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CRISPR Researchers Use Cas12a to Develop Multiplex Genome Engineering Platform

NEW YORK – Researchers from ETH Zurich in Switzerland and the University of Groningen in the Netherlands have used CRISPR-Cas12a to develop a genome engineering platform to investigate the biology of complex cell behaviors.

In a new study published today in Nature Methods, the researchers described a method they developed to encode a CRISPR array and a Cas12a enzyme in a single transcript by adding a stabilizer tertiary RNA structure, in order to enable the modification of multiple genetic elements simultaneously and thereby  elucidate and control the gene interactions and networks underlying complex cellular functions.

"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," the authors wrote. "Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors."

Current CRISPR technology allows for the use of different Cas enzymes or engineered CRISPR RNAs (crRNAs) to create distinct gene perturbations, such as gene knockouts, activations, and repressions. But the constraints of the technology as it exists today limit the scalability of CRISPR-based multiplexed genome engineering approaches.

However, cells have evolved to coordinate various processes using a limited number of cellular elements, the researchers noted. For example, protection against bacteriophages and other foreign genetic elements is mediated by Cas12a, which functions as both an RNase and a DNase, and controls both processing and maturation of its own crRNA as well as DNA target cleavage. In mammalian cells, Cas12a has been used for gene editing and transcriptional gene control, they added.

In this study, the investigators used the dual RNase/DNase function of Acidaminococcus sp. Cas12a (AsCas12a) to develop a system they called single-transcript Cas12a (SiT-Cas12a), encoding Cas12a and dozens of crRNAs in a single transcript for multiplexed genome engineering. They stabilized SiT-Cas12a transcripts by including a tertiary structural motif, which improved pre-crRNA processing and Cas12a production.

The researchers evaluated the platform using a CRISPR array containing a spacer targeting DNMT1, and noted that they observed consistent and efficient gene editing at the DNMT1 locus. They also generated conditional and inducible SiT-Cas12a platforms, termed SiT-Cas12a-[Cond] and SiT-Cas12a-[Ind], respectively. Through various experiments, the team found that SiT-Cas12a enabled either constitutive or conditional and inducible gene editing through fine temporal control of Cas12a and crRNA expression.

The investigators then evaluated the potential of the SiT-Cas12a platform for multiplexed gene editing, using a CRISPR array containing five spacers targeting different genomic loci (FANCF1, EMX1, GRIN2B, VEGF, and DNMT1). They found that gene editing efficiency was higher in cells expressing SiT-Cas12a compared to a CRISPR-Cas12a control. They also compared the gene editing efficiency of SiT-Cas12a with previously reported Cas12a platforms based on independent transcription of Cas12a and a CRISPR array from distinct promoters, and found that expression of SiT-Cas12a resulted in gene editing efficiencies equal to or higher than other tested platforms.

In further experiments, the researchers cloned a CRISPR array harboring ten distinct spacer sequences targeting the CD47 locus in the SiT-Cas12a context, either singularly or jointly, and performed gene editing quantification. They found that the gene editing efficiency of single crRNAs ranged from 2 percent to 17 percent, but that simultaneous expression of all crRNAs increased the gene editing efficiency up to 60 percent, indicating that the targeting of multiple crRNAs in the same coding gene introduced more loss-of-function mutations.

Finally, the team looked to develop a SiT-Cas12a-based platform that could facilitate orthogonal gene editing and transcriptional gene control. They assessed AsCas12a's processing efficiency using CRISPR arrays containing both long (20 base pairs) and short (15 bp) spacers, measuring three- to five-fold higher amounts of mature crRNAs in cells expressing arrays containing short spacers compared to those with long spacers. They also evaluated the SiT-Cas12a platform in an orthogonal transcriptional control and gene editing context, combining both active and inactive DNase versions of the SiT-Cas12a-based transcriptional repressor and activator with two sets of CRISPR arrays harboring spacers targeting two distinct promoters using either short or long spacers.

The researchers found that only DNase-active SiT-Cas12a effectors combined with 20-bp spacers facilitated gene editing, but that SiT-Cas12a effectors combined with 15-bp spacers induced either gene repression or gene activation with comparable efficiencies and without any detectable gene editing events. They also found that large CRISPR arrays containing both short and long spacers enabled coordinated and highly multiplexed regulation of ten distinct genes simultaneously with gene editing of another five distinct genes.

"Taken together, SiT-Cas12a effectors, based on AsCas12a, facilitated orthogonal transcriptional control and gene editing simply by altering spacer length," the team added. "Considering the mean natural length of protein-coding transcripts found within mammalian cells (13.5 kb), the potential for expressing multiple crRNAs in the SiT-Cas12a context is profound. In the future this could theoretically be used to enable massively multiplexed expression of hundreds to thousands of independent crRNAs, opening up avenues for large-scale genome engineering efforts."