CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos.
The rapid development of CRISPR technology greatly impacts the field of genetic engineering. The simplicity in design and generation of highly efficient CRISPR reagents allows more and more researchers to take on genome editing in different model systems in their own labs, even for those who found it daunting before. An active CRISPR complex contains a protein component (Cas9) and an RNA component (small guide RNA [sgRNA]), which can be delivered into cells in various formats. Cas9 can be introduced as a DNA expression plasmid, in vitro transcripts, or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP), whereas the sgRNA can be delivered either expressed as a DNA plasmid or as an in vitro transcript. Here we compared the different delivery methods in cultured cell lines as well as mouse and rat single-cell embryos and view the RNPs as the most convenient and efficient to use. We also report the detection of limited off-targeting in cells and embryos and discuss approaches to lower that chance. We hope that researchers new to CRISPR find our results helpful to their adaptation of the technology for optimal gene editing.
Horizon Discovery Group Company , Saint Louis, Missouri.
Evguenia
E
Kouranova
Horizon Discovery Group Company , Saint Louis, Missouri.
Kevin
K
Forbes
Horizon Discovery Group Company , Saint Louis, Missouri.
Guojun
G
Zhao
Horizon Discovery Group Company , Saint Louis, Missouri.
Joe
J
Warren
Horizon Discovery Group Company , Saint Louis, Missouri.
Angela
A
Bartels
Horizon Discovery Group Company , Saint Louis, Missouri.
Yumei
Y
Wu
Horizon Discovery Group Company , Saint Louis, Missouri.
Xiaoxia
X
Cui
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005910:Glioma / Q000235:genetics / Q000473:pathology; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics / Q000473:pathology; D012313:RNA / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics
D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D012313:RNA; D004247:DNA