Genome-wide CRISPR Screening In Bombyx Mori Cells

Bombyx mori is a traditional artificial domestication insect for long history.B.mori plays important roles in both economic and culture in our country.B.mori is not only an important source of income for farmers but also a model organism of Lepidoptera.The genome data of B.mori has been sequenced completely and a series of genetic manipulation tools have been established,such as transgene,RNA interference(RNAi),and genome editing.After decades of research,some basic issues in B.mori have been clarified in genetics,like sex determination,artificial domestication and so on.But,the function of vast majority B.mori genes is unknown.However,in the face of the large number of functional unknown genes of the B.mori and the urgent requirement of industry,we need a new strategy to accelerate the functional annotate in B.mori genome.After the finish of Human Genome Project(HGP),one of the major tasks in biology is to functionally annotate genomes.In recent years,high-throughput loss-of-function screens have been used successfully to identify novel genes and underlying basic biological processes.The most useful genetic screen platform is based on RNA interference(RNAi)or clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9(Cas9)(CRISPR)system.Compared with CRISPR system,there are several disadvantages that limit the application of RNA interference,such as off-target effects,incomplete loss-of-function and so on.Therefore,the genome-wide CRISPR system is the most powerful tool for loss-of-function screens,which has been used to identify essential genes,genes for drug resistance,and host-pathogen interactions.In this paper,we established a genome wide CRISPR knockout screening platform in B.mori embryonic cell line(BmE)(BmEGCKlib)by using piggyBac transposons to deliver sgRNA libraries stably into cell lines.It was the first time to construct a high-throughput genetic screens tool in B.mori.We used this platform to identified genes essential for cell viability and resistance to abiotic and biotic stresses in B.mori.In addition,our research provide piggyBac as an alternative system to deliver CRISPR library into the genome of organisms,in which lentiviral is inefficient.The main results and conclusions of our research are following:1.Construction of a transgenic knockout backbone vector,pB-CRISPR,for B.mori cellsDelivering the CRISPR library into the genome of host cells is the key step to construct a genome-wide CRISPR screen platform.The pooled CRISPR library is usually integrated into the genome by lentiviral vectors in human or mouse cells.But,the lentiviral system is inefficient in insect cells.As an alternative system,we chose piggyBac to deliver CRISPR library into the genome of B.mori.In order to integrate the CRISPR library efficiently,we constructed an all-in-one vector named as pB-CRISPR,which include three cassettes,Cas9 driven by Hsp70 promoter,sgRNA cassette driven by U6 promoter and the Zeocin antibiotic resistance gene driven by IE2promoter.Based on B.mori embryonic cell line(BmE),we next constructed an EGFP(enhanced green fluorescent protein)positive cell line(BmE-EGFP)using an alternative transposon system,Minos.In order to detect the genome editing efficacy of pB-CRISPR,we constructed three pB-CRISPR vectors contained sgRNAs target EGFP and two pB-CRISPR vectors contained nonspecific sgRNAs as controls.All the vectors were co-transfected with a piggyBac transposase expression vector(A3-Helper)into BmE-EGFP.After transfection,all the BmE-EGFP cells were selected with Zeocin for two months.The genome editing effenciency was firstly analyzed by fluorescent images,the EGFP fluorescence was nearly abolished in three tests with sgRNAs targeted EGFP.However,the nonspecific sgRNAs controls remained unchanged.We then analyzed the genome editing efficiency by flow cytometry,which showed the same consequence.At last,the indel patterns was detected using Sanger sequence analysis.The EGFP gene was amplified by PCR and performed for T-A clone.44 monoclonals were selected for Sanger sequence.Almost all EGFP gene were mutated and most of the mutation types were small fragment deletions.Furthermore,more seventy percent were frame shift.These results show that,the pB-CRISPR vector system could lose the function of protein coding gene in BmE cells efficiently.2.Establishment of genome-wide CRISPR screening library in BmE cellsIn order to establish the genome-wide CRISPR screening library in BmE cells,we first designed 1,534,227 sgRNAs of all protein coding genes in B.mori.Then,94,000sgRNAs were selected for synthesizing on the 94K arrays according to the following criteria.To increase frame shift efficiency,sgRNAs within the first three exons will be selected.The sgRNAs with high target efficiency and low off-target efficiency will be chosen.At last,about 6 sgRNAs per gene were selected.Afterwards,the pool of sgRNA was purified from 94K arrays and recombinated into the pB-CRISPR to construct the CRISPR knockout plasmid library,named as pB-CRISPR-lib.In order to make sure the high coverage and diversity of pB-CRISPR-lib,~10~8 monoclonals were grown by many parallel electrotransformations.Finally,more than 1,000 clones per sgRNA were selected.The sgRNA region of pB-CRISPR-lib was amplified by PCR and performed next generation sequence(NGS)to analyze the coverage,accuracy and diversity of the plasmid library.The NGS data showed 51,431 sgRNAs were detected in pB-CRISPR-lib on behalf of 16198 genes(97.7%of all B.mori genes)suggesting the high coverage of the plasmid library.Moreover,about 72%of sgRNAs had 11–200reads suggesting the high diversity of pB-CRISPR-lib.We next co-transfected the pB-CRISPR-lib and A3-Helper into B.mori embryonic cell line(BmE,25 cm~2 flask)at a ratio of 1/1(mass,1ug/1ug)using lipidosome according to the manufacturer's instructions.To achieve high coverage(~2,000×)and diversity,we performed about 150 parallel transfections.After two months selected with Zeocin,almost all cells were integrated with CRISPR system.At last,we constructed BmE genome-scale CRISPR/Cas9 knockout library,named as BmEGCKLib.We then collected 4×10~7 cells to evaluate the quality of BmEGCKLib through NGS.Satisfactorily,95.2%of sgRNAs(48,982 sgRNAs)in the