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Class 1 CRISPR-Cas Systems Can Be Expressed in Mammalian Cells, Study Finds

NEW YORK – An international research team led by Duke University's Charles Gersbach has found that class 1 CRISPR-Cas systems can be expressed in mammalian cells, and can also be used for DNA targeting and transcriptional control.

Class 2 CRISPR systems, such as Cas9 and Cas12, have been widely used to edit eukaryotic genomes, but class 1 systems represent about 90 percent of all CRISPR systems in nature and remain largely unexplored for genome engineering applications, the team noted.

In a study published today in Nature Biotechnology, the researchers said they were able to repurpose type I variants of class 1 CRISPR systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex called Cascade.

"We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome," the authors wrote. "By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells."

Type I CRISPR systems use the Cas3 nuclease-helicase to eliminate invading DNA. These systems can be divided into eight subtypes (I-A to I-G and I-U) based on their subtype-specific, accessory cas genes. The type I-E system from the E. coli K12 strain consists of eight cas genes and a downstream CRISPR array. Cascade is formed when five protein subunits (Cas8e, Cse2, Cas7, Cas5, and Cas6) bind to a CRISPR RNA (crRNA), the researchers explained.

To bind to a target, Cascade surveys DNA sequences to find a protospacer-adjacent motif upstream of a target sequence with complementarity to the crRNA spacer sequence. In order to repurpose Cascade for use in mammalian cells, the researchers used a cytomegalovirus (CMV) promoter to express each Cascade subunit of the E. coli K12 system (EcoCascade).

To determine whether EcoCascade complex formation occurred in human cells, the researchers co-transfected six plasmids encoding each of the five Cas subunits and the crRNA cassette into HEK293T cells. They found that although EcoCascade complexes can be purified from bacteria in the absence of a crRNA, a crRNA was necessary for EcoCascade formation in human cells.

The researchers then looked to repurpose EcoCascade for CRISPR-based programmable transcriptional activation in mammalian cells. In the natural type I CRISPR immune system, target site recognition by Cascade leads to recruitment of the Cas3 nuclease to eliminate target DNA, they explained. However, deletion of the cas3 gene from the endogenous type I-E system in E. coli has been used for a CRISPR interference strategy in bacteria by permitting Cascade to bind to target DNA and block transcription without DNA degradation.

The researchers hypothesized that Cascade could be repurposed as a programmable DNA-binding technology in eukaryotes by neglecting to express cas3. Therefore, they analyzed the potential of various Cas-effector subunits for tethering of the activation domain.

Previous research had shown that deactivated Cas9 could cause robust endogenous gene activation when fused to the catalytic core domain of the human acetyltransferase p300. For this experiment, the investigators confirmed the formation of an EcoCascade complex following heterologous expression of EcoCascade with p300 fused to Cas8e, Cse2, Cas5 or Cas6. They then generated a panel of crRNAs to test programmable endogenous gene activation in human cells, and found that EcoCascade with Cas6-p300 and individual crRNAs caused robust activation of IL1RN with many crRNAs, including more than 3,000-fold IL1RN activation with cr26.

"Importantly, cr26 with EcoCascade lacking a p300 domain, or cr26 alone, did not activate IL1RN, suggesting target-specific activation by EcoCascade-p300," the authors added.

They also explored the transactivation potential of all Cas-p300 fusions with cr26, and found that EcoCascade containing Cas8e-p300 or Cas6-p300 achieved significant transactivation of IL1RN.

In a subsequent experiment to analyze EcoCascade-p300 interactions at the target locus, the researchers observed significant enrichment of the target regions in EcoCascade-p300 samples co-transfected with cr25 or cr26. These results confirmed EcoCascade as a programmable DNA-binding platform for efficient targeting of specific loci in the human genome, they noted.

They also found a few off-target differential binding sites when comparing cr25 and cr26 to control crRNA, and said that these off-target sites all had substantially weaker binding signals than the signal at the IL1RN locus, with one exception. Further, after performing RNA sequencing to evaluate the specificity of crRNA-mediated endogenous gene activation with EcoCascade-p300, the researchers found that targeted gene activation was highly specific to the target gene when EcoCascade-p300 was co-expressed with cr26, and detected a modest change in the activation of only six other genes.

However, they also found that adding the p300 domain to EcoCascade resulted in significant off-target transcriptional changes compared to EcoCascade alone, adding that these results indicated non-specific crRNA-independent effects of overexpression of the p300 acetyltransferase fused to Cas6.

"Collectively, this genome-wide specificity analysis demonstrates highly specific crRNA-dependent targeting of EcoCascade in mammalian cells," the authors wrote.

They then looked to explore the potential for repurposing other Cascade complexes, such as the type I-B CRISPR-Cas system of the L. monocytogenes Finland_1998 (LmoCascade) strain. They fused the catalytic core domain of p300 to Cas6, and tested programmable endogenous gene activation in human cells.

"LmoCascade with Cas6-p300 and individual crRNAs revealed robust IL1RN activation with most crRNAs," the researchers wrote. "Additionally, LmoCascade containing Cas8b2-p300, Cas5-p300 or Cas6-p300 achieved significant transactivation of IL1RN."

In contrast to the panel of IL1RN-targeting crRNAs for EcoCascade, almost all of the LmoCascade crRNAs, as well as three of the four Cas-p300 effector fusions, achieved significant IL1RN activation, they added.

"Targeted transcriptional modulation is important for perturbing gene function, designing gene regulatory networks, investigating the function of distal regulatory elements, manipulating cellular and organismal phenotypes, and inducing therapeutic changes to gene expression," the team concluded. "Cascade complexes from type I CRISPR-Cas systems provide a novel and widely applicable RNA-guided platform for targeting DNA sequences in eukaryotes."

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