The CRISPR-Cas system is a key gene editing tool that has revolutionised how we manipulate DNA, providing the simplest, most efficient, and adaptable approach yet to making gene edits. These systems – originating in bacteria as a form of adaptive immunity – can be categorised into six types based on the function and structure of the Cas endonuclease, which carry out the first step in gene-editing: cutting DNA.

Of the six, the type II CRISPR-Cas9 system is the best characterised and widely used system thanks to its simplicity and efficiency in making DNA double stranded breaks (DSBs). However, because certain Cas9s can cleave DNA at off-target sites, variants of Cas9 have been engineered to improve its fidelity. Other CRISPR-Cas systems, namely type V which uses Cas12 and type IV which uses Cas13, also lend themselves well to gene editing because of their properties.

In this article, we will outline the different versions of Cas that are currently relevant to gene editing, as well as when they can be used depending on the experimental context and editing strategy.

Cas9 variants

The most common form of Cas9 used for gene editing is derived from the bacterium Streptococcus pyogenes and is thus often referred to as SpCas9. SpCas9 is directed to inflict DSBs using a guide RNA (gRNA) that is complementary to the respective target DNA sequence. SpCas9 requires the presence of a ‘protospacer adjacent motif’ (PAM) next to the target site to bind and cleave DNA. For SpCas9 the PAM sequence is 3’-NGG, and even though this sequence is highly abundant in the human genome, the need for a PAM limits the DNA sequences that can be targeted, which can make Cas9 versions with different PAM requirements preferable for certain gene-editing strategies.

To exploit different PAMs, Cas9s from other bacteria are employed, such as that from Staphylococcus aureus – SaCas9. This Cas9may also be more suited to clinical applications because of its smaller size, making it easier to package into viral vectors applicable to gene therapies.

Other Cas9s, meanwhile, are more useful for specific applications or requirements. While wild-type Cas9 has two cleavage domains (cutting the opposing strands to make a DSB), Cas9nickases have been engineered that make just one cut using one gRNA. To introduce a DSB with Cas9 nickases, it takes two separate gRNAs, one for each DNA strand. This approach can improve the efficiency of gene-editing by minimising the chance of cleaving off-target sequences.

Other engineered Cas9 variants that provide higher fidelity edits include HypaCas9, which has a mutated mismatch-sensing domain that is better at proofreading DNA sequences, and FokI-fused dCas9 that uses the FokI endonuclease to only cleave after dimerization.

dCas9 is a catalytically dead Cas9 variant, and therefore doesn’t inflict any cuts. However, it retains its DNA recognition domain to control gene expression, for example by blocking transcription through fusion to a repressor domain.

Cas12a in type V system

The type V system is becoming an increasingly popular choice for gene editing thanks to the efficiency and compactness of its Cas enzyme, Cas12a. This type possesses some distinct features, such as the ability to recognise AT-rich DNA. One key difference to Cas9 is that it makes staggered DSBs with an overhang, rendering the DNA ends more ‘sticky’ for homology-directed repair (HDR) – this can be beneficial for introducing precise edits during the DNA repair process.

In addition, the CRISPR-Cas12a system is comparatively simpler by consisting only of Cas12a and a single RNA called crispr RNA (crRNA), one of the two components of gRNA that is complementary to the target sequence. Cas9, on the other hand, requires both components of gRNA, resulting in longer gRNA molecules that are more expensive to produce. Since Cas12a has its own RNA processing activity, it is also better suited to experiments that make multiple edits in one go.

Cas13 in type IV system

In contrast to other types of Cas, Cas13 cleaves RNA instead of DNA. After activation by a single-stranded RNA, Cas13 uses its ribonuclease activity to degrade all nearby RNA, which can be harnessed to detect RNA for diagnostics. For instance, it can cleave fluorescent reporter proteins to trigger fluorescence upon recognising an RNA target. Unlike the permanent changes made from editing DNA, RNA expression is transient, offering an alternative approach that doesn’t alter the genome sequence.

Type IV-Cas 13 systems have many applications for RNA manipulation, such as RNA knockdowns to silence specific genes, live-cell transcript imaging and precise RNA base editing. It has been employed in mammalian cells as a programmable RNA editing system, for example, and a subclass of the enzyme, Cas13d, was proposed as a Covid-19 treatment that disrupts the function of the virus.

Future outlook

Though Cas nucleases from various bacteria can be utilised to increase the versatility of CRISPR-Cas applicability, there is great interest in engineering Cas nucleases that don’t depend as much – or at all - on a PAM. However, PAM-independent nucleases do have their own drawbacks – they need to integrate every sequence in a genome (not only those containing a PAM), so it takes longer to home in on the respective DNA target, as well as increasing the risk of off-target edits.

Looking more widely, other properties of Cas nucleases, such as size, thermostability, binding and cleavage rates, and applicability in different cellular contexts, are also important for developing nucleases for specific applications and advancing the field of gene editing.

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