Construction And Activity Evaluation Of STU-Cas9 Genome Editing System For Larch

Larch(Larix kaempferi)has the characteristics of strong stress resistance,rapid growth,resistance to pests and diseases,and good ecological benefits.It is an important tree species for pulp and building materials.With the decline in global forest coverage and the increasing demand for forest trees from social and economic development,more and more attention has been paid to the cultivation of fine varieties of larch through genetic trait molecular improvement strategies,and to accelerate the related basic research and application of afforestation projects.The emergence of genome editing technology has brought new opportunities to the basic research of larch genome function and its molecular breeding practice.However,due to the late start of the basic research of molecular biology and genome manipulation of larch,there is no successful report of larch genome editing.This research was based on the larch genome structure and the working mechanism of CRISPR-Cas9,and was committed to constructing a larch CRISPR-Cas9 genome editing system in order to realize the effective editing of endogenous genes of larch.Provide powerful molecular manipulation tools for the basic research of larch genome function and its molecular breeding practice.The main research contents and results are as follows:1.Through the transient expression system of larch protoplasts,it was confirmed that the Lar PE004 promoter element had high transcriptional activity in larch endogenous cells.Transient expression of rice protoplasts and stable plant transformation proved that Lar PE004 had species versatility and could work in stably transformed plants.Cloned and identified the sequence information of three types of larch endogenous genes:transcription factor-coding genes,hormone-related genes,and micro RNA-coding genes.It provided a clear target for reliable testing of larch CRISPR-Cas9 system;2.Based on different transcription unit types and promoter elements,different types of CRISPR-Cas9 systems were constructed.The editing activity was tested using the strategy of combining the transient expression system of larch protoplasts and the highthroughput sequencing of amplicons.The results showed that the single transcription unit CRISPR-Cas9(STU-Cas9)had a significantly better editing efficiency at a single site(6.1%-11.0%)than the two transcription unit CRISPR-Cas9(TTU-Cas9)(1.8%-2.2%);The STU-Cas9 system driven by Lar PE004 promoter had better editing efficiency at a single site(6.1%-11.0%)than the STU-Cas9 system driven by Ca MV 35S(1.8%-2.0%)and Zm Ubi(2.7%-3.5%);3.Based on the broad-spectrum recognition of multiple PAM sites by the Cas9 protein mutant SpRY,the Lar PE004::STU-SpRY system was constructed.It effectively realized the editing of multiple types of larch PAM sites,with an average editing efficiency of up to 60%.Effectively expand the larch genome editing system mediated by STU-Cas9;4.Confirmed that the STU-Cas9 system had good co-editing capabilities,among which: the co-editing efficiency of Lar PE004::STU-Cas9 at four sites was 1.0%-8.2%;the co-editing of Lar PE004::STU-SpRY at four sites of different PAM sequences was1.7%-8.6%;the overall co-editing activity of the Lar PE004::STU-Cas9 system was slightly better than that of the Lar PE004::STU-SpRY system in the four sites against the all PAM sequence 5’-NGG-3’.