Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria

Wang, Jian; Teng, Yuxi; Gong, Xinyu; Zhang, Jianli; Wu, Yifei; Lou, Lei; Li, Michelle; Xie, Zhong-Ru; Yan, Yajun

doi:10.1016/j.ymben.2022.10.005

Cited by 8 publications

(27 citation statements)

References 65 publications

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“…Further exploration identified the existence of the RxQ motif in multiple species, including the SeHCas9 from S. equinus HC5 (KEY47635.1) reported in our previous study, as well as SmdCas9 from S. macedonicus (WP_214302110.1), SspCas9 from S. sp.…”

Section: Resultsmentioning

confidence: 79%

“…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%

“…A potential explanation is that this mutation caused R1336 to strictly recognize dG3. T1337R or the equivalent mutation was also recruited in many Cas9s with an expanded PAM range, ,,, and could sometimes result in a preference toward G at the fourth position in PAM. , The corresponding K1340R in SeHdCas9 also demonstrated beneficial effects on PAM expanding and has been confirmed to impose no new requirement on the fourth PAM (Figure S5). To this end, we generated a SeHCas9-Q1229R variant that is highly specific to NNG PAMs and more notably a SeHdCas9-RR variant robustly recognizing all NNN PAMs that can permit versatile gene regulations in bacteria without any restrictions.…”

Section: Discussionmentioning

confidence: 95%

“…To completely determine the PAM profile of the RxQ motif-bearing Cas9, we employed the eGFP repression system established in our previous studies (Figure B). , Specifically, we used SeHdCas9 that was constructed by swapping out the PI domain of SedCas9 with that of SeHdCas9 . We also employed the Sp-sgRNA with general applicability to the SpCas9-like Streptococcus Cas9s and is highly similar to the SeH-sgRNA (Figure S2).…”

Section: Resultsmentioning

confidence: 99%

“…Investigating the PAM Interaction Mechanism. Inspired by the specific arginine−guanine and glutamine− adenine interactions presented in many previous PAM interaction studies, 17,23,24,30,31 our initial hypothesis was that SeHCas9 managed to position the arginine (R) and glutamine (Q) in the consensus RxQ PAM-binding motif to specifically interact with the dG3 and dA2 in the NAG PAM. However, by individually disrupting R1336, W1337, and Q1338 to alanine (A), we found that only R1336A significantly hindered the NAG PAM recognition in the repression test (Figure 2A).…”

Section: 1mentioning

confidence: 99%

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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: 79%