SpyCas9a

Summary

The first demonstration of RNA-guided DNA targeting by Cas9 from Streptococcus pyogenes (SpyCas9) ¹ , and consequent demonstration of its ability to edit mammalian genomes ⁴ , initiated a revolution in fundamental biology and therapeutic development.

In its native context, SpyCas9 complexes with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) to form the active ribonucleoprotein (RNP) ³ . The SpyCas9 RNP then searches for a PAM sequence 5’-NGG-3’ situated directly downstream of the target sequence in the DNA, forms an R-loop complex where one of the DNA strands is base-paired to the complementary spacer of crRNA, and uses two catalytic domains (RuvC and HNH) to cleave both DNA strands ¹ . The ease of reprogramming SpyCas9 RNPs to target various DNA sequences (when a PAM is present nearby) makes this enzyme highly attractive for genome-engineering applications. In most applications, an engineered single guide RNA (sgRNA) is used instead of the natural dual tracrRNA:crRNA guide ^{1 4} . Double-stranded breaks (DSBs) generated by SpyCas9 RNPs are usually repaired via non-homologous end-joining; however, the ultimate outcome depends on which DNA repair pathway is predominant ⁵ , which is impacted by absence/presence of a DNA template for homology directed repair (HDR), cell type, chromatin structure, cell cycle, and many other cellular factors.

Applications

SpyCas9 is the most broadly utilized and validated CRISPR editor and has been shown to be highly adept at modifying the genome of many organisms, ranging from phage to humans. Additionally, by leveraging CRISPR-Cas9’s ability to introduce targeted genetic disruptions, scientists have built successful platforms for the rapid creation of knockout animal models, genetic screening^{6 7} , and diagnostic testing ^{8 9} .

There are many ongoing CRISPR-Cas clinical trials that utilize SpyCas9. These treatments include many ex vivo cell therapies ¹⁰ and emerging systemic in vivo editing in patients ¹¹ . A comprehensive listing of these trials is outside of CasPEDIA's scope but can be found in other resources ^{link link}. A more detailed general overview of CRISPR clinical trials: ^link.

CRISPR-Cas9 has also been extensively harnessed for agricultural purposes, improving plant immunity and nutritional content for staple crops like wheat and rice. A more comprehensive listing of all agricultural CRISPR-Cas9 based engineering can be found in other resources ^{12 13 14 15} , but several examples may be found in the experimental section of CasPEDIA. A more detailed general overview of CRISPR agricultural engineering: ^link.

Experimental Considerations

When working with SpyCas9, there are several important experimental considerations to take into account.

Multiple software tools exist for SpyCas9 guide design ¹⁶ , to identify suitable high-activity target sites and minimize potential off-target events.

The size of the SpyCas9 coding sequence (CDS) poses a limitation for packaging into adeno-associated virus (AAV), as it approaches the maximum limit of approximately 4.7kb. This accounts for the inclusion of a promoter, termination signal, and sgRNA expression cassette. To accommodate the size constraint, splitting the SpyCas9 CDS into multiple AAV vectors may be necessary especially if using SpyCas9 fusion proteins ^{17 18} . Other Cas9 orthologs, including SauCas9 ¹⁹ , Nme2Cas9 ²⁰ and SluCas9 ²¹ , are more compact and therefore package more effectively into AAV. In certain cases, it can be advantageous to alter the delivery method of SpyCas9, making the size of its CDS less of a concern. For instance, one option is to deliver SpyCas9 (and its derivatives) as mRNA and sgRNA using lipid nanoparticles (LNPs) ²² , or as preassembled RNPs using virus-like particles (VLPs) ^{23 24 25} .

Engineered versions of SpyCas9 exist that broaden its PAM targeting ^{26 27} or improve its specificity ^{28 29 30 31} . These variants expand the targeting scope of SpyCas9 and may be better suited for certain genome-editing goals.

To minimize non-specific genome editing, commonly referred to as off-target sites, efforts have been undertaken to develop high-specificity variants of SpyCas9, either with rational design ^{28 29} based on structures ^{32 33} , or with directed evolution ³⁰ . These specially engineered variants preserve SpyCas9's on-target activity at a comparable level while significantly reducing the occurrence of off-target editing events. It was shown that the alteration of SpyCas9's DNA-cutting kinetics has a major effect on target specificity ³⁴ . A comprehensive evaluation of several high-fidelity variants was made in this study ³⁵ .

A number of fusion proteins have also been generated with SpyCas9, including DNA base editors ^{36 37 38 39 40 41 42} and prime editors ^{43 44 45} , which are capable of generating site-specific precise modifications such as single-base substitutions or small deletions and insertions without the need for double-stranded breaks (DSBs) and homology directed repair (HDR).

In addition to directly modifying genomic DNA, SpyCas9 (usually inactive, dCas9) fusion proteins can be harnessed for transcriptional activation ⁴⁶ or repression ⁴⁷ , targeted manipulation of epigenetic marks ^{48 49} , imaging of genomic loci in cells ⁵⁰ .

It is worth noting that SpyCas9 has tight binding to the targeted DNA and slow dissociation rate following cleavage in vitro. These characteristics make SpyCas9 behave as a single-turnover enzyme ^{1 51 52} ; however, some orthologs may demonstrate a multi-turnover nature ⁵³ . While stable product binding may be an important consideration for the design of experiments in vitro, studies report that in vivo SpyCas9 is dislodged from the cleaved site by histone chaperone FACT. This process effectively transforms SpyCas9 into a multiple-turnover enzyme within the cellular nucleus ⁵⁴ . Furthermore, even though the DNA cut produced by SpyCas9 is commonly referred to as blunt-ended double-strand break, in vitro experiments indicate that, following blunt cleavage, nucleotides are then trimmed from at least one of the newly cleaved DNA ends, though this trimming activity occurs on a timescale much slower than that of the initial cut ^{1 55 56} . In vivo cleavage products with 1–3 nt 5’ overhangs have also been reported based on DNA repair outcomes of Cas9 editing ^{56 57 56} .

Search Constructs on Addgene