Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology

Abstract

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF “terminator switch” mechanism, ≈15-fold for an ON → OFF “splinted switch” mechanism, and ≈3-fold for an OFF → ON “toehold switch” mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

Gene drive and resilience through renewal with next generationCleave and Rescueselfish genetic elements

“…Given this it is interesting to imagine ways in which the function of the Rescue transgene could be made conditional so as to bring about the death of cells, individuals, or populations under specific circumstances. For example, it may be possible to engineer essential gene function at the level of transcript or protein such that it is sensitive to the presence of viral protease activity (4), small RNAs (43), or other honest markers of infection, resulting in the death of infected host cells or individuals. One can also imagine ways in which entire ClvR-bearing populations could be suppressed in an environmental condition-specific manner.…”

Multiplexed CRISPR technologies for gene editing and transcriptional regulation

“…In the near future, we suspect that strategies leveraging anti-CRISPR proteins to rapidly turn "off" endonuclease functions will also be deployed 61 . Other strategies to control CRISPR-Cas activity rely on modifications to the structure of sgRNAs; conditional gRNAs are either constitutively active and turned "off" by RNA terminator switches, or are constitutively inactive and turned "on" by an RNA trigger 62 . This strategy has been demonstrated in both bacteria and human cells, enabling programmable CRISPR-Cas activity in vivo.…”

“…This effect was partly ameliorated in bacteria by using a dCas9-repressor fusion that recognizes a specific operator sequence and exhibits a lower toxicity, though this reduces the flexibility of DNA targeting 8 . dCas9 bottlenecking could possibly be mitigated by using conditional gRNAs, which are selectively triggered as needed in vivo 62 .…”

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA

“…The activity of (d)Cas9 protein can be induced by small molecules, [17][18][19][20] activated by light, [21][22][23][24] or controlled by tissue-specic promotors 25 or miRNAs, 26 but this approach encounters an inevitable limitation that all the targeted genes are subject to the same regulatory scope. In contrast, engineering of gRNA provides another approach to conditionally control CRISPR/Cas9 function that can manipulate specic genes via structural changes of gRNA induced by small molecules, [27][28][29][30] protein binding, 31 nuclease cleavage, 28 RNA expressions, 32,33 or miRNA/antisense RNA processing. 34,35 Besides engineering of genetically expressed gRNA, chemical modications of gRNA also provide unique tools to expand the versatility of conditional control.…”