RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

Tambe, Akshay; East-Seletsky, Alexandra; Knott, Gavin J.; Doudna, Jennifer A.; O’Connell, Mitchell R.

doi:10.1016/j.celrep.2018.06.105

Cited by 121 publications

(155 citation statements)

References 32 publications

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“…We found a critical ("seed") region for Cas13d knock-down efficacy between guide RNA nucleotides 15 to 21 with its center at nucleotide 18 relative to the guide RNA 5' end. Although seed regions have been shown for Cas13a orthologs 1,21,22 , one group reported no clear seed region for Cas13d 20 while another group showed position-dependent mismatch sensitivity for Cas13d in a cell-free assay 23 . Within the seed region, single mismatches led to diminished guide enrichment, while mismatches outside the seed region were better tolerated ( Fig.…”

Section: Mismatch Position (Nt)mentioning

confidence: 99%

Principles for rational Cas13d guide design

Wessels

Méndez-Mancilla

Guo

et al. 2019

Preprint

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AbstractType VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences.We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 – 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the model’s generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.

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Section: Mismatch Position (Nt)mentioning

confidence: 99%

Principles for rational Cas13d guide design

Wessels

Méndez-Mancilla

Guo

et al. 2019

Preprint

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“…MiRNA-17 complementary to the truncated crRNA yielded over a 50-200-fold increase in outcomes compared to those obtained with the other three miRNAs when using an equal amount of input miRNA, exhibiting a significant difference ( p <10 −5 ) in selectivity owing to sequence heterogeneity ( Fig.2e ). Cas13a has tolerance for one mismatch but is sensitive to two or more mismatches in the crRNA spacer: target duplex 12–13 . To circumvent this issue, we introduced an artificial mismatch in the crRNA spacer sequences to further enhance specificity ( Fig.2f ).…”

Section: Figurementioning

confidence: 99%

Droplet-digital Cas13a assay enables direct single-molecule microRNA quantification

Tian

Liu

et al. 2019

Preprint

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The direct quantification of microRNA at the single-molecule level is still challenging. Herein, we developed a droplet-digital Cas13a assay (ddCA) as a general approach for the amplification-free and absolute quantification of single unlabeled miRNA molecules with single-nucleotide specificity. We demonstrate its simplicity, precise quantification capability, excellent specificity and broad applicability by analyzing microRNAs from synthetic and cell lines-derived materials.

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“…However, trans-cleavage activity may be inhibited or nonspecifically activated by target-independent factors 22 .…”

Section: Introductionmentioning

confidence: 99%

CASLFA: CRISPR/Cas9-mediated lateral flow nucleic acid assay

Wang¹,

Xiong²,

Tian³

et al. 2019

Preprint

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The lateral flow assay is one of the oldest and most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the tedious and inefficient hybridization steps. Here, we have introduced a new version of the lateral flow assay, termed Cas9-mediated lateral flow nucleic acids assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a sensitivity of hundreds of copies of genome samples with high specificity within 1 h. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for analyzing genes in resource-poor or nonlaboratory environments.

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RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

Cited by 121 publications

References 32 publications

Principles for rational Cas13d guide design

Principles for rational Cas13d guide design

Droplet-digital Cas13a assay enables direct single-molecule microRNA quantification

CASLFA: CRISPR/Cas9-mediated lateral flow nucleic acid assay

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