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Adhikari 1 Utilizing the CRISPR/Cas9 method of genome engineering to generate unc-119 mutants in C. briggsae By Anand Adhikari Student #: 1450551 Under Supervision of Dr. Bhagwati P. Gupta Molecular Biology 3I03 McMaster University Submitted: April 23, 2015 Adhikari 2 Table of Contents Abstract …………………………………………………………………………………..…… 3 List of Abbreviations……………………………………………………………………….. 4 Introduction…………………………………………………………………………………. 5-8 Models of Genetic Engineering……………………………………………….…..5 Overview of CRISPR/Cas9…………………………………………….………….5-6 Plasmids and sgRNAs…………………………………………………….....…….6 CRISPR/Cas in C. elegans…………………………………………..………...7 Sister Species: C. elegans & C.briggsae…………………………………….……7-8 Brief Overview…………………………………………………………………….8 Materials & Methods…………………………………………………………………………. .9-11 Creating ssODN & Related Genomics……………………………………….……9 Mutagenesis & Transformation……………………………………………………9 Restriction Digest &DNA Prep……………………………………………………10 Microinjection, Cloning, Screening……………………………………….………10 gDNA Isolation, Sequencing, & Alignment………………………………………11 Results…………………………………………………………………………. ….…………12-16 Trial #1…………………………………………………………………………….12 Trial #2 ………………………………………………………………………..…..13-16 Target Sequence, Restriction Digest………………………………..13 DNA Concentrations, Mutation Results……………………………14 Cbr-unc-119 F2-10-1 DNA sequencing alignment…………………15 Cbr-unc-119 F2-13-2 DNA sequence alignment……………...……16 Discussion…………………………………………………………………………. ….……17-19 Trial #1 vs Trial #2……………………………………………………………17 Restriction Digest…………………………………………………………… 17-18 Trial #2 Additional Analysis…………………………………………………18 Sequencing Analysis…………………………………………………………18 Pitfalls and Future Experimentation…………………………………………19 Supplementary Tables……………………………………………………………….………..20-21 References……………………………………………………………….……………………22-23 Adhikari 3 List of Abbreviations The following list below describes the meaning of various abbreviations and acronyms used throughout the report. The page on which each one is defined or first used is also given. Abbreviation Meaning Page ApE A plasmid editor (software) 9 CRISPR Clustered regularly interspaced short palindromic repeats 4 DSB Double-stranded break 5 GFP Green fluorescent protein 7 PAM Protospacer adjacent motif 5 sgRNA Single-guide RNA 4 TALEN Transcription activator-like effector nuclease 5 ZFN Zinc-finger nuclease 5 Adhikari 4 Abstract The CRISPR/Cas9 system stands as one of the new developments in genetic engineering used to modify any genomic sequence with high levels of specificity. The system first found in bacteria allows these species to develop resistance to foreign genetic elements, providing an acquired immunity.1 More recently, the technique has been expanded to model organisms, such as C. elegans, in which modified sgRNAs are able to target genomic sequences expressed from a U6 RNA promoter.2 This technique has not been explored as extensively in C. briggsae, the sister species of C. elegans. This study was done to see whether targeted modifications of gene sequences in C. briggsae could be acquired through the CRISPR/Cas9 system. The focus of the study was the unc-119 gene, which has been shown to be involved with motor ability in C. elegans and C. briggsae.3 Additionally, the unc-119-regulated pathway and gene function is conserved to higher organisms, including humans.4 Initially, the experiment was done and yielded no mutant worms. After designing a sgRNA site ending with a “GG” motif, the mutation was visible in the F2 generation. The cbr-unc-119 gene was able to be mutated in 6 of the 28 candidates, yielding a 19% efficiency rate. None of the mutants were able to be rescued in the subsequent generation, illustrating that they followed a Mendelian model of inheritance. After sequencing the DNA of two of the obtained candidates, both illustrated a nonsense mutation resulting from an insertion. Two of the other identified candidates were unable to produce viable offspring, potentially due to additional mutations. This project has allowed additional insight towards the use of identifying mutant alleles through CRISPR/Cas9 in C. briggsae, and allows future potential for understanding evolutionary differences of development and gene function between C. elegans and C. briggsae. Adhikari 5 Introduction Models of Genetic Engineering Before CRISPR/Cas9 became an establish mode of genetic engineering, ZFN and TALEN methods were relied upon. ZFNs work by introducing a generation of DSBs at specific genomic target locations, which are based on the presence of chimeric proteins containing a DNA binding domain.5 As DNA nucleases became more efficient, ZFNs improved in localizing the specificity and efficacy of DSBs. ZFNs, however, require a very complicated recognition code and must be selected for experimentally to ensure high rate of efficiency.6 TALENs, similar to ZFNs, consist of a DNA-binding domain with a TALE protein fused to the FokI nuclease. Sequential repeats in the DNA domain correspond to the DNA targeting code