Inaccurate DSB repair amplicon sequencing data from plasmids injected into Drosophila embryos expressing Cas9
Creation of sequence randomized end joining substrates. We designed a 165nt ssDNA fragment based on R0 with randomized nucleotides adjacent to an I-SceI/CRISPR cas9 break. The PAGE purified semi randomized ssDNA oligo was made double stranded with a primer extension assay using Q5 polymerase (NEB) and an Eppendorf Vapo Protect thermocycler. The product was treated with S1 nuclease (ThermoFisher Scientific) to remove remaining ssDNA fragments. DNA inserts were purified using phenol chloroform precipitation. 600uL of phenol:chloroform:isoamyl alcohol (25:24:1) was added to each sample, followed by 15 minutes on a rocking platform. The samples were centrifuged for 15 minutes and the aqueous layer was collected. Equal volume of phenol:chloroform:isoamyl alcohol was added followed by another 15 minutes on a rocking platform and 15 minute centrifugation. The aqueous layer was collected and 1/5th volume of 8M potassium acetate was added followed by one volume of chloroform. The samples were rocked on a platform for another 15 minutes on followed by a 15 minute centrifugation. The aqueous layer was collected and 0.7 volumes of isopropyl alcohol and 1uL glycoblue was added. The samples were incubated at -80C for 30 minutes, centrifuged for 15 minutes and the pellets were washed with 200uL of 70% ethanol. The samples were centrifuged again for 15 minutes and the pellets were dried and resuspended in water Cloning of R0R was performed using the NEB PCR cloning kit (NEB) and transformed into NEB 10 beta chemically competent cells using product protocol. Transformations were grown on LB + ampicillin and diluted to yield approximately 150 colonies per plate. Each plate was washed into LB broth with ampicillin, grown overnight, and midi prepped using the Macherey-Nagel NucleoBond Xtra Midi kit. Plasmid DNA libraries were amplified by PCR with Q5 polymerase (NEB) for 19 cycles using an Eppendorf Vapo Protect thermocycler with a pooled set of primers containing one, two, or three random bases at the 5 end. AMPure bead purification was performed on the PCR products and the purified DNA was subjected to a second PCR to attach indices for amplicon sequencing. A final AMPure purification step was performed to remove all products less than 100 bp. Samples were pooled with 5% PhiX DNA and sequenced using the Illumina Nano chip for reference library generation and selection of experimental samples. Experimental libraries were chosen based on relative concentration of individual plasmids and combined to generate four distinct injection libraries with a total of 1100 plasmids. Injection libraries were diluted to 500 ng/ul in injection buffer (1 mM sodium phosphate and 50mM potassium chloride) along with 500ng/uL gRNA expression vector. Cloning of Cas9 targeting guide RNA expression vector. Cloning of gRNA expression vector was done using the pU6-BbsI-chiRNA vector (Addgene plasmid #45946, Kate OConnor-Giles Lab), and annealed complementary oligos for the R0 region of interest. Electroporation of the ligation reaction was performed and transformants were screened via sequencing. A single correct transformant was grown in LB broth with ampicillin and midi prepped using the Macherey-Nagel NucleoBond Xtra Midi kit. Fly stocks. The genotype of the fly stock used for injections was w1118, lig4169a, P(Act-5c Cas9, w+). All flies were maintained in bottles containing a cornmeal-agar medium in a 25C incubator on a 12 h lightdark cycle. Freshly eclosed flies were placed in cages and fed yeast paste on a grape agar substrate to promote embryo laying. Embryos <2 h old were recovered and dechorionated for two minutes in a 50% bleach solution. Embryos (n = 100 per experiment) were aligned uniformly on double-stick tape attached to a glass cover slip for injections of plasmids. Microinjection and DNA recovery. Embryos were desiccated for one minute and coated in halocarbon oil to prevent rupturing of the membrane when injected. The microinjections were done using a Zeiss compound microscope fitted with injection apparatus and a Parker-Hanfin Picospritzer II. Embryos were incubated at 25C for 24 h to allow for cas9 cutting and repair of the ensuing double-strand breaks. Halocarbon oil was removed using a 1% sodium dodecyl sulfate solution in 0.7% sodium chloride buffer. Plasmid DNA was extracted by grinding embryos (two slides using the same injection mix were combined) with a disposable pestle in 200 ul Buffer A (100 mM TrisHCl, pH 7.5, 100 mM EDTA, 100 mM NaCl, 0.5% SDS). After incubating at 65C for 30 min, 800 ul of LiCl/KAc solution (1 part 5 M KAc:2.5 parts 6 M LiCl) was added and tubes were incubated on ice for 10 min. The solution was centrifuged for 10 minutes and plasmids were precipitated from the supernatant with isopropanol. End joining junction sequencing. Purified plasmid DNA containing end joining junctions were enriched for error-prone repair events with in vitro I-SceI treatment to remove uncut plasmids or perfect repair events. For high-throughput amplicon sequencing, approximately 165 bp of sequence flanking the cas9 cut site was amplified from recovered plasmids by PCR with Q5 polymerase (NEB) for 19 cycles using an Eppendorf Vapo Protect thermocycler with a pooled set of primers containing one, two or three random bases at the 5 end. AMPure bead purification was performed on the PCR products and the purified DNA was subjected to a second PCR to attach indices for amplicon sequencing. A final AMPure purification step was performed to remove all products less than 100 bp. The samples were pooled with 10% PhiX DNA and sequenced on an Illumina HiSeq platform using 2  300 pair-end reads. Analysis of high-throughput amplicon sequencing. Pair-end reads corresponding to the same end joining event were merged into single consensus reads as FASTQ files using the BBMerge module in Geneious R10 (BioMatters).  The 5 and 3 ends of the resulting sequences were trimmed to the amplicon primer sequences and an additional 80 nt of chosen DNA sequence was added to each end facilitate proper alignment of the reads. Reads lacking either of chosen 10 bp portions of the amplicon primer sequences were removed as PCR artifacts as they failed to span both sides of the break. Reads were then separated into 1097 independent files based upon the presence of unique 5 and 3 barcode sequences that correspond to those of the originating DNA substrate that was injected into fly embryos. Reads lacking appropriate barcodes were excluded from further analysis. Reads for each file were subsequently aligned to the corresponding original repair substrate reference sequence using the BWA mem alignment software [31] with default settings.  The resulting SAM files where then subjected to Hi-FiBR analysis [25] using a break site of 161 and 164 bp from the 5 and 3 read ends, respectively, to classify junction events. Base substitutions within a 15 bp window surrounding the break site were interpreted as repair errors instead of sequencing artifact.  Reads were classified as either exact matches to the original repair substrate, deletions (having one or more contiguous bases deleted adjacent to the break site in the reference), insertions (having one or more contiguous inserted bases adjacent to the break site), or complex (having base substitutions, insertions, and/or deletions that are non-adjacent to the break site or contain both deleted and inserted sequences).  The number of instances of each specific repair events was counted. Junctions with <10 mapped reads were removed from consideration. Following removal of these junctions, the normalized percentage of reads that contain sequence alterations compared to the reference sequence per junction was calculated by dividing the number of reads per junction by the total number of inaccurate reads that aligned to any junction in the sample. Deletion junctions were characterized as apparent blunt joins (ABJ) or microhomology junctions (MHJ) and were analyzed to determine SD-MMEJ consistency using a novel pure Python suffix tree library, which searched for a break-spanning region that contained both microhomology and primer junctions. Insertion junctions were analyzed using an in-house R-script to determine SD-MMEJ consistency, as defined in the text.