Cas Endonuclease Overview

As CRISPR moves closer to the clinic, it becomes clear that Cas9 won’t be the only tool in the toolbox. For example, Cas13’s unique ability to degrade RNA makes it ideal for RNA-targeting therapies, while Cas12’s ability to shred ssDNA non-specifically (following a specific dsDNA cleavage event) makes it highly attractive in diagnostics.

In this overview, we highlight the Cas endonucleases that are currently in focus for therapeutic applications. This overview will be updated periodically.

By: Karen O'Hanlon Cohrt - Apr. 21, 2020

When CRISPR-Cas9 technology hit research labs in 2012, everyone was using the Cas9 endonuclease from the bacterium Streptococcus pyogenes (SpCas9).

In the years that followed, researchers isolated Cas9 enzymes from many other bacterial species. Beyond differences in protospacer adjacent motif (PAM) specificity between these naturally occurring Cas9 variants, some of the variants were attractive for other reasons, e.g. the Cas9-encoding gene from Staphylococcus aureus is significantly smaller than that from S. pyogenes, making it much easier to package into viral vectors for delivery into cells.

While Cas9 is probably the most popular Cas endonuclease in CRISPR labs today, its use is often limited by its large size (which makes it difficult to get it into cells), its PAM sequence stringency, and its propensity to cut off-target DNA sequences. Many have addressed these limitations of Cas9 by engineering derivatives with more desirable properties, in particular increased specificity and reduced PAM stringency. Alternative Cas endonucleases with overlapping as well as unique properties are also gaining traction in research, therapeutic and diagnostic applications.

Here, we provide an overview of some of the most notable Cas endonucleases in use today. We will update this overview as new discoveries are made and as new Cas variants emerge.

Cas3

First isolated in: discovered through in silico analysis of prokaryotic genomes.

Protein length: approx. 700-1,100 amino acids

Guide spacer length: N/A

gRNA length: N/A

PAM sequence: no PAM requirement. Cas3 is only activated when the CASCADE complex recognises a target through 5’-AAG PAM sequence recognition.

Cleavage type: 3’-5’ ssDNA helicase-nuclease activity. Long-range unidirectional DNA cleavage upstream of PAM site.

Notable properties: long-range and progressive DNA cleavage makes Cas3 an interesting candidate for large genome deletions. Cas3 is often described as a DNA shredder, able to make targeted genome deletions tens of kilobases in length.

Applications: study of large genomic regions, including non-coding regions. Therapeutic removal of important viruses from infected genomes, e.g., hepatitis B, Human papillomavirus, and a new type of antibacterials, e.g., as a phage therapy for urinary tract infections.

Cas9

First isolated in: Streptococcus pyogenes (SpCas9).

Protein length: 1,000 -1,600 amino acids

Guide spacer length: 18-24 nucleotides

gRNA length: approx. 100 nucleotides

PAM sequence: GC-rich PAM sequences. The prototypical SpCas9 recognises 5’-NGG-3’.

Cleavage type: blunt-ended dsDNA break that can be repaired by non-homologous end-joining or homologous recombination.

Notable properties: the first discovered and best-studied Cas to date. Wild-types generally exhibit high genome editing efficiency but are prone to off-target cleavage.

Applications: functional genomics, therapeutics.

Cas12a

First isolated in: bacteria from Prevotella and Franiscella genera.

Protein length: 1,300 amino acids

Guide spacer length: 18-25 nucleotides

gRNA length: 42-44 nucleotides

PAM sequence: AT-rich PAM sequences. Exact sequence is species-dependent.

Cleavage type: staggered dsDNA break, leaving a 5-nucleotide 5’ overhang.

Notable properties: dsDNA cleavage useful for gene edits that rely on the homologous recombination repair outcome. Following targeted dsDNA cleavage, Cas12a then cleaves ssDNA non-specifically. Smaller than Spcas9 and does not require a tracer RNA. Shorter gRNA also makes Cas12a cheaper than Cas9.

