RT Journal
A1 Kim, Sojung
A1 Koo, Taeyoung
A1 Jee, Hyeon-Gun
A1 Cho, Hee-Yeon
A1 Lee, Gyeorae
A1 Lim, Dong-Gyun
A1 Shin, Hyoung Shik
A1 Kim, Jin-Soo
T1 CRISPR RNAs trigger innate immune responses in human cells
JF Genome Research 
JO Genome Research 
YR 2018 
FD March 01 
VO 28 
IS 3 
SP 367 
OP 373 
DO 10.1101/gr.231936.117 
UL http://genome.cshlp.org/content/28/3/367.abstract 
AB 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.