Research On Homing CRISPR Barcoding Technology Coupling With Cell Cycles

Emerging cell lineage tracing techniques have led to a lot of findings,such as the immobilization pattern of leech cleavage or clonal competition in tumors.Using CRISPR/Cas9,mutations containing diversity can be gradually generated at target sites in the genome,which can be used as barcodes to record cell lineages,and cell lineage tree can be reconstructed accordingly.At present,cell lineage tracing techniques has been widely used to study tissue and organ formation,disease occurrence and evolution,damage repair and regeneration,etc.,and has made remarkable results.However,CRISPR lineage tracing technology still faces bottlenecks in terms of recording capacity and recording duration,which make it difficult to trace and accurately reconstruct complicated multicellular development processes in the long term,which hinders the in-depth understanding of key regulatory and fate determination principles.In this regard,researchers tried to solve them by using more recording sites,introducing inducible expression or changing nuclease types,which improved the ability of lineage recording to varying degrees.Among them,homing CRISPR lineage tracing technology,which can repeatedly target its own gene locus to trigger multiple rounds of mutations,stands out.In this research,we introduced cell cycle association control into the homing CRISPR system to cope with the rapid decay of information recording capacity and noise caused by the continuous activity of the CRISPR system in the current method.We constructed and verified six homing guide RNAs that can generate mutations at their own genome locus,and tried to associate Cas9 existence with cell cycle oscillators by Cas9 protein degradation,mRNA transcription and degradation control.We used Geminin sequence for Cas9 degradation control and confirmed that the fusion protein had nuclease activity and was compatible with homing guide RNA.Cas9hGem fusion protein prefered the S/G2/M phase and was weakened in the G0/G1 phase to some extent,confirming the feasibility of cell cycle conjugation to degrade Cas9.On the other hand,we found that both CCNE1 or MK167 promoter fragments could initiate cell-cycle specific transcription of downstream luciferase reporter genes.Among them,the expression level started by MKI67 promoter fragment was relatively higher,but the CCNE1 promoter activity was confined to a narrower window centered in the G1/S phase.We also tried to trigger M-G1 specific mRNA degradation by using the 3’ untranslated regions of CDK1,TOP2A,UBE2C and FBXO5 genes.However,data shows that adding those regions is not a feasible strategy for mRNA degradation control.This study preliminarily explores the feasibility of coupling cell cycle oscillator with homing CRISPR system,verifies the ability of candidate homing guide RNA to generate barcodes,and provides a new methodological framework for in-depth in vitro and in vivo exploration of process such as stem cell differentiation and tumor development.