.

• You can also use lentiviral vectors that express either Cas9 or gRNA or both.

Why do you need to screen clones when editing the genome?

When CRISPR/Cas9 is used to edit the genome, it creates a targeted DNA break that will be repaired by the endogenous DNA repair pathways in the cells. There will be a collection of different repair outcomes in a population of cells. To find the cell with a specific desired outcome, you need to screen through enough cells to find the one.

How to screen genome edited clones?

You can screen by either genotype or phenotype.

• Genomic PCR, DNA electrophoresis, sequencing can be used to screen for genotype.

• Western, FACS, Immunofluorescence, ELISA can be used to screen for phenotype.

What is Cas9 nickcase?

Cas9 nickcase is a Cas9 mutant that creates single strand DNA break instead of double strand DNA break, it contains either the D10A mutation in the RavC domain or the H840A mutation in the HNH domain.

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Prime editing is an example of using Cas9 nickcase, in this case Cas9 nickcase is linked with a reverse transcriptase.

What is dCas9?

dCas9 is a mutant form of Cas9 that doesn’t have the endonuclease activity, endonuclease dead, that’s why it’s named as dead Cas9 or dCas9.

• When complexed with the RNA component, dCas9 can still target to the specific genomic region that complementary to the gRNA sequence.

Why do you need dCas9?

dCas9 is often fused with other proteins to bring it to the desired genomic region. dCas9 can be fused with a base editor for base editing, fluorescent protein for imaging, transcription activator/inhibitor for transcription activation/inhibition, DNA methylase/demethylase for modifying epigenetics, etc.