Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs RNA
Impact Factor: 4.928

Cas9 versus Cas12a/Cpf1: Structure–function comparisons and implications for genome editing

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Cas9 and Cas12a are multidomain CRISPR‐associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best‐characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
Schematic representation of expression and interference stages in type II‐A and V‐A CRISPR–Cas systems. (a) Cas9–crRNA complex assembly and activity. The tracrRNA (magenta) base pairs with the repeat‐derived segments (gray) of the pre‐crRNA transcript. The crRNA–tracrRNA structure is recognized by Cas9 (yellow) and processed by RNase III (purple). It is currently unknown which RNase processes the 5′ end of the crRNA (indicated by the question mark). When Cas9 recognizes a 5′–NGG–3′ PAM (green) in a DNA target, the spacer‐derived segment of the crRNA (orange) base pairs with the complementary target DNA strand (black). DNA cleavage by Cas9 generates a PAM‐proximal blunt‐end cut. Note that the 5′–NGG–3′ PAM is specific for Streptococcus pyogenes Cas9. (b) Cas9–sgRNA complex formation and activity. Instead of dual‐RNA formation and processing by RNase III, a chimeric sgRNA can be expressed as a single transcript in which tracrRNA‐ and crRNA‐derived segments (green) are fused by a short loop. (c) Cas12a–crRNA complex assembly and activity. The repeat‐derived segment of the pre‐crRNA (gray) forms a pseudoknot that is recognized by Cas12a (purple). Upon pre‐crRNA binding, Cas12a itself generates the 5′ end of the crRNA by endonucleolytic cleavage (black triangle). It is currently unknown which RNase processes the 3′ end of the crRNA (indicated by a question mark). When Cas12a recognizes a 5′–TTTV–3′ PAM (green) in a DNA target (black), the spacer‐derived segment of the crRNA (orange) will base pair with complementary target DNA. Target DNA cleavage by Cas12a results in a PAM‐distal dsDNA break with 5′ overhangs
[ Normal View | Magnified View ]
Target DNA binding and cleavage. Domain colors in this figure correspond to those in Figure . (a) Schematic representation of DNA cleavage by Cas9. (b) Catalytic residues located in the HNH and RuvC domains of SpCas9 (PDB: 5F9R). Left close‐up panel: Catalytic residues in the RuvC domain. The asterisk indicates the scissile phosphate. Right close‐up panel: Catalytic residues in the HNH domain. The asterisk indicates the scissile phosphate which is located ~15 Å away from the catalytic residues. (c) Schematic representation of DNA cleavage by Cas12a. (d) Catalytic residues are located in the RuvC domain of FnCas12a (PDB: 5NFV). The black line indicates the hypothetical path of the NTS DNA. Left close‐up panel: Catalytic residues in the RuvC domain of the FnCas12aE1006Q,R1218A catalytic mutant, with TS nucleotides modeled in place based on the structure of the Cas12b–substrate DNA complex (PDB: 5U33; adapted from Swarts et al., ). The scissile phosphate is indicated with an asterisk. The pink line indicates the hypothetical path of the modeled TS DNA. Right close‐up panel: Aromatic residue Y410 caps the RNA‐TS heteroduplex in FnCas12a. The orange line indicates the hypothetical path of the 3′ end of the crRNA
[ Normal View | Magnified View ]
PAM recognition by Cas9 and Cas12a. Individual domains are colored as in Figure . (a) PAM recognition by Streptococcus pyogenes Cas9. In the close‐up, only the PAM nucleotides of the target DNA are shown. (b) Schematic representation of PAM recognition by SpCas9. Arginine residues R1333 and R1335 mediate base‐specific readout by forming hydrogen bonds to PAM residues (indicated with dashed lines). (c) PAM recognition by Francisella novicida Cas12a. In the close‐up, only the PAM nucleotides of the target DNA are shown. (d) Schematic representation of PAM recognition by FnCas12a. Lysines K613 and K671 mediate base‐specific readout by forming hydrogen bonds to PAM nucleotides (indicated with dashed lines). PAM recognition additionally relies on shape complementarity and base exclusion at specific positions. (e) Overview of Cas9 and Cas12a enzymes and mutants and the PAMs that they are able to recognize
[ Normal View | Magnified View ]
Domain architecture and conformational activation of Streptococcus pyogenes Cas9 and Francisella novicida Cas12a. (a) Schematic representation of the domain organization. Domain colors in all other panels correspond to those in this panel. (b) Guide‐free Cas9 (PDB: 4CMP). (c) Binding of a guide RNA induces conformational changes in Cas9 (PDB: 4ZT0). The seed segment of the guide RNA and the sgRNA linker segment are indicated by black circles. (d) Binding of a DNA target puts the Cas9–sgRNA complex in a cleavage‐competent conformation (PDB: 4UN3). The PAM is indicated by the black circle. (e) Hypothetical “open” model of guide‐free Cas12a, based on negative‐stain electron microscopy images of Lachnospiraceae bacterium Cas12a that point to its conformational flexibility (Dong et al., ). The predicted hinge is indicated with the pink/blue circle. (f) Binding of a guide RNA induces conformational changes in Cas12a resulting in a “closed” conformation (PDB: 5NG6). The seed segment of the guide RNA is indicated by a black circle. (g) Binding of a DNA target induces conformation changes resulting in a cleavage‐competent “active” conformation (PDB: 5FNV)
[ Normal View | Magnified View ]
Cas9 and Cas12a guide RNAs. (a) Left panel: Schematic representation of a Cas9 sgRNA. For sgRNAs, the guide RNA is transcribed as a single RNA containing a linker segment (black, in the dashed box). In a dual‐RNA (not shown), the spacer segment (gray and magenta) and repeat segments (orange) are derived from the pre‐crRNA, while the remainder of the dual‐RNA is derived from the tracrRNA. Note that mature dual‐RNA does not contain a linker segment, but instead the dsRNA segment is extended. The canonical tracrRNA and sgRNAs contain a second hairpin at the 3′ end (indicated in blue). Right panel: sgRNA binding by Streptococcus pyogenes Cas9 (PDB: 4ZT0). The seed segment (nt 11–20) is preordered in a helical conformation (magenta) and is solvent exposed, while the remainder of the spacer segment is unordered. Individual domains are colored as in in Figure . RNA segments are colored as depicted in the schematic in the left panel. The spacer derived RNA segment indicated in gray is unordered in the structure. The tracrRNA‐derived segment indicated in blue is absent from the structure. (b) Left panel: Schematic representation of a mature Cas12a guide RNA. Both the repeat (orange) and spacer segments (magenta and gray) are derived from the pre‐crRNA. Right panel: Guide RNA binding by Francisella novicida Cas12a (PDB: 5NG6). Two Mg2+ ions are involved coordination of the repeat‐derived pseudoknot (indicated by purple spheres). The guide RNA seed segment (nucleotides 1–5; magenta) is preordered in a helical conformation and solvent exposed, while the remainder of the spacer segment is unordered (gray) in the structure. Individual domains are colored as in in Figure 2. RNA segments are colored as depicted in the schematic in the left panel
[ Normal View | Magnified View ]

Related Articles

Top Ten WRNA Articles

Browse by Topic

RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts