CRISPR Cas 9 Lecture 3

Key Points

What is NHEJ? How can it be used for genome editing?

• non homologous end joining —> endogenous pathaway that repairs dsDNA breaks (DSB)

• error prone repair mechanism, often creates small deletions or insertions at the repair joint.

• these small mutations are referred to as indels.

• when indels create a frameshift at the coding region, the result can be the loss of expression of the gene (knockout)

• NHEJ can also be used to achieve deletion of the DNA sequence between 2 DSBs

Which DNA repair pathaway is needed for CRISPR knock in? what can be used as a repair template?

• HDR (homology directed repair) 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 ds or s strand of DNA

• Repair template

What are the components of CRISPR/Cas9 and what are the major functions of each component?

a. Cas9 protein

• endonuclease that can be activated by gRNA complementing with the target gene sequence and create a ds break

b. Guide RNA

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

What is the PAM? Is it necessary for CRISPR/ Cas 9 to work?

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

• for Cas 9, PAM is the 3 nucleotide sequence NGG on the target DNA 3’ to the gRNA sequence

• PAM is not the CRISPR gRNA, its on the target DNA

• 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 effects of searching for nucleases with relaxed PAM requirements.

How to reduce off target effect of CRISPR/ Cas 9

a. find unique gRNA sequence with low similarity to other sequences in the genome

b. use transient transfection to deliver CRISPR, avoid lentiviral system that integrates CRISPR into the genome

How to deliver CRISPR/ Cas9 into the cells?

• both Cas 9 and gRNA need to be present in the cells for CRISPR to work

• they can be transfectioed into the cells as plasmid, RNA or in any combination if Cas 9 protin and gRNA are in the same cell at some point

• you can use plasmids that express Cas 9 or gRNA or all in on, 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/Cas 9 is used to edit the genome it created a targeted DNA break that will be repaired by the endogenous DNA repair pathaway in the cells

• there will be a collection of different repair outcomes in a population of cells

• to find the cell with the specific desired outcome, you need to screen through enough cells to find the one

How to screen genome edited clones?

• you can scrren 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 Cas 9 nickase?

• Cas 9 nickase is Cas 9 mutant that creates single strand DNA break instead of dsBreak, it contains either D10A mutation in the Rav C domain or the H840 mutation in the HNH domain. 

• primer editing is an example of using Cas 9 nickase in this case Cas9 nickase is linked with a. reverse transcriptase 

What is dCas9? Why do you need dCas9? 

• dCas9 is a mutant form of Cas9 that doesn’t have the endonuclease activity —> endonuclease is dead thats why its names 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, dCas9 is often fused with other ptoeins to bring it to the desired genomic region.

• dCas9 can be fused with a base editor for base editing fluorescent protein for imaging, transcription activato/ inhibitor for transcription activation/ inhibition, DNA methylase/demethylase for modifying epigenetic etc.