of Tal effectors. Restriction enzymes can be engineered that are specific for any desired DNA sequence of choice with TALENs.7 Through homologous recombination, both of these methods have been used to create hereditary changes as well as sequence manipulation, but CRISPR/Cas9 has emerged as the most effective genetic engineering mechanism with the greatest potential in biotechnology and medicine.8 Overview of CRISPR/Cas9 The CRISPR/Cas9 system is an RNA/enzyme complex composed of a targeting RNA (sgRNA) and the Cas9 RNA-guided nuclease. Cas9 searches the genetic material of a cell until it recognizes the specific 3-nucleotide series (NGG) known as the PAM sequence. A 20 base pair stretch (known as the protospacer) of the sgRNA associates with the complementary DNA adjacent to the PAM sequence and allows Cas9 to unwind the DNA double helix and create a double stranded break in the DNA sequence 3 nucleotides down from the PAM sequence. Changing the targeting sequence at the 5’ end of the sgRNA allows Cas9 to cause double strand Adhikari 6 break in virtually any DNA sequence of interest in a genome. This allows for repair enzymes to repair gaps in the DNA through either error-prone non-homologous end joining (in the absence of any template) or precise homologous recombination (if a homologous sequence is provided in trans), thereby causing a deletion, insertion, or point mutation in the DNA. The Cas9 protein and specific guide RNAs can be generated into a vector that can be inserted into the germline of the model organism.9 The potential developments of CRISPR/Cas9 can be used in for many potential outlooks, including human gene therapy, screening for drug target, synthetic biology engineering, and agricultural biology.8 Plasmids and sgRNAs Through plasmid based delivery, 20bp sgRNAs can be integrated into a template plasmid through site-directed mutagenesis. Genomic software can be used to determine the idea location to insert sgRNAs into a plasmid. Construction of an expression plasmid for sgRNA is quite simple and rapid as it only requires one cloning step with a pair of partially complementary oligonucleotides. The oligo pairs encoding the 20-nt guide sequences are annealed and ligated into a plasmid through a common mutagenesis kit. Additionally, the transfected plasmids can be modified to enable virus production for in vivo delivery. In addition to PCR and plasmid-based delivery methods, Cas9 and sgRNAs can be introduced into cells as mRNA and RNA, respectively. Commonly used plasmid cloning vector in E.coli is pUC57, which has been proven to work with CRISPR/Cas9 engineering countless number of times.9 This plasmid (2710 bp in length) is also bacterially resistant to ampicillin and contains many restriction sites to confirm successful transfection.10 Adhikari 7 CRISPR/Cas in C. elegans In C. elegans, Cas9 activity was initially completed to screen for random mutations after DNA injection. Researchers were able to create mutations in two genes (unc-119 and dpy-13) that illustrated clear phenotypic defects in the F2 generation. Further studies have shown success in this mechanism with C. elegans with heat-shock promoter to drive expression of Cas9 and use of the U6 promoter to express the sgRNA to create random deletions in specific genes to show visible phenotypes.2 These changes introduce either missense or nonsense mutations in the gene of interest. PCR amplicons, like GFP reporter can be used to study in vivo expression of genes and can serve as indication of mutation carriers in the F1 generation of worms. The GFP insertion approach has the added advantage of disrupting the function of genes for which no obvious visible phenotype exists. One-fourth of the F2 generation of worms from the GFP-expressing F1s are expected to carry the mutations, following patterns of Mendelian inheritance. Expansion by using a co-inject plasmid to replace genes with GFP by screening for fluorescence has also been an adopted technique in CRISPR/Cas9.11 From these experiments, researchers have been able to garner average frequencies of gene editing ranging from 7 to 32% in C. elegans. More recently, in vivo there has been higher rates of frequency of genome editing in C. elegans with guide RNAs ending with a GG motif at the 3’ end of the target-specific sequence.12 Sister Species: C. elegans & C.briggsae C. briggsae and C. elegans are two very closely related species of nematodes from the genus Caenorhabditis. Both these organisms share a similar number of chromosomes and protein/nonprotein coding genes, interpreted as conserved genes and pathways. The wildtype strain of C. elegans is N2 whereas the wildtype strain of C. briggsae is AF16.3 From evolutionary Adhikari 8 studies, it has been determined that C. briggsae diverged from C. elegans about 30 million years ago, yet the two species have almost identical morphology. However, comparison of DNA sequences has revealed significant number of rapidly evolving and divergent genes suggesting alterations in underlying mechanisms of development