Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Slaymaker, Ian M.; Makarova, Kira S.; Essletzbichler, Patrick; Volz, Sara E.; Joung, Julia; Oost, John van der; Regev, Aviv; Koonin, Eugene V.; Zhang, Feng

doi:10.1016/j.cell.2015.09.038

Cited by 3,900 publications

(4,265 citation statements)

References 49 publications

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“…More studies on improving the fidelity and finding an alternative have been reported 1, 3, 4. To broaden the applicability of our delivery system, we explored other gene editing alternatives.…”

Section: Resultsmentioning

confidence: 99%

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Lao

Gao

et al. 2018

Advanced Science

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CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

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Section: Resultsmentioning

confidence: 99%

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Lao

Gao

et al. 2018

Advanced Science

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“…Both crRNA and tracrRNA are required for Cas9‐mediated targeting. In contrast, a single ~44‐nucleotide (nt) crRNA with a 5′‐located direct repeat sequence and a spacer sequence that complements the target are sufficient to guide the DNA cleavage of Cpf1 (Zetsche et al ., 2015). Moreover, Cpf1 recognizes the thymidine‐rich protospacer‐adjacent motif (PAM) and makes staggered cuts distal to the PAM site (Fonfara et al ., 2016).…”

Section: Introductionmentioning

confidence: 99%

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Qin

et al. 2017

Plant Biotechnology Journal

231

152

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Summary CRISPR‐Cpf1 is a newly identified CRISPR‐Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR‐Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR‐Cpf1. We found that pre‐crRNAs with a full‐length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA‐Cpf1‐induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR‐Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.

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“…In addition to Cas9, only three families of class 2 Cas effector proteins have been discovered and experimentally validated: Cpf1, C2c1, and C2c2 4,24,25 . Another gene, c2c3, which was identified only on small DNA fragments, has been suggested to also encode such a protein 4 .…”

mentioning

confidence: 99%

New CRISPR–Cas systems from uncultivated microbes

Burstein

Harrington

Strutt

et al. 2016

Nature

507

369

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CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA 1,2 . Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences 3,4 . The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research 5 . However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms 6,7 . Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in littlestudied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two

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Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Cited by 3,900 publications

References 49 publications

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

New CRISPR–Cas systems from uncultivated microbes

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