Precise and broad scope genome editing based on high-specificity Cas9 nickases
| Autor: | Jin Liu, Manuel A F V Gonçalves, Richard L. Frock, Marie Le Bouteiller, Qian Wang, Josephine M. Janssen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Genotyping Techniques
Base pair AcademicSubjects/SCI00010 Streptococcus pyogenes Induced Pluripotent Stem Cells Computational biology Transfection Genome Substrate Specificity 03 medical and health sciences Gene Knockout Techniques 0302 clinical medicine Genome editing Bacterial Proteins Genes Reporter CRISPR-Associated Protein 9 Heterochromatin Genetics CRISPR Deoxyribonuclease I Humans Gene Knock-In Techniques Gene 030304 developmental biology Gene Editing 0303 health sciences Nuclease Polymorphism Genetic biology Base Sequence Cas9 High-Throughput Nucleotide Sequencing Recombinant Proteins Clone Cells HEK293 Cells biology.protein Narese/29 CRISPR-Cas Systems Synthetic Biology and Bioengineering 030217 neurology & neurosurgery HeLa Cells RNA Guide Kinetoplastida |
| Zdroj: | Nucleic Acids Research Nucleic Acids Research, 49(2), 1173-1198. OXFORD UNIV PRESS |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount. |
| Databáze: | OpenAIRE |
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