Discovery Of A Noval Genome Editing System CRISPR/Cas12f And The Analysis Of Maize Mo17 Genome

Maize(Zea mays subsp.mays)is one of the most important food and feed crop,and it’s also an important renewable energy material.It plays a very important role in agriculture,animal husbandry and industrial development.Maize was domesticated in nearly 9000 years ago from teosinte(Zea mays subsp.parviglumis)at the Balsas River of south central Mexico.During the long domestication selection process,maize has accumulated abundant genetic diversity.B73 and Mo17 inbred lines are the most widely used materials in maize genome and genetics study and there are strong heterosis between them.The first genome map of B73 inbred was assembled through BAC Sanger sequencing method.Recently,a higher quality genome map of B73 was assembled through the third generation Pacbio sequencing technology.The completion of B73 genome played an important role in maize genetic improvement research.CRISPR/Cas is an efficient and simple gene editing system derived from archaea and bacteria.It can edit the DNA sequence which complementary to the guide RNA sequence through the complex consists of guide RNA and Cas effector protein.The development and application of CRISPR/Cas gene editing system greatly reduced the complexity of crop genetics and breeding experiments,and also shortened the breeding cycle and greatly promoted the development of crop breeding.1.In this study,through the mining of the large scale of microbial genomes and metagenomics data,a new CRISPR/Cas system was discovered which was named as CasZ(CRISPR/Cas12f)and contains six effector proteins.By the experimental verification,the CRISPR/CasZ system was found have DNA cleavage activity that only relies on a single guide RNA.and the CasZ protein can also process pre-crRNA into 44-55 nt mature crRNA.Besides.we have found that the CasZ family has very diverse PAM recognition including the 5’-TTN,5’-WTN and 5’-TTTN PAM motifs.In addition,the CRISPR/CasZ.3 system have also been proved have efficient activity in eukaryotic genome editing which means it’s the third CRISPR/Cas effector family with eukaryotic genome DNA cleavage activity after the Cas9 and Cpf1.Comparing with the SpyCas9 and LbCpfl,CasZ.3 protein has 323 and 161 amino acids less than each protein.The smaller volume of Cas effect protein means it is more easily transported to the cell for gene editing.2.We combined the Pacbio third-generation sequencing technology and Bionano optical map to complete the genome assembly of maize Mo 17 inbred line,and obtained the high-quality genome map.Through genome annotation and comparative genomic analysis,we quantitatively analyzed the genetic variation between maize B73 and Mo17,and found that the genetic variations widely existed between B73 and Mo 17 genomes.At the same time,we used the same process to analyze the genetic variation between the rice Japonica and the Indica subspecies.It is found that the variations between B73 and Mo 17 genomes of maize is more significant than that of rice subspecies.The analysis of these genetic variations will help further analyze the genetic mechanism of Heterosis in maize.3.In total,538 inbred lines of maize were used to the RNA sequencing after the pollination for 10 days,we constructed a large gene regulatory network.Furthermore,110 kernel weight associated genes and two kernel weight related network modules were discovered.The mining of these kernel development related genes has offered a good data set for the improvement of maize traits by using CRISPR/Cas gene editing technology.