Assessment and Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using Streptococcus pyogenes CRISPR-Cas9 as the model enzyme.

Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data, available on the CRISPR-Cas9 system, were also analyzed.

Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013.

Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were used for analysis. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic studies of this group of enzymes to confirm the possible amino acids involved in the active sites and catalysis.

Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. The HNH endonuclease domains are also found in DNA polymerase and reverse transcriptase genes of group I and group II introns, respectively. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, MSA of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. From the catalytic amino acids identified from them, they are grouped either -DH---N or -HH--N types. There are at least two types of metal-binding sites identified and they bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a catalytic Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs.

Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor. The N in –HNH- Cas9 confers specificity by interacting with the nucleotide at the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and zinc finger nucleases have already found applications in genome editing.