Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver-Specific Knockout Mice Using Multiplex CRISPR/Cas9 Gene Editing.

Current Protocols in Molecular Biology
Collin G JohnsonKarl-Dimiter Bissig

Abstract

Somatic liver knockout (SLiK) is a method developed to rapidly generate a liver-specific knockout of one or several genes. This technique combines the strengths of CRISPR/Cas9 gene editing and hydrodynamic tail-vein injection, a simple in vivo method for transfection of hepatocytes, to harness the powerful selection pressure of tyrosinemic livers to replace host hepatocytes with any desired gene deletion. In this protocol, we will describe sgRNA design and cloning, hydrodynamic tail-vein injection of targeting constructs, and screening and validation methods for efficient in vivo gene editing. © 2020 by John Wiley & Sons, Inc. Support Protocol 1: sgRNA design Support Protocol 2: sgRNA construction: daisy chaining multiple sgRNAs Basic Protocol: Delivery of DNA by hydrodynamic tail-vein injection and liver repopulation of edited hepatocytes Support Protocol 3: Validation of CRISPR/Cas9 cutting in vivo.

References

Feb 24, 2001·Proceedings of the National Academy of Sciences of the United States of America·J L AponteD K Johnson
Sep 22, 2006·Gene Therapy·T SudaD Liu
Dec 15, 2006·Gene Therapy·H Herweijer, J A Wolff
Dec 14, 2007·Proceedings of the National Academy of Sciences of the United States of America·Karl-Dimiter BissigInder M Verma
Feb 12, 2008·Current Protocols in Molecular Biology·D Voytas
Oct 12, 2011·Current Protocols in Molecular Biology·Barton E SlatkoFrederick M Ausubel
Oct 29, 2015·Proceedings of the National Academy of Sciences of the United States of America·Julia WeberRoland Rad
Aug 31, 2016·Nature Communications·Francis P PankowiczKarl-Dimiter Bissig
Mar 17, 2017·Scientific Reports·Kelsey E JarrettWilliam R Lagor
Sep 1, 2018·Gastroenterology·Francis P PankowiczKarl-Dimiter Bissig

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Citations

Jun 20, 2020·Expert Review of Anti-infective Therapy·Harmanjit SinghPrerna Chauhan

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