Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Cas9-mediated chromosomal rearrangements (inversions, deletions, duplications) are vital for studying genome structure and gene regulation.
• The precise mechanisms underlying these rearrangements remain largely unknown.

Purpose of the Study:

• To elucidate the mechanisms of Cas9-mediated DNA-fragment editing.
• To investigate the role of DNA repair pathways in Cas9-induced rearrangements.
• To understand the nucleotide insertion patterns during Cas9 editing.

Main Methods:

• Analyzing inserted nucleotides at DNA editing junctions.
• Characterizing Cas9 cleaved products in vitro and in vivo.
• Perturbing DNA repair genes (CtIP, FANCD2) to assess their impact on editing outcomes.
• Evaluating engineered Cas9 nucleases' cleavage profiles.

Main Results:

• Disrupting CtIP or FANCD2 enhances precise DNA-fragment deletion.
• Cas9 generates double-strand breaks with 5' overhangs (1-3 nucleotides) via endonucleolytic cleavage upstream of the PAM site.
• Engineered Cas9 nucleases exhibit distinct cleavage profiles.
• Cas9-mediated nucleotide insertions are nonrandom, correlating with sequences upstream of PAM sites.

Conclusions:

• Precise and predictable DNA-fragment editing is achievable by modulating DNA repair genes and optimizing PAM configurations.
• Understanding Cas9 cleavage and repair mechanisms is key to advancing genome editing technologies.