CRISPRi‐mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND) system

Abstract

Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time‐consuming and labor‐intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO2 uptake. CRISPRi::CA comprises a whole‐cell biosensor to monitor CO2 concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony‐forming units. Furthermore, the enzymatic performance of 5‐aminolevulinic acid synthetase (ALAS), which converts glycine and succinate‐CoA to release a molecule of CO2, has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time‐saving, and flexible technology for the screening and inspection of high‐performance enzymes, which may accelerate protein engineering in the future.

CRISPRi-Mediated NIMPLY Logic Gate for Fine-Tuning the Whole-Cell Sensing toward Simple Urine Glucose Detection

“…With the highly accessible design of CRISPRi, , we conceived that the CRISPRi could be used to reduce basal expression, thus enhancing the S/N value. The first CRISPRi was designed to target an essential gene, can gene, to offer a survival stress that would make cells express less sfGFP due to the cell growth being controlled by CRISPRi on can gene . As considering the dynamic range, the specific fluorescence must be recovered by releasing the inhibition from CRISPRi, a glucose-induced anti-CRISPRi system was designed.…”

“…First, module I was established for the functional CRISPRi targeting on essential gene to provide survival stress, whose design encompassed a constitutively expressed dCas9 as well as a sgRNA targeting to the promoter region of can essential gene. The sgRNA was driven by a synthetic promoter which has been used in CRISPRi . Second, module II was the glucose-inducible antisense sgRNA to release the survival stress from module I when glucose existed in the environment.…”

“…The first CRISPRi was designed to target an essential gene, can gene, to offer a survival stress that would make cells express less sfGFP due to the cell growth being controlled by CRISPRi on can gene. 29 As considering the dynamic range, the specific fluorescence must be recovered by releasing the inhibition from CRISPRi, a glucose-induced anti-CRISPRi system was designed. In such context, our genetic design was divided into three modules (Figure 3A) and could be analogue to electric circuit (Figure 3B).…”

“…The sgRNA was driven by a synthetic promoter which has been used in CRISPRi. 29 Second, module II was the glucose-inducible antisense sgRNA to release the survival stress from module I when glucose existed in the environment. As shown in Figure S1, NIMPLY gate was used to illustrate the relationship between binary input and output, in which the output signal (i.e., functional CRISPRi) occurred only when the A input (i.e., module I) was presented with the absence of B input (i.e., module II).…”

A direct enzymatic evaluation platform (DEEP) to fine‐tuning pyridoxal 5′‐phosphate‐dependent proteins for cadaverine production

“…Notably, manipulation of essential genes under stress is feasible, broadly applicable, and easy to select for various biocatalysts. Previous studies have shown that knocking out the essential genes successfully establishes an auxotrophic system for enzyme evolution (Atkinson et al, 2019; Tan et al, 2020). Recently, clustered regularly interspaced short palindromic repeats interference (CRISPRi) by pairing the nuclease‐deficient Cas9 mutant (dCas9) with a synthetic single‐guide RNA (sgRNA) has been exploited to program the cell, regulating the gene expression levels and tuning multicomplexed metabolic flux (Landberg et al, 2020; Lian et al, 2019; Qi et al, 2013).…”