Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition

Wang, Shuai; Chen, Tao; Mao, Huilin; Hou, Linghui; Wang, Yao; Qi, Tao; Yang, Yisu; Ong, Sang-Ging; Hu, Shijun; Chai, Renjie; Wang, Yongming

doi:10.1371/journal.pbio.3001897

Cited by 14 publications

(11 citation statements)

References 33 publications

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“…In addition, SeHdCas9 consistently recognizes NAG PAM sequences with an efficiency of nearly 100%, regardless of the fourth PAM nucleotide (Figure D), which ensures that the NNN PAM library is adequate for SeHdCas9. Overall, the SeHdCsa9 exhibited a PAM recognition pattern that is more diverse than previous Cas9 orthologs. ,,,,, …”

Section: Resultsmentioning

confidence: 91%

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Teng,

Wang,

Jiang

et al. 2023

ACS Synth. Biol.

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The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.

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Section: Resultsmentioning

confidence: 91%

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Teng,

Wang,

Jiang

et al. 2023

ACS Synth. Biol.

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“…36 However, grafting the mutations (i.e., R245A/N413A/N419A/R654A) from SaCas9-HF onto KKH SaCas9 and SaCas9 orthologs notably reduced its on-target activity. 16,17,37,38 This may be due to the different structures of DNA-guide RNA stabilizing interactions for each Cas9 or-SauriCas9-HF2 showed higher specificity at most tested sites but lower target activity at some target sites, which is similar to other variants, that is, eSpCas9, 9 SaCas9-HF, 16 and KKH-SaCas9-SAV1. 17 Further study will be performed to boost its on-target activity.…”

Section: Discussionmentioning

confidence: 86%

“…Similarly, this strategy was adopted to generate SlugCas9‐HF, improving the specificity of SlugCas9 36 . However, grafting the mutations (i.e., R245A/N413A/N419A/R654A) from SaCas9‐HF onto KKH SaCas9 and SaCas9 orthologs notably reduced its on‐target activity 16,17,37,38 . This may be due to the different structures of DNA‐guide RNA heteroduplex stabilizing interactions for each Cas9 orthologs variants.…”

Section: Discussionmentioning

confidence: 99%

Engineered Staphylococcus auricularis Cas9 with high‐fidelity

et al. 2023

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CRISPR‐Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off‐target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5′‐NNGG‐3′ protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced‐fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5′‐NNGG‐3′ than SaCas9 of 5′‐NNGRRT‐3′ PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9‐HF1, harboring the N269D mutation; SauriCas9‐HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE‐seq. At certain sites, reduced off‐target effects (approximately 61.6‐ and 111.9‐fold improvements) of SauriCas9‐HF2 compared with wild‐type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9‐HF1 and SauriCas9‐HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.

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“…So, structural information from this study could expand our understanding of the RNA-guided SaCas9 molecular mechanisms for the recognition and cleavage of DNA and may also offer guidance for the design or optimization of high-fidelity variants of SaCas9 [ 26 ], including the rational engineering of the SaCas9-mediated base editor [ 27 ], etc. In addition, the obtained structure might serve as a structural template for the homologous nucleases of SaCas9 to build their structural models in the active state [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Zhu

Qian

et al. 2023

IJMS

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Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.

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Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition

Cited by 14 publications

References 33 publications

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Engineered Staphylococcus auricularis Cas9 with high‐fidelity

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

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