Latest CRISPR Technologies for In Vivo and Ex Vivo Genome Engineering

سال انتشار: 1397
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 373

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شناسه ملی سند علمی:

NSCMRMED03_028

تاریخ نمایه سازی: 30 دی 1397

چکیده مقاله:

Background and Aim: Methods of creating genetically engineeredanimals have been developed for over three decades. However, methodsto create genetically engineered cells were not well established untilthe advent of programmable nucleases, particularly the CRISPR-Cas9system. The CRISPR-Cas9 system offers as a powerful tool for both animal(in vivo) and cellular (ex vivo) genome engineering methods. Methodsto create genetically engineered animals involve three major steps:harvesting embryos from one set of females, embryo microinjection(performed ex vivo), and transferring embryos to another set of females.Although tedious, these methods were used for over three decades tocreate animal models. Regarding cellular genome engineering, eventhough the CRISPR-Cas9 tool is very efficient in creating gene knockoutcell lines, creating knock-in cell lines is still a major challenge.Methods: We recently developed a completely in vivo method calledGONAD (Genome editing via Oviductal Nucleic Acids Delivery), thatby-passes the three critical steps. The method involves surgical exposureof oviducts of pregnant mice, installation of CRISPR reagents into theoviduct, followed by electroporation of the oviduct and finally suturingof the surgical incision. We further tested parameters of GONAD such astage of pregnancy and types of CRISPR reagents. In order to solve thechallenge of ex vivo genome editing problem, i.e., to create knock-incell lines, we tested various CRISPR reagents formats and electrophoreticconditions along with long single-stranded DNA or double-strandedDNA donors to test what formats produce better knock-in efficiencies.Human primary T cells were used for these experiments.Results: The results of our in vivo method development experimentsindicate that the 0.7-day pregnancy and CRISPR ribonucleoprotein(RNP) complexes, such as crRNA +tracrRNA + Cas9 protein along withshort or long ssDNA donors yield highest and reliable efficiencies ofgenome editing. The improved GONAD method (i-GONAD) is shown tobe suitable for the routine generation of knock-out, knock-in and largedeletionmodels at comparable efficiencies as the microinjection-basedmethods. For cellular genome engineering, we show that electroporationof CRISPR RNPs along with double-stranded or single-stranded DNA canproduce knocking in of DNA cassettes into human primary T cells at ashigh as 40% efficiency.Conclusion: i-GONAD offers several advantages over the previousmethods: it does not require the second set females (embryo recipients)and so also vasectomized males; the females used for i-GONAD canbe recycled for other experiments; i-GONAD can be easily adapted atthe laboratories lacking sophisticated microinjection equipment, and; itcan be performed by researchers not having embryo-handling skills. Inmy presentation I will discuss how i-GONAD method has simplified theprocess of creating animal models (in vivo), and how Easi-CRISPR systemcan be used for cell genome engineering (ex vivo).

نویسندگان

Channabasavaiah B Gurumurthy

Department of Genetics, Cell Biology and Anatomy, University ofNebraska, Omaha, USA