Ian L Ross, Hong Phuong Le, Sabar Budiman, Dake Xiong, Fritz Hemker, Elizabeth A Millen, Melanie Oey, Ben Hankamer
{"title":"循环标记系统使寡核苷酸定向基因编辑在衣藻中无限系列地进行。","authors":"Ian L Ross, Hong Phuong Le, Sabar Budiman, Dake Xiong, Fritz Hemker, Elizabeth A Millen, Melanie Oey, Ben Hankamer","doi":"10.1093/plphys/kiae427","DOIUrl":null,"url":null,"abstract":"<p><p>CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for nonselectable target mutations. This becomes challenging when many sequential modifications are required in a single-cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, nonselectable genes. We demonstrate that this \"Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)\" technology enables an essentially limitless series of genetic modifications to be introduced into a single-cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.</p>","PeriodicalId":20101,"journal":{"name":"Plant Physiology","volume":" ","pages":"2330-2345"},"PeriodicalIF":6.5000,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11637769/pdf/","citationCount":"0","resultStr":"{\"title\":\"A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.\",\"authors\":\"Ian L Ross, Hong Phuong Le, Sabar Budiman, Dake Xiong, Fritz Hemker, Elizabeth A Millen, Melanie Oey, Ben Hankamer\",\"doi\":\"10.1093/plphys/kiae427\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for nonselectable target mutations. This becomes challenging when many sequential modifications are required in a single-cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, nonselectable genes. We demonstrate that this \\\"Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)\\\" technology enables an essentially limitless series of genetic modifications to be introduced into a single-cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.</p>\",\"PeriodicalId\":20101,\"journal\":{\"name\":\"Plant Physiology\",\"volume\":\" \",\"pages\":\"2330-2345\"},\"PeriodicalIF\":6.5000,\"publicationDate\":\"2024-12-02\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11637769/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Physiology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1093/plphys/kiae427\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Physiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1093/plphys/kiae427","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.
CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for nonselectable target mutations. This becomes challenging when many sequential modifications are required in a single-cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, nonselectable genes. We demonstrate that this "Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)" technology enables an essentially limitless series of genetic modifications to be introduced into a single-cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.
期刊介绍:
Plant Physiology® is a distinguished and highly respected journal with a rich history dating back to its establishment in 1926. It stands as a leading international publication in the field of plant biology, covering a comprehensive range of topics from the molecular and structural aspects of plant life to systems biology and ecophysiology. Recognized as the most highly cited journal in plant sciences, Plant Physiology® is a testament to its commitment to excellence and the dissemination of groundbreaking research.
As the official publication of the American Society of Plant Biologists, Plant Physiology® upholds rigorous peer-review standards, ensuring that the scientific community receives the highest quality research. The journal releases 12 issues annually, providing a steady stream of new findings and insights to its readership.