DCas9-and DCpf1-based Genetic Circuits

As ?Nature?states,the final goal of synthetic biology is to realize novel biological systems aimed at carrying out useful functions.To reach this goal,we need to construct new biological parts,devices,and systems.Due to their programmable features,the CRISPR-(d)Cas systems would be a rather useful tool to build biological synthetic devices that can be composed of many basic logic gates.In this project,two kinds of CRISPR-d Cas systems are used,one is CRISPR-d Cas9,and the other is CRISPR-d Cpf1.Both of them lost the ability to cut open DNA strands,but they kept the ability to recognize specific DNA sequence.Hence,they can be used as gene regulatory tools as some papers reported.Moreover,they are easily re-programmed to target different DNA sequences via changes in g RNAs for d Cas9 or cr RNAs for d Cpf1.Published paper has presented the construction of layered NOR gates in S.cerevisiae,however,not many other applications of d Cas9 or d Cpf1 in yeast have been reported yet.Thus,this thesis tried to make use of these two powerful systems to engineer yeast synthetic circuits and broaden the database of biological circuits.In this project,green fluorescent protein is considered as a reporter gene(circuit output).Gibson assembly and the Golden Gate assembly are applied to construct the plasmids necessary to host the circuit transcription units.The vectors used in thesis are called shuttle plasmids since they can replicate both in yeast and E.coli.To build the main circuits based on CRIPSR-d Cas9,plasmids that express g RNAs,d Cas9,anti-CRISPR proteins,and the GFP were integrated into yeast genome.The circuit working was induced by galactose and ?-estradiol,finally,the circuit performance was described by the changes in fluorescence expression,which can be easily measured with a FACS machine.FACS data were analyzed with the R.studio software.The same strategy was applied to CRISPR-d Cpf1-based circuits.The main task of this project is to construct genetic logic gates based on CRISPRd Cas9 and CRISPR-d Cpf1.As for d Cas9,I have built eight NOT gates,two buffer gates,and a biosensor that can detect at least about 8 nmol/L of beta-estradiol in the environment.For NOT gates,they performed well when induced by 2% galactose,and the off/on ratio can reach to 0.3.As for the buffer gate,anti-CRISPRs were applied and the anti-CRISPRs seem to work rather differently.Basically all anti-CRISPR efficiencies lowered down,especially that of Lm Acr IIA4 that was supposed to have the highest effects on inhibiting d Cas9.And ?-estradiol detective biosensor can work stably during the repeat experiments.As for d Cpf1,research results verified its usage in yeast synthetic gene circuits by building eight NOT gates.They reached an almost 33% decrease in fluorescence expression(compared to a control circuit)and still need modifications,perhaps via new cr RNA design,to improve their performance.The construction of all these circuits based on d Cas9 and d Cpf1 shows that the CRISPR-d Cas9 and the CRISPR-d Cpf1 systems are useful tools for yeast synthetic biology.Moreover,the successful implementation of the beta-estradiol biosensor can be inspiring for realizing new devices with applications both in industrial processes and biomedical research.