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Rewiring Cas9 to Target New PAM Sequences
Molecular Cell
Previews Rewiring Cas9 to Target New PAM Sequences Virginijus Siksnys1,* and Giedrius Gasiunas1 1Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius LT-02241, Lithuania *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.03.002
Papers by Anders et al. (2016) and Hirano et al. (2016b), published in this issue of Molecular Cell, show that SpCas9 uses an induced fit mechanism to recognize altered protospacer adjacent motif (PAM) sequences. Cas9-guide RNA system has been suc- hybridize to any DNA sequence by evolution experiments revealed that cessfully reprogrammed to cleave, nick, altering crRNA component, PAM se- changing amino acid residues in the vicinor bind desired chromosomal DNA tar- quence becomes the major constrain ity of PAM alters Streptococcus pyogenes gets (Hsu et al., 2014; Sternberg and that limits the available sequence space (Sp) Cas9 PAM specificity (Kleinstiver Doudna, 2015). The DNA target site for genome editing applications. Directed et al., 2015). Papers by Anders et al. for Cas9 is composite and (2016) and Hirano et al. consists of two nucleotide (2016b), published in this sequences (boxes) that are issue of Molecular Cell, proread by two different modvide a structural mechanism ules (Figure 1). The major of the altered PAM recognibox (20 nt) termed a prototion by the SpCas9 protein. spacer is recognized through SpCas9 reads out the the Watson-Crick base canonical 50 -NGG-30 PAM sequence through the bidenpairing between a spacer tate hydrogen bonding intercomponent of the guide actions of two consecutive RNA (crRNA or sgRNA) and Arg1333 and Arg1335 resithe complimentary strand of dues with acceptor atoms the target DNA. The minor on G bases. Substitution of box (2–5 nt) termed a protoArg1335 residue by other spacer adjacent motif or amino acids by the sitePAM represents a nucleotide directed mutagenesis did not signature uniquely associalter PAM specificity arguing ated with each Cas9 protein. against a naive stereochemiPAM is recognized by the cal code between amino Cas9’s PI (PAM interaction) acid residue and DNA base motif through the hydrogen (Anders et al., 2014). Howevbonding and Van der Waals er, directed evolution experi(vdW) interactions between ments revealed Cas9 variants amino acid residues of the that can interact with nonCas9 protein and atoms on canonical PAM sequences the DNA base edges. Literally (Kleinstiver et al., 2015). It speaking, the Cas9 target is turned out that changes resecured by two combination sulting in the altered PAM locks that are controlled by specificity occurred in the PI two different codes. Impormotif in the vicinity of the tantly, unlocking the PAM critical Arg residues. Crystal sequence by the Cas9 prostructures of SpCas9 variants tein is absolutely required recognizing non-canonical to initiate crRNA-mediated PAM variants solved by Anbase pairing to the comders et al. (2016) and Hirano plimentary DNA sequence Figure 1. Structural Mechanism of a Protospacer Adjacent Motif et al. (2016b) now demonfollowed by the R-loop forSequence Recognition by SpCas9 The DNA target site for SpCas9 is composite and consists of two nucleotide strate that SpCas9 recogmation and subsequent sequences termed a protospacer and protospacer adjacent motif (PAM), nizes altered PAM sequences cleavage (Szczelkun et al., respectively. Protospacer interaction motif (PIM) recognizes the 50 -GG-30 PAM by an induced fit mechanism. 2014). Since guide RNA can in the WT SpCas9. Mutations in PIM sequence enable recognition of altered PAM by an induced fit mechanism. Mutations at the Cas9-PAM be easily reprogrammed to Molecular Cell 61, March 17, 2016 ª2016 Elsevier Inc. 793
Molecular Cell
