tag:blogger.com,1999:blog-2203096312729800474.post7916316089495913966..comments2022-04-01T10:32:42.048-07:00Comments on A CRISPR blog / bibliography: #CRISPR #Cas9 protospacer sequence considerations - some current thoughts.Unknownnoreply@blogger.comBlogger1125tag:blogger.com,1999:blog-2203096312729800474.post-77430350284519648092015-08-17T13:41:57.402-07:002015-08-17T13:41:57.402-07:00Regarding the "first base G" issue - as ...Regarding the "first base G" issue - as per the last paragraph in this post...I've since read Koike-Yusa et al's paper from Nat. Biotechnol. 2014 v.32 (30 pp. 267-273 in which they made a lentivral expression mouse CRISPR library with >80,000 guide RNAs. They wound up always placing a G in the first position of the guide RNA, even if it was mismatched to the actual target. I.e. if the 20 base protospacer did not have a G normally, they synthesize it with a G in position 1 followed by the remaining normal 19 bases. They reported increased efficiencies likely due to increase sgRNA transcription thanks to the G and also because a single mismatch at position 1 doesn't bother Cas9 much, apparently. This is sort of like truncating by 1 base and sticking a G on the end. While Fu et al showed that truncation to 18 or 17 bases can reduce off-target effects, this tiny "truncation" probably doesn't have much effect on off-targeting.Douglas Mortlockhttps://www.blogger.com/profile/11500150356385406841noreply@blogger.com