CRISPR

Programming Cas9 with single-guide RNAs (sgRNAs)

Researches wanted to simplify the system and generate a system where there's a single RNA with Cas9. They linked crRNA and tracrRNA to make sgRNA ("single-guide RNA"). sgRNA is basically a crRNA-tracerRNA chimera

How can CRISPR technology be used against a infectious viral disease like HIV? Activating provirus in latent cells followed by cART clearance

cART = combined antiretroviral therapy. A combination of 30 known approved drugs. However, none of the current drugs can get rid of the latent infection. Using CRISPR to switch on the latent genes (what is meant by "shock")

After the dsDNA break is generated, the cell can repair via two types of pathways:

dsDNA breaks are repaired by two types of pathways: 1) Non-homologous end joining (NHEJ): => the ends of the DNA are chemically ligated back together => this is an imprecise repair pathway that usually generates a small insertion or deletion at the site of the break **A good way to mutagenize DNA at specific locations 2) Homologous Recombination => a donor DNA is inserted that has sequences that match those flanking the site of the ds break. This DNA can be integrated into the genome at the site of the break => this method introduces new genetic information into the genome **BRCA1 uses this pathway to repair dsDNA breaks **This pathway is more precise but it requires the presence of another DNA that is very similar to the one that is cleaved (can be another chromatid or a piece of DNA that we can provide)

Summary

=> The proviral genome is amenable to CRISPR (meaning it can be affected by CRISPR) => Can disrupt HIV expression by using CRISPR to target LTR regions in proviral genome. => Two opposing nickase targeted to the TAR region of LTR can disrupt HIV gene expression. => Nickase activity provides an extra level of safety and specificity.

Programmed Cas9 cleaves DNA at specific sites

An experiment that tested the ability to program Cas9 to cleave at specific sites: Constructed different sgRNAs that are specific to different regions on a plasmid. Programmed Cas9 to cute at designated sites and then digested the cleaved DNA with Sal1 cutting enzyme. Then ran the gel and found different lengths of DNA fragments were made. The lengths corresponded to the different target sites of the sgRNA constructs. This confirmed that Cas9 can be programmed to cleave DNA at specific sites.

What is CRISPR?

CRISPR is a bacterial system that bacteria use to fight viruses. It consists of an enzyme called Cas9 and a guiding RNA. Cas9 works together in a complex with the guide RNA to be directed to the complementary sequence of a gene that is being targeted where a ds break will be generated. Target sequence must have a PAM sequence, which is just a 3 nt sequence and varies per bacteria.

D

CQ: Guide RNA...

A

CQ: True or Falst? In bacteria, CRIS PR system consists of two RNA molecules, crRNA and tracrRNA. Jennifer Doudna and colleagues combined the two in one guide RNA molecule.

A

CQ: What is CRISPR system?

B

CQ: What is Cas 9?

D

CQ: Which of the following can be done using CRISPR?

How do we inhibit transcription of HIV provirus using CRISPR?

Can target the repeats at the 5' LTR and 3' LTR in proviral gene. This is an excellent option because it is the same sequence on both ends so you can program CRISPR to simply target that one sequence. Targeting different regions within the LTR, which will cause insertions and deletions in regions that are important for transcription (ex TAR element). This will prevent the virus from expressing its genes. **Both achieve the objective of preventing the virus from expressing its genes.

How can CRIPSR activate the virus?

Can use a "dead" Cas9 (dCas9) to target DNA with a guide RNA but this mutated Cas9 will not cleave the target site. **We can use this construct to bring protein complexes to a specific locus in the genome! This particular construct has VP16 activator complex attached to it, which is a protein that can activate dormant promoters. We bring this construct upstream to a transcription start site.

CRISPRs

Clusters of Regularly Interspaces Short Palindromic Repeats (CRISPRs) **Short palindromic repeats (the black boxes) are interspaced with sequences that are derived from viruses (the colored boxes) CRISPRs are the hallmarks of acquired immunity in bacteria

Why are the current drugs against HIV not "functional" cure?

Current drugs require you to take them lifelong to suppress latent infection but none of the current drugs can get rid of the latent infection.

Cas9 is a dual-RNA-guided dsDNA endonuclease

Has the ability to interact with DNA and generate a ds break in DNA at sequences that match with the guide RNA (crRNA). This guide RNA interacts with the second RNA called tracrRNA and this complex forms a structure that recruits Cas9 protein. ***So these 2 RNAs and a single protein (crRNA, tracrRNA, and Cas9) are required for the CRISPR system to function in nature.

Nickase

Is a mutated Cas9 enzyme that only nicks (cuts a single strand at target site) instead of cutting both strands and generating dsDNA break. dsDNA break is still achieved by using two different guiding strands. Nickase activity provides an extra level of safety and specificity.

T7 Endonuclease 1 Assay aka "Surveyor Assay" ***you can probably skip this card...

Is a way to test for INDEL formation. INDEL is insertions/deletions. T7E1 is an endonuclease that targets at mismatch areas, which are present in heteroduplexes. Heteroduplexes can be the result of CRISPR. So a surveyor assay can tell show you if INDELS were generated because if fragments from the T7E1 cut are present, then INDELS were there because T7E1 targeted INDELS. If this occurs then it means CRISPR did form INDELS.

NHEJ vs HR

NHEJ can serve for a very good purpose bc if we want to introduce a knock out mutation or some sort of mutation, we are going to screen for those events of non-homologous end joining. So if we just let the CRISPR system generate this cleavage in the DNA, at a fairly high rate, the cell will repair it incorrectly generating mutations that can result in a knock out of a gene or a point mutation so it's a really good way to mutagenize in very specific locations In HR, addition to the Cas9 and guide RNA, you can also introduce the DNA that you want the cell to use for homology based repair - your construct that you want to be introduced instead of that original sequence. And this is where you can either introduce loxP sites and that will be introduced in the cells DNA. Or you can generate an insertion. so if a cell contains a mutated copy of a gene, you can introduce a wild-type copy for that gene.

Three steps to acquire immunity in bacteria

Step 1) Adaptation: CRISPR system allows integration of short pieces of the viral DNA into the CRISPR locus Step 2) crRNA ("CRISPR RNA") biogenesis: CRISPR sequences are transcribed and the crRNA interact with Cas9 to form "Interference Complexes" Step 3) Interference: base pairs with matching sequences in viral DNA to interfere with it.

How can CRISPR technology be used against a infectious viral disease like HIV? Excising or deactivating integrated provirus

Target the proviral genes: the genes that produce the viral proteins Proviral editing approach: excise out the entire virus from the genome.

dCas9 system vs chemical reactivators

The "dead" dCas9 enzyme acts as a RNA-directed homing device for activator proteins such as Herpes Simplex Virus type 1 VP16 trans-activator. Global gene expression analysis shows that dCas9-VP64 activation of genomic HIV is more specific than chemical latency reactivators such as TNF-alpha.

What is the challenge with using chemical reactivators of viruses?

These drugs are nonspecific and other genes get activated causing massive amounts of side-effects.

Transcriptionally modulating HIV

Two approaches that involve modulating transcription: Transcriptional inhibition => long-term proviral suppression Shock and Kill => shocking viral reactivation then killing the latent reservoir **We will look at transcription inhibition first then the shock and kill method.

How can CRISPR technology be used against a infectious viral disease like HIV? Blocking infection and integration

Virus RNA produces dsDNA, so that is a target at the incoming stage. We can also target genes that are really important for the virus like CCR5.