CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

Chen, Janice S.; Ma, Enbo; Harrington, Lucas B.; Costa, Maria Da; Tian, Xinran; Palefsky, Joel M.; Doudna, Jennifer A.

doi:10.1126/science.aar6245

Cited by 2,942 publications

(3,486 citation statements)

References 42 publications

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“…Recently, the targeting range for Cas12a has been expanded greatly by engineered versions that recognize 5 0 -TYCV-3 0 and 5 0 -TATV-3 0 PAMs without sacrificing efficiency or specificity [71]. The latest findings about Cas12a surprisingly revealed that after target binding of either dsDNA or ssDNA Cas12a induces the indiscriminate cleavage of ssDNA in the surrounding solution [83]. In these studies it could be shown that via the CRISPR/Cas12a system targeted mutagenesis can be accomplished and that the appropriate mutations are heritable [72,73].…”

Section: Using Cas12a (Formerly Named Cpf1) In Plantsmentioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

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Section: Using Cas12a (Formerly Named Cpf1) In Plantsmentioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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“…The proposed viral detection system, named Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHER-LOCK), uses a first step of RPA (or RT-RPA for DNA), a T7 transcription step, and a Cas13a/crRNA against the virus of interest. [137,138] The advantage of these approaches is the single-molecule nucleic acid detection ability, making them a promising tool for the detection of very low-titer viruses; however, further investigation in this field is necessary. [133] The newest versions, SHER-LOCKv2 and heating unextracted diagnostic samples to obliterate nucleases (HUDSON), allows the simultaneous detection on-field of four different viruses of the same family: ZKV, Dengue virus, West Nile virus, and Yellow fever virus (all Flaviviridae, Flavivirus).…”

Section: High-throughput Sequencing (Hts) or Next-generation Sequencimentioning

confidence: 99%

Viral Detection: Past, Present, and Future

et al. 2019

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Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting‐edge technologies that may be playing important roles in the years to come are described.

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“…Some enzymes simply fail to work and some choose their substrates promiscuously, necessitating thorough biochemical characterization [59][60][61][62][63][64][65] . Nevertheless, orthologous Cas9 enzymes with different PAM requirements increase targeting flexibility and allow multiplexing for combinatorial genetic screens, as demonstrated for SpCas9 and SaCas9 6,11,66 .…”

Section: Article Preprintmentioning

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

Cited by 2,942 publications

References 42 publications

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

Viral Detection: Past, Present, and Future

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

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