Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

Tak, Yu Gyoung; Kleinstiver, Benjamin P.; Nuñez, James K.; Hsu, Jonathan Y.; Gong, Jingyi; Weissman, Jonathan S.; Joung, J. Keith

doi:10.1101/150656

Cited by 2 publications

(3 citation statements)

References 37 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We used a nuclease-dead dCas12a (D832A) from Lachnospiraceae bacterium fused with tripartite VPR for targeted gene activation (Tak et al, 2017). To minimize the size of the construct, we used a minimized VPR (miniVPR) (Vora et al, 2018), which showed comparable activation in a TRE3G-GFP HEK293T reporter cell line (Figures S1A and S1B; Tables S1, S2, S3, and S4).…”

Section: The Split Cas12a System Enables Construction Of and Gates In Mammalian Cellsmentioning

confidence: 99%

“…Standard molecular cloning techniques were used to assemble constructs in this paper. Nuclease-dead (D832A) dCas12a from Lachnospiraceae bacterium and its crRNA backbone were modified from plasmids obtained from Addgene (#104567 and # 78742) (Tak et al, 2017;Kleinstiver et al, 2016). The leucine zipper was amplified from plasmids obtained from Addgene (#15304 and #15305) (Luan et al, 2006).…”

Section: Plasmid Cloningmentioning

confidence: 99%

“…By splitting the Cas12a protein into distinct components that can be individually controlled, it theoretically could provide a functional core for a variety of synthetic circuits. Despite recent efforts to develop dimerizing variants of Cas12a (Tak et al, 2017;Nihongaki et al, 2019), there is limited work on repurposing this system for complex cellular circuits that can sense, integrate, and respond (Liu et al, 2014). Prior work has engineered split Cas9 proteins, and a functional split Cas12a could use its unique RNA processing features to expand the toolbox for complex synthetic Cas-based circuits (Ma et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multiple Input Sensing and Signal Integration Using a Split Cas12a System

et al. 2020

View full text Add to dashboard Cite

show abstract

Section: The Split Cas12a System Enables Construction Of and Gates In Mammalian Cellsmentioning

confidence: 99%

Section: Plasmid Cloningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Multiple Input Sensing and Signal Integration Using a Split Cas12a System

et al. 2020

View full text Add to dashboard Cite

show abstract

CRISPR-Cas12a target binding unleashes single-stranded DNase activity

Chen¹,

Ma²,

Harrington³

et al. 2017

Preprint

View full text Add to dashboard Cite

CRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12acan be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics.Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPRCas13 enzymes.One Sentence Summary: Cas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/226993 doi: bioRxiv preprint first posted online Nov. 29, 2017; 3 Main Text:CRISPR-Cas adaptive immunity in bacteria and archaea uses RNA-guided nucleases to target and degrade foreign nucleic acids (1, 2). The CRISPR-Cas9 family of proteins has been widely deployed for gene editing applications (3, 4) based on the precision of double-stranded DNA (dsDNA) cleavage induced by two catalytic domains, RuvC and HNH, at sequences complementary to a guide RNA sequence (5, 6). A second family of enzymes, CRISPR-Cas12a (Cpf1), uses a single RuvC catalytic domain for guide RNA-directed dsDNA cleavage (7-12) (Fig. 1a). Distinct from Cas9, Cas12a enzymes recognize a T-rich protospacer adjacent motif (PAM) (7), catalyze their own guide RNA (crRNA) maturation (13) and generate a PAM-distal dsDNA break with staggered 5' and 3' ends (7), features that have attracted interest for gene editing applications (14-16). However, the substrate specificity and DNA cleavage mechanism ofCas12a are yet to be fully elucidated.While investigating substrate requirements for Cas12a activation, we tested Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) for guide RNA-directed singlestranded DNA (ssDNA) cleavage, a capability of diverse CRISPR-Cas9 orthologs (17, 18).Purified LbCas12a or Streptococcus pyogenes Cas9 (SpCas9) proteins (fig. S1) were assembled with guide RNA sequences targeting a circular, single-stranded M13 DNA phage. In contrast to SpCas9, we were surprised to find that LbCas12a induced rapid and complete ...

show abstract

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

Cited by 2 publications

References 37 publications

Multiple Input Sensing and Signal Integration Using a Split Cas12a System

Multiple Input Sensing and Signal Integration Using a Split Cas12a System

CRISPR-Cas12a target binding unleashes single-stranded DNase activity

Contact Info

Product

Resources

About