Practice question

CRISPR-Cas9, ZFN, TALEN constitute programmable site-specific endonucleases enabling precise genome manipulation. CRISPR uses single-guide RNA 20 nt spacer complementary to genomic target adjacent NGG PAM recognized Streptococcus pyogenes Cas9 possessing HNH domain cleaving complementary strand RuvC noncomplementary generating blunt double-strand break 3 bp upstream PAM. Zinc-finger arrays modules each recognizing 3 bp triplets linked FokI nuclease dimerize cutting within spacer, while TALEN repeats repeat-variable di-residues HD recognizing cytosine NI adenine NG thymine NN guanine provide single-base flexibility. Cellular repair via error-prone nonhomologous end-joining introduces indels knocking out CCR5 for HIV resistance, while homology-directed repair donor template 400 bp homology arms flanking break precisely corrects sickle HBB glutamate 6 valine mutation or inserts therapeutic transgene into safe harbor AAVS1 chromosome 19 constitutive expression under endogenous promoter. Permanent genomic alteration distinguishes editing from transient augmentation offering curative potential single intervention approved exagamglogene autotemcel for sickle cell. This mechanistic insight guides vector optimization, dosing strategies, and clinical safety monitoring essential for translational development and regulatory evaluation.