CRISPR-CasФ: A new all-in-one hypercompact genome editor

Bacteriophages (also known as ‘phages’), which are viruses that infect bacteria, are the most abundant biological entities on the planet [1]. As a response to the constant threat of phage infection, a huge arsenal of defense mechanisms has evolved in bacterial hosts, and as a consequence, the phages evolved to rapidly counter these defense mechanisms. That is why an ‘arms race’ between phages and their bacterial hosts has been established. Prokaryote innate immunity systems interfere at different levels of the phage’s infection cycle via receptor masking, superinfection exclusion, restriction-modification, bacteriophage exclusion, toxin antitoxin modules, abortive infection, prokaryotic Argonaut molecules, production of anti phage chemicals and defense island system associated with restriction-modification systems [2]. Adaptive (and heritable) immunity is provided by CRISPR-Cas systems. 

CRISPR-Cas systems are mostly found in prokaryotes, providing adapting immunity against mobile genetic elements (MGEs) such as bacteriophages or plasmids. The continuous arms-race between prokaryotic hosts and their cognate MGEs is speculated to be responsible for the rapid evolution of highly diverse CRISPR-Cas systems. In response to these antiviral defense mechanisms, phages have evolved numerous mechanisms to overcome prokaryotic CRISPR Cas immunity. The first discovered mechanism rely on mutational drifts occurring in regions that require perfect complementarity between the crRNA and the seed region for interference, or in the PAM sequence [3-5]. Phages can also modify their bases with hydroxymethylcytosine and its glycosylated form to reduce target binding affinity and avoid CRISPR mediated targeting  [6]. But the most surprising mechanism to counteract the CRISPR defense system was the CRISPR-Cas system itself; some phages encode their own CRISPR locus targeting host antiviral genomic regions [7]. 

Biggiephage injecting the CasΦ into bacterial cells to turn the bacteria against the phage’s competitor

UC Berkeley image by Basem Al-Shayeb and Patrick Pausch

The laboratory of Jennifer A. Doudna, a pioneer in adapting the CRISPR-Cas system as a gene editing tool, has published an article in Science describing a new CRISPR-Cas system found in the genome of a huge phage which belongs to the Biggiephage clade [8]. Bacteriophages CRISPR Cas systems lack the CRISPR spacer acquisition machinery and generally harbor compact CRISPR arrays (~5 spacers per array), some of which target the genes of competing phages or phage hosts. Doudna’s lab identified CasФ (also known as Cas12j), encoded in the genome of the Biggiephage clade, which contains a unique C-terminal RuvC domain. CasФ has an unusual small size of ~70-80 kDa, about half the size of Cas9 or Cas12a. Authors described three divergent CasФ orthologs, named CasФ-1, CasФ-2 and CasФ-3, and tested whether they can function as a bona fide CRISPR-Cas system. 

They described that CasФ is able to target double-stranded DNA (dsDNA) in a crRNA dependent manner, and that binding of this crRNA is dependent on the presence of a 5’ TBN 3’ PAM, where B is G, T or C. Furthermore, through RNA-seq analysis they found that the casФ gene and the reduced CRISPR array are transcribed, but there was no evidence of other noncoding RNA such as the transactivating CRISPR RNA (tracrRNA) within the locus. Therefore, CasФ is a functional phage nuclease that can act as a bona fide CRISPR-Cas effector cleaving crRNA complementary DNA in the absence of tracrRNA, being much more compact than other active CRISPR-Cas systems. 

RNA-seq also revealed that CasФ DNA cleavage requires the presence of the abovementioned PAM and a crRNA spacer of ≥14 nt long. CasФ generates staggered 5’ overhangs of 8-12 nt, similar to the staggered DNA cuts observed for other type V CRISPR nucleases such as Cas12a and CasX. Therefore, CRISPR-CasФ system will be useful for knock-in experiments due to the generated overhangs. CasФ-2 and -3 were more active in vitro than CasФ-1, with a preference for the non target strand, and the most interesting characteristic of CasФ is that it is capable to cleave single-stranded DNA (ssDNA) in cis or trans, suggesting that may also target ssDNA MGEs or ssDNA intermediates. The collateral trans cleavage activity of CasФ, coupled with a unique crRNA and minimal PAM requirements may be useful for broader nucleic acid detection as previously demonstrated for Cas12a and Cas13 in diagnostic methods such as DETECTR, SHERLOCK or CARMEN [9-11].

Figure 1: Scheme illustrating the differences in RNA processing and DNA cutting for Cas9, Cas12a, CasX, and CasФ.
 Pausch, P., et al., CRISPR-CasΦ from huge phages is a hypercompact genome editor. 2020. 369(6501): p. 333-337.

As CRISPR-CasФ systems must produce mature crRNA to guide foreign DNA cleavage, and there is an absence of detectable tracrRNA in this system, the authors tried to address the mechanisms underlying crRNA maturation. The RuvC active site of CasФ is responsible for pre-crRNA processing, for dsDNA cleavage of the target sequence and for collateral ssDNA cleavage of non-target sequences. Furthermore, the authors explored the suitability of CRISPR CasФ system for human genome editing performing a gene disruption assay using CasФ co-expressed with several crRNA targeting the eGFP sequence in HEK293 cells. CasФ 2 nuclease, with an individual crRNA was able to edit up to 33% of cells, being comparable to levels initially reported for CRISPR-Cas9, -Cas12a and -CasX [12-14]. They also demonstrated that CasФ-2 can be delivered as ribonucleoprotein (RNP) into plant protoplasts, thus improving the efficiency of gene editing. 

The small size of the CasФ, in combination with its minimal PAM requirements, will be particularly advantageous for both vector-based delivery into cells and a wide range of targetable genomic sequences. Nevertheless, the collateral activity displayed by the CasФ nuclease has to be assessed both in vitro and in vivo to ensure the absence of off target effects. 


Related articles

Searching for clues, CRISPR-Cas system becomes a pathogen’s detective

Beyond gene editing: the diversity of CRISPR-Cas system applications

What are the advances in CRISPR technology?

Recent advances in CRISPR technology-PART I: CRISPR-Cas9 Alternatives

Recent advances in CRISPR technology-PART II: Innovations

Nature Biotechnology: New base editors change C to A in bacteria and C to G in mammalian cells


References:

1. Cobian Guemes, A.G., et al., Viruses as Winners in the Game of Life. Annu Rev Virol, 2016. 3(1): p. 197-214.

2. Trasanidou, D., et al., Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs. FEMS Microbiol Lett, 2019. 366(9).

3. Bondy-Denomy, J., et al., Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins. Nature, 2015. 526(7571): p. 136-9.

4. Sun, C.L., et al., Phage mutations in response to CRISPR diversification in a bacterial population. Environ Microbiol, 2013. 15(2): p. 463-70.

5. Deveau, H., et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol, 2008. 190(4): p. 1390-400.

6. Vlot, M., et al., Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes. Nucleic Acids Res, 2018. 46(2): p. 873-885.

7. Seed, K.D., et al., A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity. Nature, 2013. 494(7438): p. 489-91.

8. Pausch, P., et al., CRISPR-CasΦ from huge phages is a hypercompact genome editor. 2020. 369(6501): p. 333-337.

9. Gootenberg, J.S., et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 2018. 360(6387): p. 439-444.

10. Gootenberg, J.S., et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017. 356(6336): p. 438-442.

11. Chen, J.S., et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 2018. 360(6387): p. 436-439.

12. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.

13. Liu, J.J., et al., CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature, 2019. 566(7743): p. 218-223.

14. Zetsche, B., et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015. 163(3): p. 759-71


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