A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
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TLDR
This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.Abstract:
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.read more
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Journal ArticleDOI
Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System
Woong Y. Hwang,Yanfang Fu,Deepak Reyon,Morgan L. Maeder,Prakriti Kaini,Jeffry D. Sander,J. Keith Joung,Randall T. Peterson,Randall T. Peterson,Jing-Ruey J. Yeh +9 more
TL;DR: It is demonstrated that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100% and the utility of the CRISPR-Cas system in the zebrafish expands beyond somatic indel formation to heritable and precise genome modifications.
Journal ArticleDOI
A CRISPR/Cas system mediates bacterial innate immune evasion and virulence
Timothy R. Sampson,Sunil D. Saroj,Anna C. Llewellyn,Anna C. Llewellyn,Yih-Ling Tzeng,David S. Weiss,David S. Weiss +6 more
TL;DR: It is demonstrated that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein.
Journal ArticleDOI
Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.
Jason B. Miller,Shuyuan Zhang,Petra Kos,Hu Xiong,Kejin Zhou,Sofya S. Perelman,Hao Zhu,Daniel J. Siegwart +7 more
TL;DR: It is shown that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression, and represents a promising step towards improving the safety and utility of gene editing.
Journal ArticleDOI
Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells
Chen Yu,Yanxia Liu,Tianhua Ma,Kai Liu,Shaohua Xu,Yu Zhang,Honglei Liu,Honglei Liu,Marie La Russa,Marie La Russa,Min Xie,Sheng Ding,Lei S. Qi +12 more
TL;DR: A reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR) is developed and small molecules that can enhance CRISPR-mediated HDR efficiency are identified.
Journal ArticleDOI
Cas9-Guide RNA Directed Genome Editing in Soybean
Zhongsen Li,Zhan-Bin Liu,Aiqiu Xing,Bryan P. Moon,Jessica P. Koellhoffer,Lingxia Huang,R. Timothy Ward,Elizabeth Clifton,S. Carl Falco,A. Mark Cigan +9 more
TL;DR: High-frequency and heritable targeted mutagenesis, transgene integration, and endogenous gene editing in soybean is realized using CRISPR Cas9-gRNA.
References
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CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III
Elitza Deltcheva,Krzysztof Chylinski,Krzysztof Chylinski,Cynthia M. Sharma,Karine Gonzales,Yanjie Chao,Zaid Ahmed Pirzada,Maria R. Eckert,Jörg Vogel,Emmanuelle Charpentier,Emmanuelle Charpentier +10 more
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