• CRISPR-Cas9 Cleavage Specificity:
• The Cas9 endonuclease from Streptococcus pyogenes (SpCas9) requires two distinct molecular cues to recognize and cleave target DNA:

• Guide RNA (sgRNA) matching a 20-nucleotide target protospacer sequence.
• mandatory Protospacer Adjacent Motif (PAM): SpCas9 strictly requires the motif $5'\text{-NGG-}3'$ immediately adjacent to the $3'$ end of the protospacer target.
  • Without the correct PAM sequence, Cas9 cannot unwind the DNA duplex or activate its RuvC and HNH nuclease cleavage domains.

• nalysis:
• (PAM sequence): Correct.

• Inverse PCR: A variant of PCR used to amplify unknown DNA flanking regions that lie on either side of a known core sequence.
• Mechanism: The target genomic DNA is digested with a restriction enzyme cutting outside the known sequence, circularized by self-ligation using T4 DNA ligase, and then amplified using primers pointing outwards (divergent primers) from the known segment.
• Other PCR Variants:
• Nested PCR: Uses two successive sets of primers (outer and inner) targeting the same region to enhance specificity and yield.
• RT-PCR: Reverse Transcription PCR, uses reverse transcriptase to synthesize cDNA from an RNA template before PCR amplification.
• Anchored PCR: Amplifies sequence when only one end has a known sequence by attaching an artificial poly-G or adaptor anchor.

Correct option: (2) Inverse PCR

• Target Gene Amplification Workflow:

• Primer Synthesis: Before initiating PCR amplification, oligonucleotide primers flanking the target gene sequence must be designed and synthesized.
• Separating Two Strands of DNA (Denaturation): The double-stranded template DNA is heated to $94\text{--}95^\circ\text{C}$ to break hydrogen bonds and produce single-stranded templates.
• Annealing with Template: The reaction is cooled to $50\text{--}65^\circ\text{C}$ to allow forward and reverse primers to hybridize specifically to their complementary target sequences.
• DNA Polymerase Action (Extension): Thermostable DNA polymerase (e.g., Taq polymerase) extends the primers from $5'$ to $3'$ at $72^\circ\text{C}$ by incorporating dNTPs.
  • Correct Order: (B) $\to$ (A) $\to$ (D) $\to$ (C).