Previews sequence interface did not change the overall structure of PI-motif but enabled subtle DNA backbone distortions that resulted in rewiring of H-bonding and vdW interactions between the altered amino acid residues and mutated PAM sequence. For example, the EQR and VRER variants achieved recognition of longer PAM sequences, 50 -NGNG-30 and 50 -NGCG-30 , respectively, by building new interactions with an additional arginine residue. This arginine residue is partly solvent exposed and therefore can adopt conformations suitable for making contacts with the guanine base located at fourth position of extended PAM sequence. Although such changes are difficult to predict, Anders et al. (2016), guided by the crystal structure, designed the SpCas9 variant that recognized the 50 -NAAG-30 PAM. This example illustrates the potential of rational design in the engineering of SpCas9 variants with altered PAM preferences. Structural and functional studies of the Francisella novicida Cas9 (FnCas9) published recently in Cell also revealed the important role of PI-motif plasticity in 50 -NGG-30 PAM sequence recognition by FnCas9 (Hirano et al., 2016a). Despite the structural and size differences between SpCas9 and FnCas9 proteins, the structural mechanism of 50 -NGG-30 PAM recognition is conserved and relies on the interaction of two consecutive Arg residues with acceptor atoms on G bases. Structure-guided engineering of PI-motif resulted in the FnCas9 variant specific for the relaxed 50 -YG-30 PAM. Taken together, these data demonstrate the feasibility of structure-guided
rational design approaches for engineering of Cas9 variants with altered PAM specificity that bypass the limits in genome targeting imposed by the PAM sequence. However, it remains to be established how many new PAM specificities the SpCas9 scaffold can accommodate. Although the directed evolution experiments supported by structural studies of SpCas9 variants implies the plasticity of PI-motif, the specificity of Cas9 variants with altered PAM preferences still needs to be rigorously evaluated. Mutations of amino acid residues at the protein-DNA interface often result in a shift of the target site preference and relaxed sequence specificity pattern rather than a ‘‘true’’ specificity change. Indeed, some of Cas9 variants with altered PAM specificities in vitro still interacted with the canonical PAM sequence at increased Cas9 concentrations (Anders et al., 2016; Hirano et al., 2016b). Furthermore, biochemical experiments show that PAM specificity is dependent on Cas9-guide RNA complex concentration (Karvelis et al., 2015). It would be helpful to have an index that quantitatively compares PAM recognition by Cas9 variants, similar to the fidelity index used to quantify the specificity of engineered restriction enzymes. Keeping these limitations in mind, alternative strategies for generating Cas9 variants with new PAM specificities have to be exploited. Cas9 orthologs are abundant with over 1,000 Cas9 sequences available in sequence databases. Exploring these Cas9 orthologs (or distant relatives) may provide variants with novel PAM specificities and expand the repertoire of Cas9s available for genome target-
794 Molecular Cell 61, March 17, 2016 ª2016 Elsevier Inc.
ing applications. Tapping into the natural diversity of Cas9 proteins already identified Cas9 enzymes with new PAM specificities from Staphylococcus aureus (Ran et al., 2015) and Brevibacillus laterosporus (Karvelis et al., 2015). Directed evolution and combinatorial approaches relying on the existing natural diversity of Cas9 proteins in the future may enable targeting of any DNA sequence in the genome with a single nucleotide precision. REFERENCES Anders, C., Niewoehner, O., Duerst, A., and Jinek, M. (2014). Nature 513, 569–573. Anders, C., Bargsten, K., and Jinek, M. (2016). Mol. Cell 61, this issue, 895–902. Hirano, H., Gootenberg, J.S., Horii, T., Abudayyeh, O.O., Kimura, M., Hsu, P.D., Nakane, T., Ishitani, R., Hatada, I., Zhang, F., et al. (2016a). Cell 164, 950–961. Hirano, S., Nishimasu, H., Ishitani, R., and Nureki, O. (2016b). Mol. Cell 61, this issue, 886–894. Hsu, P.D.D., Lander, E.S.S., and Zhang, F. (2014). Cell 157, 1262–1278. Karvelis, T., Gasiunas, G., Young, J., Bigelyte, G., Silanskas, A., Cigan, M., and Siksnys, V. (2015). Genome Biol. 16, 253. Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., Topkar, V.V., Nguyen, N.T., Zheng, Z., Gonzales, A.P.W., Li, Z., Peterson, R.T., Yeh, J.R., et al. (2015). Nature 523, 481–485. Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., et al. (2015). Nature 520, 186–191. Sternberg, S.H., and Doudna, J.A. (2015). Mol. Cell 58, 568–574. Szczelkun, M.D., Tikhomirova, M.S., Sinkunas, T., Gasiunas, G., Karvelis, T., Pschera, P., Siksnys, V., and Seidel, R. (2014). Proc. Natl. Acad. Sci. USA 111, 9798–9803.
