CRISPR Artificial Splicing Factors

Jillette, Nathaniel; Cheng, Albert W.

doi:10.1101/431064

Cited by 5 publications

(6 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Type VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for target gene knock-down without altering the genome. In addition to target RNA knock-down 1 – 10 , Cas13 proteins have been used for viral RNA detection 7 , 9 , 11 , 12 , site-directed RNA editing 13 , demethylation of m 6 A-modified transcripts 14 , RNA live-imaging 15 , 16 , and modulation of splice site choice as well as cleavage and polyadenylation site usage 5 , 17 , 18 . Cas13 proteins are guided to their target RNAs by a single CRISPR RNA (crRNA) composed of a direct repeat (DR) stem loop and a spacer sequence (guide RNA or gRNA) that mediates target recognition by RNA-RNA hybridization.…”

mentioning

confidence: 99%

Massively parallel Cas13 screens reveal principles for guide RNA design

et al. 2020

View full text Add to dashboard Cite

Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knock-down without altering the genome. To define rules for the design of Cas13d guide RNAs, we conducted massively-parallel screens targeting mRNAs of a 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 RNAs with and without mismatches relative to the target sequences. Knock-down efficacy is driven by guide RNA-specific features and target site context. Single mismatches generally reduce knock-down to a modest degree, but spacer nucleotides 15 – 21 are largely intolerant to target site mismatches. We developed a computational model to identify optimal guide RNAs and confirm its generalizability testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 guide RNAs for all protein-coding transcripts in the human genome.

show abstract

mentioning

confidence: 99%

Massively parallel Cas13 screens reveal principles for guide RNA design

et al. 2020

View full text Add to dashboard Cite

show abstract

“…The same study also demonstrated the therapeutic relevance of RNA knockdown for frontotemporal dementia with Parkinsonism using the CasRx system. Jillette et al [29] engineered inducible artificial splicing factors (iCASFx) based on CRISPR/dCasRx in human 293T cells. Although there are currently few reports on usage of the CasRx system, it is a promising alternative tool for post-transcriptional gene silencing and shows no detectable side effects in previous research, including toxicity, cell growth or cell death.…”

Section: Resultsmentioning

confidence: 99%

Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi’s sarcoma-associated herpesvirus ORF52

Pan

Zhang

2019

Bioscience Reports

View full text Add to dashboard Cite

Precise genome editing with directed base insertion or targeted point mutations can be achieved by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance in clinical disease therapy. However, HDR efficiency, compared with non-homologous end-joining (NHEJ), is inherently low. To enhance HDR, enabling the insertion of precise genetic modifications, we compared two strategies during surrogate reporter assays in mouse N2A cells: the suppression of DNA ligase IV, a key molecule in NHEJ, using the CasRx (Ruminococcus flavefaciens Cas13d) system, and co-expression of Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF52 proteins. We found that suppression of DNA ligase IV promotes HDR efficiency by 1.4-fold. When co-expressed with the Cas9 system, ORF52 improved HDR efficiency by up to 2.1-fold. In addition, we used ORF52 co-expression to modify the ACTB and Tubb3 genes of mouse N2A and E14 cells, which further increased HDR efficiency by approximately two- to four-fold. In conclusion, our data suggest that ORF52 co-expression is effective for enhancing CRISPR/Cas9-mediated HDR, which may be useful for future studies involving precise genome editing.

show abstract

“…* * * Type VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for target gene knock-down without altering the genome. In addition to target RNA knock-down 1-9 , Cas13 proteins have been used to enable viral RNA detection systems 7,9-11 , site-directed RNA editing 12 , demethylation of m 6 Amodified transcripts 13 , RNA live-imaging 14,15 , and modulation of splice site choice as well as cleavage and polyadenylation site usage 5,16,17 .Cas13 proteins are guided to their target RNAs by a single CRISPR RNA (crRNA) composed of a direct repeat (DR) stem loop and a spacer sequence (guide RNA) that mediates target recognition by RNA-RNA hybridization. Although Cas13 enzymes exert some non-specific collateral nuclease activity upon activation 4-6,10,18 , they have greatly reduced off-target activity in cultured cells compared to RNA interference 2,5,12 .…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…cross-validation folds (top/bottom N = 2, 4,8,16,32, 64, 128 or 256 guide RNAs). To compute the null distribution, we calculated the median predicted guides scores of randomly selected guide RNAs across 1000 samplings for each N. For the leave-one-out cross-validation we trained on all data from three tiling screens and performed Spearman rank correlation of the predicted the guide efficiency of the held-out fourth screen to the observed log2FC enrichments.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Principles for rational Cas13d guide design

Wessels

Méndez-Mancilla

Guo

et al. 2019

Preprint

View full text Add to dashboard Cite

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.

show abstract

CRISPR Artificial Splicing Factors

Cited by 5 publications

References 35 publications

Massively parallel Cas13 screens reveal principles for guide RNA design

Massively parallel Cas13 screens reveal principles for guide RNA design

Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi’s sarcoma-associated herpesvirus ORF52

Principles for rational Cas13d guide design

Contact Info

Product

Resources

About