SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes.

Area of Science:

• Genetics
• Molecular Biology
• Epigenetics

Background:

• Programmable nucleases like CRISPR/Cas9 enable precise genome editing (PGE) via DNA double-stranded breaks (DSBs) and homology-directed repair (HDR).
• Alternative methods utilize nickase variants to induce single-strand DNA nicks, facilitating PGE through pathways like synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI).
• While SDSA is understood, the mechanism of ssDI remains unclear, prompting investigation into its regulatory factors.

Purpose of the Study:

• To elucidate the genetic factors regulating the ssDI pathway in precise genome editing.
• To identify genes whose absence enhances ssDI efficiency.
• To understand the role of chromatin modifiers in regulating ssDI.

Main Methods:

• Conducted a genome-wide CRISPR knockout screen in human cells to identify genetic modifiers of ssDI.
• Utilized transgenic reporters and single-copy genes (HUSH-regulated and non-HUSH regulated) to assess ssDI efficiency.
• Investigated the function of identified regulators (SETDB1/ATF7IP and HUSH complex) in ssDI.

Main Results:

• The screen identified the SETDB1:ATF7IP heterodimer and the HUSH complex as significant negative regulators of ssDI.
• SETDB1/ATF7IP and HUSH specifically inhibited ssDI at transgenic reporters and HUSH-regulated single-copy genes.
• This negative regulation was not observed at other endogenous single-copy loci, suggesting locus-specific effects.

Conclusions:

• Chromatin modifiers, specifically SETDB1/ATF7IP and the HUSH complex, play a critical role in regulating the ssDI subpathway of HDR.
• Chromatin structure can act as a barrier to genetic recombination, influencing PGE outcomes.
• These findings support strategies involving chromatin modulation to improve PGE efficiency at endogenous single-copy loci.