Applications: unlike Cas9, Cas12a is able to process its own gRNAs, making it suitable for multiplex gene editing. Multiple gRNAs are expressed from a single transcriptional unit (known as a crRNA array), where each crRNA directs Cas12a to a specific genomic target. Diagnostics: once Cas12 recognises a target dsDNA (e.g. disease gene), it can then degrade an exogenously supplied labelled ssDNA substrate to release a detectable signal. Twelve BIO is using this property to develop Cas12a as a diagnostic tool for lung cancer.

Cas12d (CasY)

First isolated in: identified through metagenomic analysis of groundwater bacteria.

Protein length: 1,200 amino acids

Guide spacer length: variable; most within 17-19 nucleotides

gRNA length: 122 nucleotides

PAM sequence: 5’-TA

Cleavage type: staggered dsDNA break.

Notable properties: requires a novel RNA in additional to a gRNA to cleave dsDNA.

Applications: genome editing for research and therapeutics.

Cas12e (CasX)

First isolated in: identified through metagenomic analysis of groundwater bacteria.

Protein length: approx. 980 amino acids

Guide spacer length: 20 nucleotides

gRNA length: 20 nucleotides

PAM sequence: 5’-TTCN

Cleavage type: staggered dsDNA break.

Notable properties: its small size makes it easier to package into viral delivery vectors.

Applications: genome editing for research and therapeutics.

Cas13

First isolated in: discovered through in silico predictions in bacterial and archaeal genomes.

Protein length: 900-1,300 amino acids

Guide spacer length: 28-30 nucleotides

gRNA length: 64 nucleotides

PAM sequence: none

Cleavage type: cuts ssRNA

Notable properties: Cas13 cuts RNA and not DNA. Once it is activated by a ssRNA sequence that bears complementarity to its crRNA spacer, it begins to degrade all nearby RNA non-specifically.

Applications: target transcript knockdown and live-cell transcript imaging. Ability to non-specifically degrade ssDNA in being exploited for in vitro precision diagnostics, most notably SHERLOCK, developed in the Zhang Lab at the Broad Institute (USA). SHERLOCK became the first FDA-authorized CRISPR application as a COVID-19 diagnostic kit and is also key to the CARMEN chip for massive pathogen surveillance. Specific RNA-editing capacity makes Cas13 a candidate for RNA-editing therapies to influence gene expression without altering genome sequence.

Cas14

First isolated in: uncultivated archaea.

Protein length: 400-700 amino acids

Guide spacer length: 18-25 nucleotides

gRNA length: approx. 140 nucleotides

PAM sequence: none for ssDNA cleavage. For dsDNA cleavage, Cas14 requires T-rich PAM sequences, e.g., TTTA.

Cleavage type: ssDNA is first cleaved in a sequence-specific manner. Cas14 then cleaves ssDNA non-specifically.

Notable properties: non-specific ssDNA cleavage.

Applications: high-fidelity single nucleotide polymorphism (SNP) genotyping

Sources:

1. F. Jiang, J. A. Doudna, CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys46, 505-529 (2017).

2. T. Sinkunas et al., Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J30, 1335-1342 (2011).

3. A. E. Dolan et al., Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Mol Cell74, 936-950.e935 (2019).

4. B. P. Kleinstiver et al., Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol37, 276-282 (2019).

5. J. S. Chen et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science360, 436-439 (2018).

6. D. B. T. Cox et al., RNA editing with CRISPR-Cas13. Science358, 1019-1027 (2017).

7. O. O. Abudayyeh et al., RNA targeting with CRISPR-Cas13. Nature550, 280-284 (2017).

8. M. J. Kellner, J. G. Koob, J. S. Gootenberg, O. O. Abudayyeh, F. Zhang, SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc14, 2986-3012 (2019).

9. L. B. Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science362, 839-842 (2018).

10. J. J. Liu et al., CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature566, 218-223 (2019).

11. R. Cloney, Metagenomics: Uncultivated microbes reveal new CRISPR-Cas systems. Nat Rev Genet18, 146 (2017).

12. D. Burstein et al., New CRISPR-Cas systems from uncultivated microbes. Nature542, 237-241 (2017).

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