Inducible dCpf1 Activators for Tunable Gene Regulation
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Solution Overview
Problem
Current RNA-guided CRISPR nucleases, such as Cas9, have limitations in efficiently activating endogenous genes in human cells, particularly when targeting specific DNA sequences with associated guide RNAs and protospacer adjacent motifs (PAMs).
Innovation Solution
Development of constitutively active and chemically inducible dCpf1-based transcriptional activator platforms, which include fusion proteins with catalytically inactive Cpf1 from Lachnospiraceae bacterium ND2006 (dLbCpf1) fused to activation domains like VPR, p65, or conditional dimerization domains, allowing for multiplex and tunable activation of endogenous genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas9 is used to target specific DNA sequences with guide RNAs and PAMs, then DNA cleavage activity is achieved, but efficiency in activating endogenous genes in human cells is limited
Solution Approach 1:
The Cas9 protein is divided into two separate components: a catalytically inactive Cpf1 fragment (dLbCpf1) that provides DNA binding and recruitment, and a separate activation domain (such as VPR, p65, or other transcriptional activators) that provides the gene activation function. This segmentation allows each component to be optimized independently for its specific function, improving overall activation efficiency while reducing off-target effects.
Solution Approach 2:
The patent introduces an intermediary system where dLbCpf1 acts as a mediator that recruits activation domains to specific genomic loci through guide RNA-directed DNA binding. This intermediary approach decouples the DNA targeting function from the activation function, allowing for more reliable and efficient gene activation in human cells compared to direct Cas9 usage.
2Productivity
If multiple activation domains are fused to Cpf1 to enhance activation strength, then gene activation efficiency improves, but device complexity increases
Solution Approach 1:
Multiple activation domains (such as VP16, p65, and Rta in the VPR configuration) are merged into a single composite activation domain that functions as a unified module. This merged activation domain can be separately expressed and then recruited by dLbCpf1, avoiding the need to create increasingly complex multi-domain fusions while still achieving synergistic activation effects.
Solution Approach 2:
The system employs conditional dimerization domains (such as FKBP and FRB) that allow dynamic assembly of the activation complex in response to small molecule inducers. This dynamic approach enables control over activation timing and intensity without permanently increasing the structural complexity of the fusion proteins themselves.
3Speed
If constitutively active activators are used, then gene activation is immediate, but tunability and control over expression levels are lost
Solution Approach 1:
The system uses inducible dimerization domains that respond to periodic or controlled addition of small molecule inducers (such as rapamycin analogs). This allows the activation process to be initiated on demand, providing both rapid activation when needed and the ability to tune expression levels by controlling the timing and concentration of inducer addition.
Solution Approach 2:
The system enables parameter control through varying the concentration of small molecule inducers, which directly modulates the dimerization efficiency and consequently the activation level. This allows for tunable gene expression across a wide range, from basal to maximal activation, while maintaining the capacity for rapid activation when inducer is added.
4Adaptability or versatility
If drug-inducible systems are implemented for control, then tunability is achieved, but device complexity increases
Solution Approach 1:
Small molecule inducers act as intermediaries that trigger dimerization of conditional dimerization domains without requiring complex regulatory circuits or additional protein components. This simple chemical induction mechanism provides sophisticated control capability while adding minimal complexity to the overall system architecture.
Data Source
AI summary
Drug-inducible, tunable, and multiplexable Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1)-based activators, and methods of use thereof.