Fig. 2

Mechanisms of CRISPR/Cas9 based gene editing. A. Schematic representation of mechanism of CRISPR/Cas9 based gene editing. Cas9 scans the DNA for the specific PAM sequence with the help of PAM interacting (PI) domain. Once the PAM sequence is identified sgRNA melts the target nucleotides upstream to PAM sequence and pairs them with crRNA. Then Cas9 cuts the target DNA 3 base pairs upstream of PAM sequence. Double strand breaks are repaired by Non homologous end joining (NHEJ) (majority) and Homology directed repair (HDR) resulting in random and precise deletions and insertions respectively B. CRISPR Cas9 based transcriptional regulation. Dead Cas9 (dCas9) is fused with specific transcription regulatory domains. Upon binding to the target DNA site, dCas9- regulatory domain complex recruits the corresponding gene activators/repressors to carry out the regulatory function C. CRISPR/Cas9 based base editing. dCas9 or Cas9 nickase (nCas9) are used for base editing. Cytosine base editors deaminate cytosine to uracil which is recognised by the DNA repair mechanisms and is substituted with Adenosine, whereas Adenosine base editors deaminate Adenosine to inosine which is substituted with Guanine by repair mechanisms