Enhancing the openness of chromatin improves the efficiency of editing in Cas9 | Genome Biology

Title: Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhance Cas9 editing efficiency in vivo
journal:
Guanwen Liu, Kangquan Yin, Qianwei Zhang, Caixia Gao and Jin-Long Qiu
Published time: 26 Jun, 2019/07/26
DOI:
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A recent study published on
proved for the first time in plants that the openness of chromatin is positively correlative with the editorial efficiency of Cas9. More importantly, the study improved chromatin openness through Cas9-TV/dsgRNA strategy to enhance the editing efficiency of Cas9 in vivo, and provided a new method to improve the editing efficiency of Cas9 in vivo, especially for the editing of genes located in close areas of chromatin.
Genomic editing technology is the latest revolutionary technology in life science, especially CRISPR/Cas9 system with its simple, efficient and low-cost characteristics in basic theoretical research, gene therapy and crop breeding and other fields have been widely used. However, a growing number of researchers have found that CRISPR/Cas9 has significantly different editing efficiencies at different points in the genome in different types of endocellular cells or individuals. Very low editing efficiency at some sites limits the application of CRISPR/Cas9 at the genome-wide level. Therefore, finding the reasons behind this problem and providing solutions will help expand the breadth and depth of CRISPR/Cas9 applications in different species.
CRISPR/Cas9 system is derived from primary nuclear organisms and faces a completely different cellular environment when applied to the nucleocytes. Unlike primary nucleocyte DNA, the genomic DNA of the endocysts is wrapped around histones and further compressed to form advanced chromosome structures. Chromatin states at different genomic site are likely to affect the editing efficiency of CRISPR/Cas9 systems. In recent years, some studies in animal cell line have shown a correlation between the openness of chromatin and the editing efficiency of Cas9. Recently,
teamed up in plant cells to study the relationship between CRISPR/Cas9 genome editing efficiency and chromatin openness, and developed a new method to improve editing efficiency in Cas9 by increasing chromatin openness.
researchers first analyzed the indel frequency of gene-editing plants with a total of 70 sgRNA target points in 41 rice genes, combined with the chromatin state of the target point, and found that the sgRNA editing efficiency in the open area of the targeted chromatin was significantly higher than that of the sgRNA editing in the close area of the targeted chromatin. By designing a series of sgRNAs targeting different chromatin regions, the efficiency of Cas9 genome editing and the openness of chromatin were positively correlated in rice protons. In order to rule out the effect of sgRNA sequence composition on genome editing efficiency, the study also selected several sgRNAs that can target different chromatin state points, and found that the editing efficiency of the same sgRNA in the open region of chromatin was significantly higher than that of chromatin tight region. These results show that Cas9 genome editing is more efficient in chromatin open areas in rice cells.
genes that are active are in the area where chromonromosomes are open, transcription activaters can induce the opening of chromosomes. The researchers fused a synthetic transcription activater with Cas9 to form the fusion protein Cas9-TV. Compared with Cas9, the genome editing efficiency of this fusion protein is significantly improved. dsgRNA (dead sgRNA) binds to the genome without double-stranded fractures, and the introduction of dsgRNA near the target site may help Cas9-TV further improve the openness of chromatin, and researchers have found that Cas9-TV/dsgRNA can increase the editing efficiency of Cas9 genomes several times. The researchers also demonstrated through experiments that both Cas9-TV and dsgRNA enhance chromatin openness at target points, and Cas9-TV combined with dsgRNA to enhance chromatin openness. The chromatin state in the body is in dynamic change, and the openness of chromatin is relative. In contrast, Cas9-TV and Cas9-TV/dsgRNA improve genome editing efficiency in both open and tight areas of chromatin. In addition, Cas9-TV and Cas9-TV/dsgRNA did not increase the off-target effect of Cas9.
the first experiment in plants to show that the openness of chromatin is positively correlative with the editing efficiency of Cas9. More importantly, the study improved chromatin openness through Cas9-TV/dsgRNA strategy to enhance the editing efficiency of Cas9 in vivo, and provided a new method to improve the editing efficiency of Cas9 in vivo, especially for the editing of genes located in close areas of chromatin. The findings were recently published online by Genome Biology under the title "Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhance Cas9 editing efficiency in vivo". China
National Key Laboratory of Plant Genomics
and
are co-first authors and
co-authors of the paper. The study was funded by the Ministry of Agriculture, the Ministry
Science and Technology.The CRISPR/Cas9 system is unable to edit all targetable genomic sites with full efficiency in vivo. We show that Cas9-mediated editing is more efficient in open chromatin regions than in closed chromatin regions in rice. A construct (Cas9-TV) formed by fusing a synthetic transcription activation domain to Cas9 edits target sites more efficiently, even in closed chromatin regions. Moreover, combining Cas9-TV with a proximally binding dead sgRNA (dsgRNA) further improves editing efficiency up to several folds. The use of Cas9-TV/dsgRNA thus provides a novel strategy for obtaining efficient genome editing in vivo, especially at nuclease-refractory target sites.
(Source: Science.com)