Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts

Campa et al. (2019). Nat Methods DOI: 10.1038/s41592-019-0508-6 Citations: 313

CRISPR multiplexing needed separate promoters for Cas9 and guides, limiting scale. Cas12a and a CRISPR array were fused on one transcript via a Malat1 triplex, forming SiT-Cas12a. Ten crRNAs per gene raised CD47 knockout to 60%.

Parts used

AsCas12aKRAB DomainVPR

Key findings

  • The researchers developed SiT-Cas12a encoding Acidaminococcus sp. Cas12a and CRISPR arrays on single Pol II transcripts by inserting a 110-nt Triplex tertiary structure from Malat1 between Cas12a and the CRISPR array. The Triplex stabilized transcripts after Cas12a RNase-mediated processing, preventing poly(A) tail removal and mRNA destabilization. Golden Gate assembly enabled cloning of arrays with up to 25 spacers on single plasmids, with gene editing efficiencies equal to or higher than platforms using separate promoters.

  • SiT-ddCas12a fused to three tandem KRAB domains achieved 40-80% transcriptional repression across ten genes using 20-spacer arrays, while ddCas12a-[Activ] with p65-HSF1 produced 10-1000-fold activation at ten targets. Small RNA-seq confirmed processing of all 20 mature crRNAs from single transcripts. The platform enabled coordinated regulation of ten genes simultaneously with editing of five additional loci, outperforming VPR activator by 10-fold at limiting spacer concentrations.

  • Spacer length modulation enabled orthogonal gene editing and transcriptional control without requiring different Cas proteins. Arrays with 15-bp spacers produced 3-5-fold higher mature crRNA levels than 20-bp spacers and directed transcriptional regulation without detectable gene editing. Conversely, 20-bp spacers enabled catalytically active Cas12a to perform gene editing (2-25% indels across five loci) while maintaining transcriptional control, allowing simultaneous editing and regulation of fifteen genes total.

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Abstract

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.