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Jemima Kamran

Mentor: Dr. Desiree Tillo, Frederick National Laboratory for Cancer Research.

Date/Time: August 24, 2021 at 3:00pm

Abstract: Cultured regularly interspaced short palindromic repeats (CRISPR) technology is a tool for editing genomes. It allows investigators to alter DNA sequences and modify gene function. CRISPR systems have two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas) protein. This RNA-protein complex introduces a double stranded break (DSB) in the genomic target defined by the gRNA.  The double-strand break is then repaired by the cellular machinery via two repair pathways: 1) the non-homologous end joining (NHEJ) pathway, which leads to small indels in target DNA and is useful for generating gene knock-outs, and 2) the homology directed repair (HDR) pathway, which is utilized by investigators to incorporate a sequence of choice at the cleavage site.    There is no standard analytic tool for the interpretation of CRISPR-Cas9 edited sequences. The goal of this project was to evaluate two software tools, CRISPResso2 (in both amplicon and pooled mode) and CRISPRpic in analyzing NHEJ and HDR experiments.

For the NHEJ experiment, the CRISPRpic assigned fewer reads as deletions, insertions, and substitutions compared to CRISPResso and CRISPRessoPooled.  For the HDR experiment, CRISPRpic classified fewer HDR reads than CRISPRessoPooled. While CRISPRpic classified fewer HDR reads, more of the HDR reads were classified as substitutions.  When the window parameters were modified in both programs, CRISPRpic assigned more reads to the modified class when the window was small around the cleavage site.   As expected, the kmer approach of CRISPRpic took less time and fewer computational resources than the global pairwise alignment approach of CRISPResso.   The differences between the two programs can be attributed to the alignment based technique of CRISPResso and the kmer counting technique of CRISPRpic.  More investigation is needed to evaluate which software tool is best for evaluating CRISPR data. Future paths of study should be to more closely compare the reads aligned between both approaches, and to examine additional CRISPR data sets to more accurately compare the two programs.