Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations

C C-S Hsiung; C M Wilson; N A Sambold; R Dai; Q Chen; N Teyssier; S Misiukiewicz; A Arab; T O'Loughlin; J C Cofsky; J Shi; L A Gilbert

doi:10.1038/s41587-024-02224-0

Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations

Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02224-0. Online ahead of print.

Authors

C C-S Hsiung^{1

2

3

4}, C M Wilson^#^{2

3

4

5}, N A Sambold^#⁴, R Dai^{2

3

4

6}, Q Chen⁷, N Teyssier⁸, S Misiukiewicz^{2

3

6}, A Arab⁴, T O'Loughlin^{2

3}, J C Cofsky⁹, J Shi⁷, L A Gilbert^{10

11

12}

Affiliations

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
² Department of Urology, University of California, San Francisco, CA, USA.
³ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
⁴ Arc Institute, Palo Alto, CA, USA.
⁵ Tetrad Graduate Program, University of California, San Francisco, CA, USA.
⁶ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁷ Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Biological and Medical Informatics Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁹ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
¹⁰ Department of Urology, University of California, San Francisco, CA, USA. luke@arcinstitute.org.
¹¹ Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. luke@arcinstitute.org.
¹² Arc Institute, Palo Alto, CA, USA. luke@arcinstitute.org.

^# Contributed equally.

PMID: 38760567
DOI: 10.1038/s41587-024-02224-0

Abstract

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting one to three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein-DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

Abstract

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