Gene editing: an instrument for practical application of gene biology to plant breeding / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.

Analysis of full-length sequence and 18 point mutations of HLA-B in a leukemia patient and her family / 中国组织工程研究

BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly. OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology. METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes. RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3’UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.

Gene editing: an instrument for practical application of gene biology to plant breeding / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.

Sequence analysis and 3D molecular structure simulation of a novel HLA allele B*51: 159 / 中华医学遗传学杂志

Objective@#To identify a novel human leukocyte antigen (HLA) B allele in a Chinese Han individual and construct its three-dimensional structure.@*Methods@#The initial HLA genotyping was performed by PCR-sequence-based typing (PCR-SBT). The ambiguous allele was confirmed with single-strand DNA sequencing. The DNA sequence was analyzed to identify the difference between the novel allele and its closest matching allele. Finally, the three-dimensional molecular structure of the novel allele was constructed using a Swiss-Model.@*Results@#One allele of the subject at the HLA-B locus was B*44: 03: 01, whilst the other was a novel allele which differed from the closest matching allele B*51: 01: 01: 01 by nucleotide (nt) 329 A>C in exon 2, resulting in an amino acid change at codon 86 (p.Asn86Thr).@*Conclusion@#A novel HLA-B allele has been identified and officially named as HLA-B*51: 159 by the WHO Nomenclature Committee for Factors of the HLA System. The three-dimensional structure of B*51: 159 was simulated.

Sequence Analysis and Identification of A Novel HLA-A* 2 4∶3 2 7 Allele Been Designated by the HLA Nomenclature Committee of WHO / 现代检验医学杂志

Objective To do analysis of sequence and identify a novel HLA-A allele in Chinese hematopoietic stem cell do-nors.Methods A rare HLA-A allele was initially detected by Luminex PCR-SSO typing,then the sample was sequenced by sequence-based typing (SBT)and the group-specific sequencing primer (GSSP)to confirm the mutation allele and locus.Re-sults The sequence of the sample results showed that the allele compared with the highest homologous allele HLA-A*24∶02∶01∶01 was the difference in the exon 3 at position 544 G>A,resulting in an amino acid sequence of HLA-A*24∶02∶01∶01 at position 158 change Ala to Thr.Conclusion This allele is a new HLA-A allele and has been designated as HLA-A*24∶327 by the HLA Nomenclature Committee of World Health Organization (WHO).

Sequence analysis of a novel HLA-A?01 ∶130 allele and modeling of three-dimensional structure of HLA molecule / 中国免疫学杂志

Objective:To confirm the novel allele HLA-A*01∶130 and analyzed the nucleotide sequence of the abnormal reaction pattern.Methods: The HLA typing of sample DNA was performed by PCR-SBT.The ambiguous novel HLA allele was confirmed with single stranded SBT method,then DNA sequencing was performed to identify the difference between the novel allele and HLA-A?01:66 allele.Finally, it was modeled by Swiss-Model to three-dimensional structure of HLA Molecule.Results: The novel allele was not the same with all known HLA-A allele sequence.After analysis,there was one nucleotide differed from the A?01:66 at position 368 where A→G( codon 99 TAT→TGT) resulting in a coding change,99 Tyr was changed to Cys.The amino acid substitution at residue 99 the HLA polypeptide was located in a beta-sheet of antigenic peptide-binding region.Conclusion: The allele is a novel allele that has now been officially named as HLA-A*01∶130 by the World Health Organization( WHO) HLA Nomenclature Committee.

Identification of a novel allele, HLA-B * 54∶26 / 中华微生物学和免疫学杂志

Objective To identify a novel HLA-B allele in Chinese Han population.Methods No full matched result was obtained in HLA-B locus in HLA typing for China marrow donor program(CMDP) using bi-allelic sequence-based typing(SBT).A confirmatory test for novel HLA allele was performed with mono-allelic SBT.Results B * 15∶05∶01 was confirmed and another allele should be a B * 54new.The B * 54new has 2 nt changes from the closest matching HLA-B * 54∶01∶01 at nt 559,560 where AC→GA(codon163ACG→GAG),led to a coding change,163T→E.Conclusion A novel HLA-B allele was confirmed and officially named HLA-B * 54∶26 under the Genbank Accession number JN209963 by the WHO Nomenclature Committee for Factors of the HLA System in February 2012.

Identification of a novel human leukocyte antigen allele, HLA-A * 31 ∶ 22 / 中华微生物学和免疫学杂志

Objective To identify and confirm a novel HLA allele.Methods A new human leukocyte antigen A allele was found during routine HLA genotyping by polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSOP) and sequencing-based typing (SBT).HLA-A locus was amplified from exon 1 through exon 8,and the nucleotide sequence of exon 2 to exon 4 for HLA-A were sequenced in both directions.Results The novel HLA-A * 31 allele is identical to A * 31 ∶ 01 ∶ 02 with an exception of one base substitution at position 245 of exon 2 where an ' A' change to ' C' resulting in codon 82 changed from GAG to GCG.Conclusion A novel HLA allele,A * 31 ∶ 22,was identified,and was named officially by the WHO Nomenclature Committee for factors of the HLA system.

Identification and sequence analysis of a novel allele HLA-B~* 9537 / 中国输血杂志

Tin Exon3,resulting in 4 amino acid changed from Glu to Asp(E103D),Thr to Lys(T113K),Gln to Glu(Q114E) and Ser to Phe(S116F),respectively.Conclusion The novel allelewas identified,and was assigned the name B*9537 officially by the WHO Nomenclature Committee.

Serologic heterogeneity of HLA-A24 correlates with allelic types in the Korean population

HLA-A24 is the second most frequently expressed HLA-A type in Koreans (GF 22.8%). Four different serologic reaction patterns were observed in Korean A24 positive samples using a commercial serologic typing kit. To clarify the nature of serologic heterogeneity, thirteen A24 positive DNA samples representing the four different serologic reaction patterns were subjected to DNA sequencing analysis of the amplified HLA-A genes from each sample. Four A*24 alleles (A*2402101, A*2403, A*2408, and A*2421) were associated with the four unique serologic reaction patterns. During this study, a novel allele, A*2421, was characterized. The new sequence is similar to A*2402101, differing at codon 127 (AAA-->AAC; K-->N). By comparing putative amino acid sequences and serologic reaction patterns of A*24 allelic products identified in this study, several crucial sites for A24- and A9-specific antibody binding were predicted: 127K for A24 antibody binding, and 62E-65G and 166D-167G for A9 antibody binding. This information will be helpful for accurately assigning HLA-A24 types by serology and for predicting serologic types of new alleles.

Identification of a novel HLA class Ⅰ allele,B~*1316 / 中国输血杂志

Objective To analyze the nucleotide sequences of novel HLA class I le,B*1316.Methods Routine sequence-specific oligonucleotide(SSO) typing and sequencing based typing(SBT) was used.Results The B*1316 allele differs from B*1302 by one nucleotide substitution in exon 3: T to A at nt position 184,which results in an amino acid substitution at codon 62 from Val to Glu.Conclusion A novel HLA class I allele,B*1316 has been identified,and was officially recognized by WHO Nomenclature Committee in April 2006.