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Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.

C C-S Hsiung1,2,3,4, C M Wilson2,3,4,5, N A Sambold4

  • 1Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

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Researchers developed a new CRISPR interference (CRISPRi) tool, multiAsCas12a-KRAB, enabling simultaneous targeting of multiple genomic sites. This advancement facilitates efficient pooled screening for genetic interactions and regulatory element function.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Multiplexed genetic perturbations are essential for studying gene interactions.
  • CRISPR interference (CRISPRi) is a genotoxicity-free method for perturbing genetic elements, especially non-coding ones, in pooled screens.
  • Current CRISPRi methods are limited to targeting only a few genomic sites per cell.

Purpose of the Study:

  • To engineer a novel CRISPR interference system capable of multiplexed transcriptional repression.
  • To enhance the efficiency and scope of pooled genetic screening using CRISPRi.
  • To enable the discovery and functional dissection of regulatory elements through combinatorial perturbations.

Main Methods:

  • Engineering of an Acidaminococcus Cas12a (AsCas12a) variant (multiAsCas12a) with a stabilizing mutation (R1226A) for enhanced DNA nicking.
  • Fusion of multiAsCas12a with the KRAB effector domain for potent transcriptional repression.
  • Development of 6-plex CRISPR RNA (crRNA) arrays for multiplexed CRISPRi.
  • Application of the multiAsCas12a-KRAB system in high-throughput pooled screens in human cells.

Main Results:

  • The multiAsCas12a-KRAB system demonstrated improved CRISPRi activity compared to DNase-dead AsCas12a-KRAB fusions.
  • The engineered system successfully rescued the activity of previously inactive lentivirally delivered crRNAs.
  • multiAsCas12a-KRAB supported multiplexed CRISPRi using 6-plex crRNA arrays in pooled screens.
  • The system was utilized to identify enhancer elements and analyze combinatorial functions of cis-regulatory elements.

Conclusions:

  • The multiAsCas12a-KRAB system provides a powerful tool for multiplexed transcriptional interference.
  • This technology enables efficient group testing of numerous chromatin perturbation combinations for biological discovery.
  • The developed framework advances the field of genetic screening and engineering by allowing large-scale combinatorial perturbation analysis.