Latest revision as of 11:50, 20 October 2021

PspCas13b

PspCas13b is a CRISPR-associated protein originating from Prevotella sp and can be expressed in mammalian cells to target and cleave messenger RNA with compatible guide RNA. To note, PspCas13b is a novel part in the iGEM Biobrick Parts Registry. The sequence also contains a nuclear export signal (NES) and a 3x hemagglutinin (HA) tag.

Characterisation

Expression of Cas13b in mammalian cells

To verify the expression of the Cas13b part in the mammalian system, HEK293T cells were transfected with px330A-PspCas13b using Lipofectamine 2000. Cas13b is able to target and cleave messenger RNA strands with the aid of a compatible guide RNA, unlike in dCas13b, whereby the nuclease activity has been inactivated through the mutation of the enzymatic catalytic sites. An inactivated form of Cas13b is important for our RESCUE editor as we aim to make a site-directed change rather than cleave the target strand.

Transfected cells were lysed and cell extract was separated on an SDS-PAGE gel. As our PspCas13b includes an HA epitope tag at the C-terminus, we were able to detect for either Cas13b protein using a monoclonal HA antibody. As Cas13b protein is 3402 base pairs long, we expect a protein size of approximately 124 kDa to be expressed.

Figure 1: Western blot of PspCas13b and dPspCas13b. Both Cas13b and dCas13b are approximately 124 kDa and can be identified as a band between 150 kDa and 100 kDa.

From Figure 1, the appearance of a band between 150 kDa and 100 kDa showed that both Cas13b and dCas13b were expressed at the right molecular weight. However, we note that the different intensities in protein expression across the three replicate samples may be attributed to variation in transfection efficiency across each sample.

Contribution of iGEM Moscow_City 2021

Usage

Reducing off-target activity

A possible solution to off-target activity by engineering dCas13b (catalytically inactive nuclease) fused to an RNase domain (such as the PINc domain) to generate a dCas13–PIN platform capable of precise targeting and controlled RNA degradation [1].

It is also worth paying attention to spacers: data indicated that crRNA requires >21-nt base-pairing with its target to trigger the necessary CRISPR-Cas conformational change and nuclease activation necessary for target degradation and predicts low probability of off-target activity transcriptome-wide due to the extensive (>21-nt) spacer-target base-pairing required. Importantly, the ability of PspCas13b to tolerate up to 3-nt mismatches, especially in internal regions, indicates its potential to remain effective against the majority of variants with single-nucleotide polymorphisms in the target sequence, conferring protection against viruses such as SARS-CoV-2. [2]

Use on viruses

Some data showed that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays [3].

Optimizing gene size

Also, when the length of Cas13b gene needs to be reduced, e.g. to not exceed size limits of AAV's cargo capacity, HEPN2 gene can be removed - as it is appears to be significantly less integrated into the overall structure than the other domains, Cas13b enzyme with HEPN2 removed would retain RNA targeting activity for base editing [4].

References

[1] Ali et al. CRISPR/Cas13 as a Tool for RNA Interference. Trends Plant Sci. 2018 May;23(5):374-378.
[2] Fareh et al. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. Nat Commun 12, 4270 (2021).
[3] Mahas et al. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol 20, 263 (2019).
[4] Slaymaker et al. High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage. Cell Rep. 2019 Mar 26;26(13):3741-3751.e5.

Moscow City Contribution

Sequence and Features