Flexibility in PAM recognition expands DNA targeting in xCas9
xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring oth...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2025-02-01
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| Online Access: | https://elifesciences.org/articles/102538 |
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| author | Kazi A Hossain Lukasz Nierzwicki Modesto Orozco Jacek Czub Giulia Palermo |
| author_facet | Kazi A Hossain Lukasz Nierzwicki Modesto Orozco Jacek Czub Giulia Palermo |
| author_sort | Kazi A Hossain |
| collection | DOAJ |
| description | xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9’s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements. |
| format | Article |
| id | doaj-art-2081b2e769c2434da3b3e4e12a09afbf |
| institution | Kabale University |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | eLife Sciences Publications Ltd |
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| series | eLife |
| spelling | doaj-art-2081b2e769c2434da3b3e4e12a09afbf2025-02-10T17:37:53ZengeLife Sciences Publications LtdeLife2050-084X2025-02-011310.7554/eLife.102538Flexibility in PAM recognition expands DNA targeting in xCas9Kazi A Hossain0https://orcid.org/0000-0002-1149-964XLukasz Nierzwicki1Modesto Orozco2https://orcid.org/0000-0002-8608-3278Jacek Czub3Giulia Palermo4https://orcid.org/0000-0003-1404-8737Department of Bioengineering , University of California Riverside, Riverside, United States; Department of Physical Chemistry, Gdańsk University of Technology, Gdańsk, PolandDepartment of Bioengineering , University of California Riverside, Riverside, United StatesInstitute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain; Departament de Bioquímica i Biomedicina, Facultat de Biologia, Universitat de Barcelona, Barcelona, SpainDepartment of Physical Chemistry, Gdańsk University of Technology, Gdańsk, Poland; BioTechMed Center, Gdańsk University of Technology, Gdańsk, PolandDepartment of Bioengineering , University of California Riverside, Riverside, United States; Department of Chemistry, University of California Riverside, Riverside, United StatesxCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9’s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.https://elifesciences.org/articles/102538CRISPR-Cas9RNAgenome editingentropyprotein-DNAprotein dynamics |
| spellingShingle | Kazi A Hossain Lukasz Nierzwicki Modesto Orozco Jacek Czub Giulia Palermo Flexibility in PAM recognition expands DNA targeting in xCas9 eLife CRISPR-Cas9 RNA genome editing entropy protein-DNA protein dynamics |
| title | Flexibility in PAM recognition expands DNA targeting in xCas9 |
| title_full | Flexibility in PAM recognition expands DNA targeting in xCas9 |
| title_fullStr | Flexibility in PAM recognition expands DNA targeting in xCas9 |
| title_full_unstemmed | Flexibility in PAM recognition expands DNA targeting in xCas9 |
| title_short | Flexibility in PAM recognition expands DNA targeting in xCas9 |
| title_sort | flexibility in pam recognition expands dna targeting in xcas9 |
| topic | CRISPR-Cas9 RNA genome editing entropy protein-DNA protein dynamics |
| url | https://elifesciences.org/articles/102538 |
| work_keys_str_mv | AT kaziahossain flexibilityinpamrecognitionexpandsdnatargetinginxcas9 AT lukasznierzwicki flexibilityinpamrecognitionexpandsdnatargetinginxcas9 AT modestoorozco flexibilityinpamrecognitionexpandsdnatargetinginxcas9 AT jacekczub flexibilityinpamrecognitionexpandsdnatargetinginxcas9 AT giuliapalermo flexibilityinpamrecognitionexpandsdnatargetinginxcas9 |