pB-CRISPR-lib were detected in BmEGCKLib,suggesting the quality of BmEGCKLib was similar to pB-CRISPR-lib.The compositions of the BmEGCKLib and pB-CRISPR-lib(correlation coefficient=0.99)proved the same conclusion.In brief,we constructed the genome-wide CRISPR screening library in BmE cells with high coverage,accuracy,and diversity for the functional gene screening.3.Identifying of essential genes and growth-restricting genes in B.mori using the genome-wide knockout screeningIn order to annotate the basic functional genes of B.mori,we next identified the essential genes and growth-restricting genes in BmE cell line using the BmE genome-scale CRISPR/Cas9 knockout library,BmEGCKLib.To achieve high coverage,4×10~7 BmEGCKLib cells were collected every month for three time points:BmE-Lib1,BmE-Lib2,BmE-Lib3.Afterwards,all the three groups genomic DNA was extracted and the sgRNAs region was amplified by PCR for next generation sequence(NGS)analyzing.We then calculated the number of sgRNAs and the abundance of sgRNAs for every time point.The results show that the number of sgRNAs was reduced and the majority sgRNAs depleted,while few were enriched,suggesting the BmEGCKlib cell was selected over time.We then used the MAGeCK program(the model-based analysis of genome-wide CRISPR-Cas9 knockout)to calculate the the negative(sgRNA depleted)scores or positive(sgRNA enriched)scores for each gene and rank genes to identified essential genes(sgRNA depleted)and growth-restricting genes(sgRNA enriched)in B.mori.We also performed the biological replicates,which exhibited the high correlation in sgRNA-level(r=0.87)and gene-level(r=0.76).We identified 1006 essential genes and 838 growth-restricting genes respectively in BmE cell line.The overlap of essential genes between B.mori and eukaryotic species were significantly.Interestingly,the transcriptional levels of essential genes were higher than all genes average transcriptional levels in both BmE cell line and silk gland.Besides,all the essential genes and growth-restricting genes of BmE cells were distributed across all 28chromosomes.The Gene Ontology(GO)classification showed that,essential genes of BmE cells were related to core cell components.To identify the gene functions,essential genes were mapped to the Kyoto encyclopedia of genes and genomes(KEGG)biological pathways.The most enriched KEGG pathways for essential genes were Ribosome,RNA transport,mRNA surveillance,Spliceosome,Ribosome biogenesis in eukaryotes,Pyrimidine metabolism,Oxidative phosphorylation,Metabolic pathways,RNA degradation,TOR signaling pathway,Purine metabolism and DNA replication.Which were primarily relevant to DNA metabolism,RNA metabolism and protein metabolism.Furthermore,we performed subcellular localization analysis of essential genes and growth-restricting genes.The significantly enriched subcellular localization for essential genes were nucleus and cytoplasm(~38.2%and~34.4%,respectively).Interestingly,essential genes than growth-restricting genes were found subcellular localization in the core organelle,nucleus and mitochondrion.4.Identifying of genes responsible for temperature challenge in B.mori using the genome-wide knockout screeningThe natural environment is crucial to normal organisms.In order to identify genes responsible for extreme environment challenges,we performed the genome-wide knockout screening.We chose an abnormal temperature challenge as an example.Three groups of BmEGCKLib cells(4×10~7 BmEGCKLib cells every group)were selected for testing.Two of them were exposed to 4?or 30?as a test,the other one was exposed to 27?as control.20 days later,all the surviving cells were collected,afterwards,all the three groups genomic DNA was extracted and the sgRNAs region was amplified by PCR for next generation sequence(NGS)analyzing to identify genes responsible for temperature challenge.We next mapped all the selected genes to Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways for biological annotating.Surprisingly,the most enriched KEGG pathway in the 4?was highly overlapped with essential genes,that were primarily relevant to RNA processing and protein processing.Besides,the negative selected genes were enriched in the steroid biosynthesis pathway fatty acid biosynthesis and fatty acid metabolism pathways,suggesting that maintaining the membrane fluidity is crucially when cells respond to abnormal temperature challenge.We then performed subcellular localization analysis of selected genes.We found that,more negative selected genes than positive selected genes were located on cytomembrane in both the 4?and 30?groups.However,more positive selected genes than negative selected genes were located on mitochondria in both the 4?and 30?groups.5.Identifying of genes response for BmNPV infection in B.mori using the genome-wide knockout screeningBmNPV(Bombyx mori Nucleopolyhedrovirus)is the most harmful pathogen of B.mori.In order to identify genes responsible for BmNPV infection in B.mori,we next performed genome-wide knockout screening in BmEGCKlib cells.The BmEGCKLib was divided into two groups,each group contained about 4×10~7 cells.One of the groups was infected with BmNPV 4 times(once every 2 days),afterwards,all the cells survived were collected,and the genomic DNA was extracted for next generation sequence(NGS)analyzing.However,the other group used as control.We identified811 positive selected genes and 809 negative selected genes response for BmNPV infection.Furthermore,genes response for BmNPV infection in B.mori were classified into KEGG pathways.The most enriched KEGG pathways were phagosome,Notch signaling pathway,Dorso-ventral axis formation,MAPK signaling-fly and Wnt signaling pathway.At last,we chosed three positively screened genes to construct three konckout cell lines to validate our screen.Compare with control,all the three konckout cell lines showed BmNPV resistance.In conclusion,we established a genome wide CRISPR knockout platform in B.mori for the first time.Afterwards,we identified genes essential for cell viability and resistance to abiotic and biotic stresses in B.mori by CRISPR screening.We not only provided a novel tool to annotate the functional of B.mori genome but also afford an alternative strategy for high-throughput screens in non-model organisms.