Question

ASAP please ..
Hey, I need help answering these questions, about the introduction of an article about HIV inhibition using adenovirus-delivered CRISPR/CAS9.  Thaanks!!

strand, resulting in double-stranded breaks (DSBs) that trigger cellular repai mechanisms. In eukaryotes, the DSBs are more commonly repaired by the mechanism ot error prone non ho mologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletians (indels), around the DSBs. Owing to the simplicity of manipulation and versati ity, the CRISPR/Cas9 system has been utized as an attractive tool tor various applications, such as genome widc screening, gene repression and activation, targeted fluorescence imaging and novel approaches against pathogens including hepatitis B virus, human papillomavirus, Epstein-Barr virus, malaria and INTRODUCTION More than 33 years have elapsed since the first reported case of AIDS. Although the introduction of effective antiretro- viral therapy (ART) has resulted in huge reductions in rates ot human immunodeficiency virus type 1 (IV-1) illness, IHlV-1 in- fection remains incuable. In addition, ART encounters a num ber of challenges, including a persistent latent viral reservoir, the requirement for lifelong adherence, and the potential de velopment of drug resistance and toxicity. Thus the develop ment of novel therapeutic methods that may enhance current therapeutic options and even lead to curative approaches would significantly expand our portfolio of strategies to coun- teract HIV-1/AIDS. 1, what was the main idea of paragraph #1? 2. what are the main ideas of paragraph #2? 3. There are three significant challenges listed in this paragraph. List them and briefly describe why they are challenges End of document Using a pai of sBRNAs targeting the LTR of HIV-1, it was shown that HIV-1 provirus can be removed from the genone of infected cell lines. By combining TALEN or CRISPR/Cas9 with PiggyBac technology, researches have generated induced pluripotent stem cells (iPSC hamozygous for the naturally oc- curring CCR5D3 variant resistant to HIV-1 infection. How- ever, owing to the large size of the Cas9 codin sequence with a total length of more than 5 kb when combining with sgRNA, promoters and other essential clements for etficient expres sion, delivery of CRISPR/Ca for instance, CD4T-lymphocytes, the primary target for HIV 1 infection i vivo, reains challeging. In the cuent study after optimized design and screening of a panel of sgRNAs in cell lines, we obtained several sgRNA sequences with high po tency to target CCRS. By utilizing a chimeric adenoviral we demonstrated the efficacy of CCR5 editing and established HIV-1 resistance in primary CD4+ T-cells. Nuclease-mediated genome editing represents one promising strategy for HIV-1 therapy. Nucleases including zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) recognize genome locus based on prot DNA interactions. By construction of a tandem array of vari ous artiticially engineered modules, ZFN and TALEN can target virtually any DNA sequence. However, these two technologies to some extent present limitations and remain not only tech- nically complex but al laborious and time-csurning to ma- nipulate. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein 9 (Cas9), derived from a type II CRISPR/Cas system naturally existent in bacteria, has been harnessed as a novel nuclease tool to me- diate genome editingi mnalian cells. By simple delivery of two essential components, a human codon-optimized Cas9 protein and a single guided RNA (SgRNA), the CRISPR/Cas9 complex formed can be programmed to target any genomic lacus followed by a 59-protospacer adjacent motif (PAM) se- quence of NGG, with the specificity determined by the sgRNA containing a 20 nt guide sequence complementary to the nome locus of interest. Upon the guidance of sgRNA, Cas9 protein is programmed to cleave the targeted DNA at each s9 components into primary cells,

Answer 1

Ans 1: The main idea of paragraph 1 is to carry out nuclease mediated genome editing using CRISPR/Cas9 technique so as to introduce double stranded break in the targeted DNA using Cas9 which will result in errors during repairing mechanism in the viral genome which is integerated in human genome.

And 2 : Using pair of sgRNAs, LTR of HIV virus (which are the sequences through which HIV intergerate in human genome) can be chopped off resulting   in removal of provirus which makes cells to become resistant to HIV infection.