Previews Rewiring Cas9 to Target New PAM Sequences Virginijus Siksnys1,* and Giedrius Gasiunas1 1Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius LT-02241, Lithuania *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.03.002
Papers by Anders et al. (2016) and Hirano et al. (2016b), published in this issue of Molecular Cell, show that SpCas9 uses an induced fit mechanism to recognize altered protospacer adjacent motif (PAM) sequences. Cas9-guide RNA system has been suc- hybridize to any DNA sequence by evolution experiments revealed that cessfully reprogrammed to cleave, nick, altering crRNA component, PAM se- changing amino acid residues in the vicinor bind desired chromosomal DNA tar- quence becomes the major constrain ity of PAM alters Streptococcus pyogenes gets (Hsu et al., 2014; Sternberg and that limits the available sequence space (Sp) Cas9 PAM specificity (Kleinstiver Doudna, 2015). The DNA target site for genome editing applications. Directed et al., 2015). Papers by Anders et al. for Cas9 is composite and (2016) and Hirano et al. consists of two nucleotide (2016b), published in this sequences (boxes) that are issue of Molecular Cell, proread by two different modvide a structural mechanism ules (Figure 1). The major of the altered PAM recognibox (20 nt) termed a prototion by the SpCas9 protein. spacer is recognized through SpCas9 reads out the the Watson-Crick base canonical 50 -NGG-30 PAM sequence through the bidenpairing between a spacer tate hydrogen bonding intercomponent of the guide actions of two consecutive RNA (crRNA or sgRNA) and Arg1333 and Arg1335 resithe complimentary strand of dues with acceptor atoms the target DNA. The minor on G bases. Substitution of box (2–5 nt) termed a protoArg1335 residue by other spacer adjacent motif or amino acids by the sitePAM represents a nucleotide directed mutagenesis did not signature uniquely associalter PAM specificity arguing ated with each Cas9 protein. against a naive stereochemiPAM is recognized by the cal code between amino Cas9’s PI (PAM interaction) acid residue and DNA base motif through the hydrogen (Anders et al., 2014). Howevbonding and Van der Waals er, directed evolution experi(vdW) interactions between ments revealed Cas9 variants amino acid residues of the that can interact with nonCas9 protein and atoms on canonical PAM sequences the DNA base edges. Literally (Kleinstiver et al., 2015). It speaking, the Cas9 target is turned out that changes resecured by two combination sulting in the altered PAM locks that are controlled by specificity occurred in the PI two different codes. Impormotif in the vicinity of the tantly, unlocking the PAM critical Arg residues. Crystal sequence by the Cas9 prostructures of SpCas9 variants tein is absolutely required recognizing non-canonical to initiate crRNA-mediated PAM variants solved by Anbase pairing to the comders et al. (2016) and Hirano plimentary DNA sequence Figure 1. Structural Mechanism of a Protospacer Adjacent Motif et al. (2016b) now demonfollowed by the R-loop forSequence Recognition by SpCas9 The DNA target site for SpCas9 is composite and consists of two nucleotide strate that SpCas9 recogmation and subsequent sequences termed a protospacer and protospacer adjacent motif (PAM), nizes altered PAM sequences cleavage (Szczelkun et al., respectively. Protospacer interaction motif (PIM) recognizes the 50 -GG-30 PAM by an induced fit mechanism. 2014). Since guide RNA can in the WT SpCas9. Mutations in PIM sequence enable recognition of altered PAM by an induced fit mechanism. Mutations at the Cas9-PAM be easily reprogrammed to Molecular Cell 61, March 17, 2016 ª2016 Elsevier Inc. 793
Molecular Cell
