26 THE USE OF PAIRED CRISPR GUIDE RNAs AND THE Cas9 SYSTEM DOES NOT ALWAYS PRODUCE SITE SPECIFIC DELETIONS OF GENE SEQUENCE IN PORCINE CELL AND EMBRYO CULTURE
Authors: Spate A. M. , Whitworth K. M. , O'Gorman C. W. , Byrne A. K. , Prather R. S. , Wells K. D. 
Published: December 2016

The use of the CRISPR/Cas9 system has become increasingly popular for creating gene edits in both cell and embryo culture. High specificity and efficiency of editing as well as low cost and ease of use has helped to promote its use. We hypothesised that by using multiple CRISPR guides at one time, we could quickly create exact deletions spanning greater areas of sequence. A total of 5 candidate genes (A, B, C, D, E) were targeted for deletions ranging in size of 74 to 551 bp. All modifications were created through the co-injection of 2 CRISPR guide RNAs with Cas9 RNA into in vitro-produced presumptive porcine zygotes. The CRISPR guides were created using gBlocks containing the T7 promoter sequence, 18–24 bp of CRISPR guide RNA, and 85 bp of tracer RNA. The RNA structure of each guide was reviewed using RNA Folding Form as well as offsite cutting using NCBI Blast. CRISPR guide RNA pairs (20 ng μL−1) and Cas9 RNA (20 ng μL−1) were co-injected (1–3 ρl) into the cytoplasm of IVF produced porcine zygotes using the FemtoJet 4i injector. Following injections, the zygotes were cultured in vitro for 5–6 days, and viable blastocyst or morula were selected for embryo transfer into recipient gilts. Resulting pigs were assayed for expected modifications using PCR. Pigs were considered modified if an insertion or deletion was measured by gel electrophoresis and DNA sequencing. Only one pair of CRISPR guides was injected per zygote, resulting in an individual PCR assay for the gene of interest. In total, 42 live piglets were born, 24 of which were edited, yielding 57% modification. When expected modifications v. observed were analysed, only 4 of 24 pigs (16%) produced the predicted modification on at least one allele. Of the remaining 20 pigs, several showed more than one form of modification. Insertions of ranging from 1 to 400 bp were detected in 10 pigs, 9 pigs formed biallelic modifications, 6 pigs produced altered sequence for greater than 2 alleles (mosaic), 6 pigs had deletions larger than the expected ranging from 11 to 1739 bp, and 14 had deletions smaller than the expected. Due to the absence of plasmid during injections, the insertions observed contained repetitive elements from the gene being modified as well as random additional bases. Additionally, CRISPR pairs were used in cell culture of porcine fibroblast modifying gene F, where they produced 6 different deletions ranging from the expected 63b to 617 bp. We recognise that the cutting efficiency of each CRISPR guide was not measured, as our goal was to create the expected deletions from pairs of CRISPR guides. We acknowledge our hypothesis was incorrect, as this data indicates that the CRISPR/Cas9 system is a very useful tool for gene editing, however it can induce unexpected modifications when used in pairs, in cell and embryo culture.
Study was supported by funding from Food for the 21st Century and NIH (U42OD011140).