research article (crispr-cas9) exonic disruption

a comprehensive overview of both basic miRNA mechanisms and the specific role of miRNAs in herpes viruses, suitable for an informative presentation.This paper would likely highlight the innovative use of CRISPR-Cas9 to target HIV-1 directly within infected cells, potentially offering a new strategy for treating HIV infection and addressing limitations of current antiretroviral therapies.Summary of key findings
Emphasis on the potential of CRISPR-Cas9 exonic disruption as a novel approach to HIV-1 treatment.

1. CRISPR-Cas9 Mediated Exonic Disruption for HIV-1
Elimination
Jonathan Herskovitz, Mahmudul Hasan, MilankumarPatel, Wilson R. Blomberg, Jacob D. Cohen, Jatin Machhi,
Farah Shahjin, R. Lee Mosley, JoEllynMcMillan, Bhavesh D. Kevadiya, Howard E. Gendelman
Impact Factor -11·205
November 10, 2021
S. Jaishree (227798)
Ph D. in Biomedical sciences
Advisor: Prof. Song Jong Keun

2. HIV (Human Immuno-Deficiency Virus)
Toxins2022, https://doi.org/10.3390/toxins14020138

3. HIV Infection & Provirus Replication

4. HIV Genome & Proteins
https://www.onlinebiologynotes.com/structure-genome-proteins-hiv/
Genome:
•HIV is ss RNA virus.
•The genome consists of two identical copies of
+SS RNA and protein which are linked at their 5’
end.
•Genome of HIV consists of 9 gene, 3 structural
gene and 6 non-structural gene (regulatory gene).
•Structural gene (env,gag and pol), regulatory gene
(tat,rev,nef,vif,vpr and vpu in HIV-I and vpx in
HIV-2)

5. Genome Editing
Genome
Editing

6. BioTech2021,10(3), 14;https://doi.org/10.3390/biotech10030014
CRISPR-CAS9

7. Hypothesis
The hypothesis of the study is that exonic disruption using CRISPR-Cas9 can be used to eliminate proviral
DNA in infected CD4+ leukocytes, which is a barrier to HIV-1 cure.
Lentiviral and lipid nanoparticle (LNP) delivery of the CRISPR-Cas9 HIV-1 gene editing systems successfully
block HIV-1 reactivation from prior latently infected CD4+ T cell and monocyte-macrophage cell lines.

8. In Silico Design of HIV-1 tat gRNAs
Figure-1 HIV-1 CRISPR-Cas9 Mosaic guide RNA (gRNA) Design
•Conserved-Low variance of
nucleotides
•Entropy-High degree of variance in
nucleotides
LTR-Long terminal repeats
tat-Trans activator of Transcription

9. (a)AgRNAlibrarywasscreenedagainstapanelofHIV-1molecularclones
byco-transfectionintoHEK293FTcells.Progenyvirionproductionwas
measuredbyreversetranscriptase(RT)activityinculturefluids.
(b)PearsoncorrelationbetweengRNAtargetconservationamong4004
proviralDNAsequencesandRTknockdownwereassessed.
(c)PCRtestswerecompletedonDNAextractedfromamplifieduntreatedor
CRISPR-TatDEplasmid-treatedcells.Thewhitearrowindicatesthe
expectedmolecularsizeoftheTatDEexcisionband.
Tat/rev Directed gRNAs Suppress HIV 1 Replication
On-target CRISPR-
Cas9 TatDE specificity

10. LentiviralTatDECRISPR Inactivâtes Latent HIV-1
Figure 3 Lentiviral TatDE CRISPR Inactivates Latent HIV-1
MOI-Multiplicities of Infection
ACH 2 cells -human T-lymphocyte cell line infected with the HIV.

11. Exonic Disruption is The Potential Mechanism for HIV-1 Replication
Attenuation
Figure 4 Exonic Disruption and HIV-1 Replicative Fitness
•Non-frameshift point mutations
Two (TatD), Three (TatE) or five (TatDE) exons altered.
•Locus targeted by TatEgRNA is critical for maximal
CRISPR activity.
•The high efficacy of TatDE CRISPR-Cas9 therapy against
numerous HIV-1 strains results from disrupting five viral
exons simultaneously.

12. Figure 5 Synthesis, Characterization, and Antiretroviral Activity of CRISPR-Cas9 LNPs in Primary Human Monocyte-Derived Macrophages (MDM).
TatDE Lipid Nanoparticles (LNPs) Protects Macrophages From
HIV-1ADAInfection
Thin film hydration method
Characterizationofnanoparticles:
B&CTEMimages
D-Atomicforcemicroscopy
E-DynamiclightscatteringGraph
F-MonocytederivedmacrophagestreatedwithLNPsindose
dependentmanner.

13. Figure 6 CRISPR LNPs Cell Trafficking
TatDE LNP intracellular trafficking in primary human MDMs
Two sets of Experiments
1.TatDEs labelled with Rhodamine DPHE phospholipid.
Rab5 & Rab7-(Ras-related proteins)
Early and late endosomal markers
LAMP1 –(Lysosomal-associated membrane protein 1)
Lysosomal degradation
2.Nucleic acid labelled LNPs.
Z-stack analysis : LNPs location

14. Figure 7 HIV-1 RNP Delivery for Virus Editing.
TatDE ribonucleoprotein (RNP) excision of proviral DNA
•pNL4-3 & pCH040.c/2625 :HIV clones

15. Figure 8 CRISPR mRNA Delivered by LNPs is in HIV-1 Infected Cells.
mRNA delivered by rLNPs are expressed in HIV infected cells
Clean cap mRNA:
•Cap structure is a modified nucleotide that is added to the 5' end of
eukaryotic mRNA during transcription.
•It is important for mRNA stability, translation initiation, and the
efficiency of protein expression.
(f) GFP LNP treatment to the cells showed readily detectable GFP
expression in both U1 and JLat cell lines affirmed by the shift of
population intensities for GFP recorded cells
U1-Human T-cell lymphoma cell line
Jlat–Human T-cell leukemia cell line
GFP-Green fluorescence protein
FLuc-Firefly Luciferase

16. Figure 9 mRNA LNP carriage (rLNP) of HIV-1 TatDE and Cas9 Attenuate Viral Replication.
TatDE rLNPCas9 mRNA and TatDE sgRNA and elimination of
HIV-1 infection
U1-Human T-cell lymphoma cell line
Jlat–Human T-cell leukemia cell line

17. Strengths &Limitations Of The Study
•gRNA approach of using CRISPR -inactivate
genetically heterogeneous viral targets.
•Correlation with reference controls.
•CRISPR on-target cleavage efficiency, target
DNA conservation, excision length, and
numbers of disrupted exons during CRISPR-
Cas9 treatment.
•Variant HIV strains.
•Lack of off-target editing within the human
genome is an inherent limitation.

18. Conclusion
•The combination of “diverse” strain-inactivating TatDand “Tri-exon”-directed TatEgRNAs prompted maximal
suppression of HIV-1 replication by CRISPR-Cas9.