Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor
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Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor. / Bryson, James W.; Auxillos, Jamie Y.; Rosser, Susan J.
In: Nucleic acids symposium series, Vol. 50, No. 1, 2022, p. 549-560.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor
AU - Bryson, James W.
AU - Auxillos, Jamie Y.
AU - Rosser, Susan J.
PY - 2022
Y1 - 2022
N2 - The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5' of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.
AB - The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5' of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.
KW - STRUCTURAL BASIS
KW - IN-VIVO
KW - RNA
KW - CPF1
KW - ENDONUCLEASE
U2 - 10.1093/nar/gkab1191
DO - 10.1093/nar/gkab1191
M3 - Journal article
C2 - 34908140
VL - 50
SP - 549
EP - 560
JO - Nucleic acids symposium series
JF - Nucleic acids symposium series
SN - 0261-3166
IS - 1
ER -
ID: 346779131