Journal: Nature Communications
Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii
doi: 10.1038/s41467-021-27004-1
Figure Lengend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.
Article Snippet: Then, 200 ng (2 µL) of annealed ssODNs was digested with either Bfa I or PvuII-HF (1 U, New England Biolabs) in CutSmart buffer (1×) in a total volume of 10 µL and incubated at 37 °C for 1 h. Immediately after digestion, reactions were separated on an agarose gel (1.5%) stained with SYBR Safe (1×), imaged (UVP BioDoc-It) and analysed semi-quantitatively by gel densitometry using ImageJ (Supplementary Data ).
Techniques: Sequencing, Transfection