Previews sequence interface did not change the overall structure of PI-motif but enabled subtle DNA backbone distortions that resulted in rewiring of H-bonding and vdW interactions between the altered amino acid residues and mutated PAM sequence. For example, the EQR and VRER variants achieved recognition of longer PAM sequences, 50 -NGNG-30 and 50 -NGCG-30 , respectively, by building new interactions with an additional arginine residue. This arginine residue is partly solvent exposed and therefore can adopt conformations suitable for making contacts with the guanine base located at fourth position of extended PAM sequence. Although such changes are difficult to predict, Anders et al. (2016), guided by the crystal structure, designed the SpCas9 variant that recognized the 50 -NAAG-30 PAM. This example illustrates the potential of rational design in the engineering of SpCas9 variants with altered PAM preferences. Structural and functional studies of the Francisella novicida Cas9 (FnCas9) published recently in Cell also revealed the important role of PI-motif plasticity in 50 -NGG-30 PAM sequence recognition by FnCas9 (Hirano et al., 2016a). Despite the structural and size differences between SpCas9 and FnCas9 proteins, the structural mechanism of 50 -NGG-30 PAM recognition is conserved and relies on the interaction of two consecutive Arg residues with acceptor atoms on G bases. Structure-guided engineering of PI-motif resulted in the FnCas9 variant specific for the relaxed 50 -YG-30 PAM. Taken together, these data demonstrate the feasibility of structure-guided
rational design approaches for engineering of Cas9 variants with altered PAM specificity that bypass the limits in genome targeting imposed by the PAM sequence. However, it remains to be established how many new PAM specificities the SpCas9 scaffold can accommodate. Although the directed evolution experiments supported by structural studies of SpCas9 variants implies the plasticity of PI-motif, the specificity of Cas9 variants with altered PAM preferences still needs to be rigorously evaluated. Mutations of amino acid residues at the protein-DNA interface often result in a shift of the target site preference and relaxed sequence specificity pattern rather than a ‘‘true’’ specificity change. Indeed, some of Cas9 variants with altered PAM specificities in vitro still interacted with the canonical PAM sequence at increased Cas9 concentrations (Anders et al., 2016; Hirano et al., 2016b). Furthermore, biochemical experiments show that PAM specificity is dependent on Cas9-guide RNA complex concentration (Karvelis et al., 2015). It would be helpful to have an index that quantitatively compares PAM recognition by Cas9 variants, similar to the fidelity index used to quantify the specificity of engineered restriction enzymes. Keeping these limitations in mind, alternative strategies for generating Cas9 variants with new PAM specificities have to be exploited. Cas9 orthologs are abundant with over 1,000 Cas9 sequences available in sequence databases. Exploring these Cas9 orthologs (or distant relatives) may provide variants with novel PAM specificities and expand the repertoire of Cas9s available for genome target-
794 Molecular Cell 61, March 17, 2016 ª2016 Elsevier Inc.
ing applications. Tapping into the natural diversity of Cas9 proteins already identified Cas9 enzymes with new PAM specificities from Staphylococcus aureus (Ran et al., 2015) and Brevibacillus laterosporus (Karvelis et al., 2015). Directed evolution and combinatorial approaches relying on the existing natural diversity of Cas9 proteins in the future may enable targeting of any DNA sequence in the genome with a single nucleotide precision. REFERENCES Anders, C., Niewoehner, O., Duerst, A., and Jinek, M. (2014). Nature 513, 569–573. Anders, C., Bargsten, K., and Jinek, M. (2016). Mol. Cell 61, this issue, 895–902. Hirano, H., Gootenberg, J.S., Horii, T., Abudayyeh, O.O., Kimura, M., Hsu, P.D., Nakane, T., Ishitani, R., Hatada, I., Zhang, F., et al. (2016a). Cell 164, 950–961. Hirano, S., Nishimasu, H., Ishitani, R., and Nureki, O. (2016b). Mol. Cell 61, this issue, 886–894. Hsu, P.D.D., Lander, E.S.S., and Zhang, F. (2014). Cell 157, 1262–1278. Karvelis, T., Gasiunas, G., Young, J., Bigelyte, G., Silanskas, A., Cigan, M., and Siksnys, V. (2015). Genome Biol. 16, 253. Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., Topkar, V.V., Nguyen, N.T., Zheng, Z., Gonzales, A.P.W., Li, Z., Peterson, R.T., Yeh, J.R., et al. (2015). Nature 523, 481–485. Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., et al. (2015). Nature 520, 186–191. Sternberg, S.H., and Doudna, J.A. (2015). Mol. Cell 58, 568–574. Szczelkun, M.D., Tikhomirova, M.S., Sinkunas, T., Gasiunas, G., Karvelis, T., Pschera, P., Siksnys, V., and Seidel, R. (2014). Proc. Natl. Acad. Sci. USA 111, 9798–9803.