Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics

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

doi:10.1101/2023.09.18.558350

bioRxiv · 10.1101/2023.09.18.558350

Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics

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

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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 1-3 genomic sites per cell. To develop a tool for higher-order (>3) combinatorial targeting of genomic sites with CRISPRi in functional genomics screens, we engineered an Acidaminococcus Cas12a variant -- referred to as multiplexed transcriptional interference AsCas12a (multiAsCas12a). multiAsCas12a incorporates a key mutation, R1226A, motivated by the hypothesis of nicking-induced stabilization of the ribonucleoprotein:DNA complex for improving CRISPRi activity. multiAsCas12a significantly outperforms prior state-of-the-art Cas12a variants in combinatorial CRISPRi targeting using high-order multiplexed arrays of lentivirally transduced CRISPR RNAs (crRNA), including in high-throughput pooled screens using 6-plex crRNA array libraries. Using multiAsCas12a CRISPRi, we discover new enhancer elements and dissect the combinatorial function of cis-regulatory elements. These results instantiate a group testing framework for efficiently surveying potentially numerous combinations of chromatin perturbations for biological discovery and engineering.

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Hsiung, C. C.- S., Wilson, C. M., Sambold, N. A., Dai, R., Chen, Q., Misiukiewicz, S., Arab, A., Teyssier, N., O'Loughlin, T., Cofsky, J., Shi, J., Gilbert, L. A.. 2023-09-18. Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics. https://doi.org/10.1101/2023.09.18.558350

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