CRISPR RNAs trigger innate immune responses in human cells

Genome Research
Sojung KimJin-Soo Kim

Abstract

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

References

Mar 3, 2004·Nature Biotechnology·Dong-Ho KimJohn J Rossi
Oct 14, 2006·Science·Andreas PichlmairCaetano Reis e Sousa
Feb 11, 2010·Cell·Jan RehwinkelCaetano Reis e Sousa
Apr 2, 2011·Nature·Elitza DeltchevaEmmanuelle Charpentier
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jan 31, 2013·Nature Biotechnology·Seung Woo ChoJin-Soo Kim
Nov 21, 2013·Genome Research·Young Hoon SungJin-Soo Kim
Apr 3, 2014·Nature Reviews. Genetics·Hyongbum Kim, Jin-Soo Kim
Jun 30, 2015·Nature Biotechnology·Ayal HendelMatthew H Porteus
Oct 20, 2015·Nature Biotechnology·Je Wook WooJin-Soo Kim
Jun 9, 2016·Nature Biotechnology·Daesik KimJin-Soo Kim
Jun 9, 2016·Nature Biotechnology·Junho K HurJin-Soo Kim
Jul 28, 2016·Nature Reviews. Immunology·Martin Schlee, Gunther Hartmann
Nov 8, 2016·Nature·Daniel P DeverMatthew H Porteus
Feb 17, 2017·Nature Communications·Hyeran KimSang-Gyu Kim
Mar 1, 2017·Nature Biotechnology·Kyoungmi KimJin-Soo Kim
May 22, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Lei XuHongkui Deng
Aug 8, 2017·Nature·Hong MaShoukhrat Mitalipov

❮ Previous
Next ❯

Citations

Apr 17, 2018·Human Molecular Genetics·Cui ZhangJinfu Wang
Feb 9, 2019·Biomaterials Science·Joon Eoh, Luo Gu
Jan 31, 2019·Annual Review of Immunology·Dimitre R Simeonov, Alexander Marson
May 23, 2019·Human Genetics·Minyoung Lee, Hyongbum Kim
Jan 26, 2020·International Journal of Molecular Sciences·Quynh NguyenToshifumi Yokota
Apr 2, 2020·International Journal of Molecular Sciences·Caroline F PeddleRobert E MacLaren
May 2, 2020·The Journal of Experimental Medicine·Emily C FreundAditya Murthy
Feb 27, 2020·Briefings in Functional Genomics·Qi ZhouJing Yang
Sep 1, 2018·Science·Gavin J Knott, Jennifer A Doudna
Apr 26, 2019·The CRISPR Journal·Romain RouetRoss C Wilson
Apr 4, 2019·Frontiers in Immunology·Jie LiuQi Zhao
Nov 7, 2019·Experimental & Molecular Medicine·Su Bin MoonYong-Sam Kim
Jun 2, 2020·The Journal of Clinical Investigation·Francesco ChemelloEric N Olson
Jul 1, 2020·Nature Biotechnology·Joost van HaasterenDavid V Schaffer
Jan 17, 2020·Chembiochem : a European Journal of Chemical Biology·Cody M PalumboPeter A Beal
Mar 14, 2020·Genes·Thomas M LaniganThomas L Saunders
Jun 13, 2020·Frontiers in Immunology·Rih-Sheng HuangSteven Lin
Aug 8, 2018·Nature Communications·Jungjoon K LeeJin-Soo Kim
Jun 2, 2020·Infection & chemotherapy·Hyoung Shik Shin
May 6, 2020·Scientific Reports·Carlos Pinzon-ArteagaCharles R Long
Mar 25, 2020·Bioscience Reports·Matthew P HirakawaKimberly S Butler
Feb 6, 2020·Nature Cell Biology·Aaron A SmargonGene W Yeo
Sep 11, 2020·Evolutionary Applications·Virginie Courtier-OrgogozoChristophe Boëte
Apr 11, 2019·Frontiers in Cellular and Infection Microbiology·Qiaoqiao XiaoShuliang Chen
Oct 9, 2020·International Journal of Molecular Sciences·Dmitry KostyushevVladimir Chulanov
Mar 24, 2020·The Journal of Investigative Dermatology·Auke B C Otten, Bryan K Sun
Sep 16, 2020·Advanced Drug Delivery Reviews·Jie LiNiren Murthy
Nov 17, 2019·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Eileen McGowanYiguang Lin
Nov 26, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Garima SharmaChiranjib Chakraborty
Feb 5, 2021·Bioanalysis·Mikko HölttäNeil Henderson
Nov 3, 2020·Clinical and Translational Medicine·Hengji ZhanYuchen Liu
Feb 11, 2021·European Journal of Human Genetics : EJHG·Vratko Himič, Kay E Davies
Feb 17, 2021·Pathogens and Global Health·Kyle E WattersGregory D Koblentz
Apr 2, 2020·Bioconjugate Chemistry·Daniel A GlazierWeiping Tang

❮ Previous
Next ❯

Related Concepts

Related Feeds

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.