Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.

Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.

[1]  Haopeng Yu,et al.  Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease. , 2022, Cell reports.

[2]  Do Yon Kim,et al.  Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus , 2021, Nature biotechnology.

[3]  Haopeng Yu,et al.  Programmed genome editing by a miniature CRISPR-Cas12f nuclease , 2021, Nature Chemical Biology.

[4]  Lei S. Qi,et al.  Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. , 2021, Molecular cell.

[5]  C. Beisel,et al.  The tracrRNA in CRISPR Biology and Technologies. , 2021, Annual review of genetics.

[6]  P. Bork,et al.  Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation , 2021, Nucleic Acids Res..

[7]  Jonathan Y. Hsu,et al.  Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA , 2021, Nature Protocols.

[8]  H. Nishimasu,et al.  Structure of the miniature type V-F CRISPR-Cas effector enzyme. , 2020, Molecular cell.

[9]  Shiraz A Shah,et al.  CRISPRCasTyper: Automated Identification, Annotation, and Classification of CRISPR-Cas Loci. , 2020, The CRISPR journal.

[10]  Silvio C. E. Tosatto,et al.  Pfam: The protein families database in 2021 , 2020, Nucleic Acids Res..

[11]  Joshua K Young,et al.  PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage , 2020, Nucleic acids research.

[12]  Lei S. Qi,et al.  Multiple Input Sensing and Signal Integration Using a Split Cas12a System. , 2020, Molecular cell.

[13]  Stan J. J. Brouns,et al.  Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants , 2019, Nature Reviews Microbiology.

[14]  Eugene V Koonin,et al.  RNA-guided DNA insertion with CRISPR-associated transposases , 2019, Science.

[15]  E. Koonin,et al.  Origins and evolution of CRISPR-Cas systems , 2019, Philosophical Transactions of the Royal Society B.

[16]  Christine L. Sun,et al.  Clades of huge phages from across Earth’s ecosystems , 2019, bioRxiv.

[17]  G. Gao,et al.  Adeno-associated virus vector as a platform for gene therapy delivery , 2019, Nature Reviews Drug Discovery.

[18]  David A. Scott,et al.  Functionally diverse type V CRISPR-Cas systems , 2019, Science.

[19]  Jennifer A. Doudna,et al.  Programmed DNA destruction by miniature CRISPR-Cas14 enzymes , 2018, Science.

[20]  Robert D. Finn,et al.  HMMER web server: 2018 update , 2018, Nucleic Acids Res..

[21]  Kira S. Makarova,et al.  Diversity and evolution of class 2 CRISPR–Cas systems , 2017, Nature Reviews Microbiology.

[22]  Jennifer A. Doudna,et al.  New CRISPR-Cas systems from uncultivated microbes , 2016, Nature.

[23]  Eric S. Lander,et al.  C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector , 2016, Science.

[24]  Chris M. Brown,et al.  CRISPRDetect: A flexible algorithm to define CRISPR arrays , 2016, BMC Genomics.

[25]  Yang Yang,et al.  A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice , 2016, Nature Biotechnology.

[26]  Wenyan Jiang,et al.  CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems. , 2015, Annual review of microbiology.

[27]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[28]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[29]  Feng Zhang,et al.  A split-Cas9 architecture for inducible genome editing and transcription modulation , 2015, Nature Biotechnology.

[30]  Alexandros Stamatakis,et al.  RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies , 2014, Bioinform..

[31]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[32]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[33]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[34]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[35]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[36]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[37]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..