Applications of genome editing ultimately depend on DNA repair triggered by targeted double-stran... more Applications of genome editing ultimately depend on DNA repair triggered by targeted double-strand breaks (DSBs). However, repair mechanisms in human cells remain poorly understood and vary across different cell types. Here we report that DSBs selectively induced on a mutant allele in heterozygous human embryos are repaired by gene conversion using an intact wildtype homolog as a template in up to 40% of targeted embryos. We also show that targeting of homozygous loci facilitates an interplay of non-homologous end joining (NHEJ) and gene conversion and results in embryos which carry identical indel mutations on both loci. Additionally, conversion tracks may expand bidirectionally well beyond the target region leading to an extensive loss of heterozygosity (LOH). Our study demonstrates that gene conversion and NHEJ are two major DNA DSB repair mechanisms in preimplantation human embryos. While gene conversion could be applicable for gene correction, extensive LOH presents a serious s...
Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function... more Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.
Genome editing has potential for the targeted correction of germline mutations. Here we describe ... more Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains ...
Here, we report the successful design, construction, and characterization of a 770-kilobase synth... more Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromo... more We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.
Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generat... more Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites), and then use a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% non-synthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes re...
The forfeiting of photosynthetic capabilities has occurred independently many times throughout eu... more The forfeiting of photosynthetic capabilities has occurred independently many times throughout eukaryotic evolution. But almost all non-photosynthetic plants and algae still retain a colorless plastid and an associated genome, which performs fundamental processes apart from photosynthesis. Unfortunately, little is known about the forces leading to photosynthetic loss; this is largely because there is a lack of data from transitional species. Here, we compare the plastid genomes of two…
Applications of genome editing ultimately depend on DNA repair triggered by targeted double-stran... more Applications of genome editing ultimately depend on DNA repair triggered by targeted double-strand breaks (DSBs). However, repair mechanisms in human cells remain poorly understood and vary across different cell types. Here we report that DSBs selectively induced on a mutant allele in heterozygous human embryos are repaired by gene conversion using an intact wildtype homolog as a template in up to 40% of targeted embryos. We also show that targeting of homozygous loci facilitates an interplay of non-homologous end joining (NHEJ) and gene conversion and results in embryos which carry identical indel mutations on both loci. Additionally, conversion tracks may expand bidirectionally well beyond the target region leading to an extensive loss of heterozygosity (LOH). Our study demonstrates that gene conversion and NHEJ are two major DNA DSB repair mechanisms in preimplantation human embryos. While gene conversion could be applicable for gene correction, extensive LOH presents a serious s...
Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function... more Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.
Genome editing has potential for the targeted correction of germline mutations. Here we describe ... more Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains ...
Here, we report the successful design, construction, and characterization of a 770-kilobase synth... more Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromo... more We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.
Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generat... more Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites), and then use a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% non-synthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes re...
The forfeiting of photosynthetic capabilities has occurred independently many times throughout eu... more The forfeiting of photosynthetic capabilities has occurred independently many times throughout eukaryotic evolution. But almost all non-photosynthetic plants and algae still retain a colorless plastid and an associated genome, which performs fundamental processes apart from photosynthesis. Unfortunately, little is known about the forces leading to photosynthetic loss; this is largely because there is a lack of data from transitional species. Here, we compare the plastid genomes of two…
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Papers by Jianhui Gong