Closely related type II-C Cas9 orthologs recognize diverse PAMs

Wei, Jingjing; Hou, Linghui; Liu, Jingtong; Wang, Ziwen; Gao, Siqi; Qi, Tao; Gao, Song; Sun, Song; Wang, Yongming

doi:10.7554/elife.77825

Cited by 24 publications

(13 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our development of PID-chimeric Nme2 Smu -ABEs that recognize a single-cytidine PAM, further increase the utility of these compact BEs. While our work was in progress, others also demonstrated that the SmuCas9 PID can be used to create chimeric Cas9 nucleases with a N4CN PAM, even though their Nme2 Smu Cas9 nuclease exhibited poor efficiency at the target sites they assessed 34 . Although our Nme2 Smu -ABEs had detectable activity at all N4CN PAM target sites tested, we observed a reduction in activity at sites with N4CC PAMs when compared to domaininlaid Nme2-ABE with a wildtype PID.…”

Section: Discussionmentioning

confidence: 82%

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Bamidele

Zhang

Dong³

et al. 2023

Preprint

View full text Add to dashboard Cite

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we have engineered Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first used domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibited shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expanded the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We used these enhancements to correct two common MECP2 mutations associated with Rett syndrome with little or no bystander editing. Finally, we validated domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

show abstract

Section: Discussionmentioning

confidence: 82%

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Bamidele

Zhang

Dong³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Guide RNAs, naturally occurring aptamers, can be swapped between CRISPR species within the same class and can still work fine with Cas proteins from another CRISPR system. [69] In theory, CRISmers might be better suited to target intracellular proteins as they are localized within the cellular nucleus in the system in order to activate a selection reporter gene. However, on the other hand, as the delivery technologies for exogenous RNA evolve, which is a key contributing factor to the success of mRNA vaccines, intracellular targets will become more accessible to RNA therapeutics.…”

Section: Discussionmentioning

confidence: 99%

“…Guide RNAs, naturally occurring aptamers, can be swapped between CRISPR species within the same class and can still work fine with Cas proteins from another CRISPR system. [ 69 ]…”

Section: Discussionmentioning

confidence: 99%

Repurposing CRISPR/Cas to Discover SARS‐CoV‐2 Detecting and Neutralizing Aptamers

Zhu

Mei

et al. 2023

Advanced Science

View full text Add to dashboard Cite

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

show abstract

“…Moreover, reconstructed Cas9 ancestors (Alonso‐Lerma et al, 2023 ) and laboratory‐evolved (Kleinstiver et al, 2015 ; Miller et al, 2020 ) or engineered (Collias & Beisel, 2021 ; Gasiunas et al, 2012 ; Hirano et al, 2019 ; Karvelis et al, 2013 ; Vicencio et al, 2022 ; Walton et al, 2020 ; Yamada et al, 2017 ) Cas9 variants with smaller sizes and different PAM requirements have been harnessed to the CRISPR technology. Yet, the search for Cas9 orthologs in prokaryotic genomes (Fedorova et al, 2020a , 2020b ; Gasiunas et al, 2020 ; Wei et al, 2022 ) and metagenomes (Burstein et al, 2017 ; Zhang et al, 2014 ) is expanding the catalogue of CRISPR tools and, allegedly, will continue to provide new alternatives to circumvent their limitations.…”

Section: Introductionmentioning

confidence: 99%

Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

Esquerra-Ruvira

Baquedano

Ruiz

et al. 2023

Microbial Biotechnology

View full text Add to dashboard Cite

Non‐coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR‐associated (Cas) proteins of the prokaryotic CRISPR‐Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas‐catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR‐based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA‐targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5′‐NGG‐3′ PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR‐Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5′‐NGG‐3′ PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

show abstract

Closely related type II-C Cas9 orthologs recognize diverse PAMs

Cited by 24 publications

References 46 publications

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Repurposing CRISPR/Cas to Discover SARS‐CoV‐2 Detecting and Neutralizing Aptamers

Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

Contact Info

Product

Resources

About