Construction Of Pig Genome-scale CRISPR/Cas9 Knock-out Library And Screen For Key Host Factors For Virus Resistance

Nowadays,there are still various infectious diseases in pig industry that seriously affect the breeding process and production efficiency,which has tremendous economic impact on pork industry.As a result,it is of great importance to prevent the spread of infectious diseases in swine.Long-term use of vaccines and drugs may lead to great genetic variation of virus and gradually become less effective.Instead,genetic modification of key genes for viral infection in host may overcome this problem,and has shown promising results in preventing Porcine Reproductive and Respiratory Syndrome Virus?PRRSV?.However,it is still a great challenge to discover more key host genes involved in disease resistance.CRISPR/Cas9 is a power genome editing technology and has broad applications in many fields by manipulating DNA at single cell or embryo stage.CRISPR/Cas9high-throughput screening strategy combined with loss of gene function enables the discovery of key genes associated with disease resistance in a genome-wide scale.However,sg RNA with high on-target and low off-target activities are critical to evaluate the accuracy of CRISPR screening.Therefore,we firstly explored the efficiency and specificity of sg RNA with a full length of 20 nts and then truncated to 19,18,and 17 nts.Finally,we selected the sg RNA with a length of 20 nts due to the fewest potential off-target sites to design and construct the pig genome-wide CRISPR/Cas9 knockout library.Combining with CRISPR high-throughput screening strategy and Japanese Encephalitis Virus infected PK-15 cell model,we successfully screened for host factors leading to a significant inhibition of JEV replication.Thus,genome-wide CRISPR knockout screening provided a new strategy for disease resistance breeding in the pig.The results are as follows:1. The length of 5?-end of sg RNAs can affect the activity and specificity of sg RNAIn this study,we found that 5'-end lengths of sg RNAs can affect the activity of sg RNA.We predicted the potential off-target sites of sg RNA targeting the same genomic site but in different lengths by CRISPR-offinder software.We observed significantly increased number of predicted off-target sites after sg RNA length truncation.2. Rare off-target mutations in the CRISPR/Cas9-edited cells by using sg RNAs in different lengthsTo evaluate the off-target effect of sg RNA in different lengths,we firstly compared six methods for detecting the activity of sg RNA.We found that Target Seq strategy allowed highly sensitive detection of sg RNA activity.Then,we employed this method to evaluate the off-target effect of sg RNA in different lengths.We designed and synthesized 6,549capture probes in total for candidate on target and off-target sites which were shared by different sg RNA lengths.Finally,approximately 70%potential off-target sites of sg RNA were successfully captured.Although the number of predicted off-target sites significantly increased after sg RNA length truncation,only 4?9 predicted off-targets had detectable cleavage activity.This result indicated that the specificity of sg RNA in different lengths is higher than predicated.3. Microsatellite located in predicted off-target sites can interfere with accurate sg RNA specificity assessmentHigh Indels mutation frequency was found even in the control group by Target Seq.To explore the possible reasons,we analyzed the sequence of predicted off-target sites.We observed that numerous microsatellites with different sequence feature located in predicted off-target sites.The proportions of microsatellites corresponding to the predicted off-target sites of the selected sg RNAs in 20,19,18,and 17 nts were 25.9%,28.6%,15.4%,and 5.0%,respectively.When the sg RNA length was 20 nts,the proportion of?CA?₈?GA?₂ reached 79.2%.As the length of the sg RNA shortened,this microsatellite type derived three new microsatellites,such as A?CA?₈GA,?CA?₈GA,and A?CA?₇GA.One of them randomly selected for further validation by T7ENI cleavage assay.The result showed that multiple cut bands can be detected for the selected microsatellite targets even in the control group.These results show that microsatellite located in predicted off-target sites can interfere with accurate sg RNA specificity assessment.4. Design pig genome-wide CRISPR/Cas9 knockout libraryWe selected sg RNA with length of 20 nts to design pig genome-wide CRISPR/Cas9knockout library according to the result of the off-target study of sg RNA in different length.The sg RNA library was designed by CRISPR-offinder software,with the selection of following parameters:sg RNAs with length of 20 nts,the PAM sequence with NGG,the maximum number of mismatches allowed up to 3,and minimizing for potential off-target sites.We designed in total 85,674 sg RNAs targeting all annotation protein coding gene,linc RNA,mi RNAs,with additional 1,000 scrambled-sequence sg RNAs serving as negative controls.Each gene was covered with 3 sg RNAs.5. Construction of a genome-wide sg RNA library plasmidWe generated more than 200 colonies per sg RNA in the library.The sg RNA plasmid pool was constructed by PCR and Gibson assembly approaches.The coverage of library was determined by high-throughput sequencing with up to 96.2%coverage of sg RNAs in the plasmid pool.To generate a genome-wide mutant cells library,we packaged a sg RNA library lentivirus pool.The Cas9 stable expressed PK-15 cells were transduced by the lentiviral sg RNA library?>500 cells per sg RNA?.Our library coverage reach 94.7%in the transduced cells.6. We successfully found that Heparan Sulfate Proteoglycan?HSPG? pathway-related genes can significantly inhibit JEV replication in the pig genome-wide CRISPR/Cas9 screen.Our screening strategy is performed by multiple rounds of JEV infection,and the survival cells of the third and fourth rounds were collected for deep sequencing.We calculated the abundance of sg RNAs and analyzed overlap sg RNAs in the third and fourth rounds.We observed 44 overlap sg RNAs the number of sg RNA reads greater than10,000 and 162 overlap sg RNAs with the number of sg RNA reads greater than 1,000.Next,the significantly enriched GO terms of candidate genes in biological process includes nitrogen compound metabolic process,protein folding in endoplasmic reticulum?ER?and heparan sulfate proteoglycan biosynthetic process,etc.The mainly cellular components of these enriched genes were ER membrane protein complex,Golgi apparatus and cis-Golgi network.The enriched KEGG pathways were protein processing in endoplasmic reticulum,Glycosaminoglycan?GAG?biosynthesis,and Heparan sulfate proteoglycan?HSPG?biosynthesis,etc.Moreover,10 genes with at least one sg RNA involved in HSPG biosynthesis were significantly enriched,including EXT1,EXT2,GLCE,HS6ST1,B3GAT3,B4GALT7,XYLT2,EXTL3,SLC35B2 and GAA.Therefore,we selected four key genes involved in HSPG synthesis to assess their impact on JEV replication,including SLC35B2,HS6ST1,GLCE,and B3GAT3.Finally,we confirmed the knockout of these four genes can significantly inhibit JEV replication,assessed by Plaque-forming units assay,absolute quantification PCR,and immunofluorescence?IF?.