and behaviour. Consistent with this, recent comparative studies of vulval development between C. elegans and C. briggsae have revealed differences in the competence of certain vulval precursor cells.13 There are many genes responsible for regulating cell competence, or the ability of cells to respond to intercellular signals. Additionally, the striking homology between these nematodes and humans makes them an ideal organism for genetic and developmental studies.12 Brief Overview This study looked at whether targeted modifications of unc-119 in C. briggsae could be acquired through the CRISPR/Cas9 system. Through the creation of a plasmid vector that contained an sgRNA target site of the unc-119 gene in C. briggsae and introducing it into the AF16 worms, visible mutants were able to be seen in the F2 generation of worms. Sequencing potential candidates allowed for alterations in gene sequences to be identified as insertions that led to nonsense mutations, immediately preceding the chosen sgRNA target site. Adhikari 9 Materials & Methods: Creating ssODN & Related Genomics To create the single-stranded oligonucleotide for cbr-unc-119, the transcript for unc-119 gene in C. briggsae was found on Wormbase. The transcript excluding introns and untranslated regions was copied into ZiFit in FASTA format in order to check for potential target sites.14 A target site was chosen that had a G/C content >50%. For Trial #2, a target site was chosen that had a similar G/C content and additionally ended with a “GG” motif.12 The chosen target site was then entered on the NCBI BLAST software in order to confirm the identity of the chosen targetsite on the unc-119 gene in C. briggsae.15,16 Primers were created that matched the targetsite using ApE software.17 The forward primer included the 5’ end of the pUC57 plasmid recognition site were ordered via MOBIX Lab Online Oligio Synthesis at McMaster University. Mutagenesis & Transformation Q5 Site-Directed Mutagenesis was performed to add the cbr-unc-119 sgRNA site into the pUC57 plasmid vector.17 Differences in the protocol were that the heat shock lasted for 45 seconds, the reaction mix was incubated on ice for two minutes following heat shock, and the SOC/bacteria mix was incubated for two hours. The mix was plated and incubated overnight at 37ºC for colony formation. Three colonies were isolated and were each placed in a separate test tube containing 4 µl of SOC medium. Additionally a 1:1000 dilution of carbenicillin was added to the test tubes to reduce the formation of satellite colonies. Colonies were each transferred into a separate test tube and they were placed in the incubated shaker for 16 hours. Adhikari 10 Restriction Digest &DNA Prep The plasmid was then isolated in 1.5mL tubes using the Presto Mini Prep Kit.18 DNA Spectrophotometer was used to determine the initial concentrations of the plasmids (shown in Table #4). Restriction enzyme digest was then performed using HindIII, EcoRI , and Acl1. Double digest was set up using HindIII and EcoRI. Separate digest was done with Acl1. Additionally, lin-10 was used as a control since it is cut by Acl1 at the target site. Gel results are shown below in Image 1.19 After confirming successful mutagenesis via gel electrophoresis (Image 1), plasmid DNA concentrations were increased using vacufuge centrifugation to create sufficient concentrations for microinjection. The final DNA concentrations are shown below in Table 4. Microinjection, Cloning, Screening Injection mix was then created by using appropriate concentrations of sgRNA, Cas9, myo2::GFP, and dH20.20 16 parental AF16 hermaphrodites were injected in the gonad with the injection mix, and the worms were given three days to grown F1s.21 F1s from each plate were screened for GFP-expression. F1 worms that expressed GFP were plated 2 per plate and labeled accordingly. In Trial #2, non-fluorescent worms from each plate that illustrate GFP expression were also plated 2 per plate, since it was unsure whether the mutation corresponded with GFP expression. The picked F1s were given three days to grow and give rise to an F2 generation. The F2 generation of worms was then screened for the visible unc-119 mutant phenotype. Potential candidates were plated one worm per plate. Primers were created for the F2 worms outside of the range of the target site and were ordered via Mobix.14,15,16 Adhikari 11 gDNA Isolation, Sequencing, & Alignment gDNA of F2 worms was isolated using standard gDNA isolation protocol.22 Single worm PCR was done on an F2 from each candidate plate, using High Fidelity protocol.23 PCR product was run on a 1% gel electrophoresis, and successful PCR product formation was extracted via using gel extraction.24 2µl of the extracted product was run on a gel alongside 2 µl of ladder, and the intensity of the band compared to the ladder was used to gauge the concentration of the DNA. 5 µl of DNA was then sent for sequencing. Alignment was done on Clustal Omega program to compare the sequenced DNA to the WT (Shown below, Figures 1 &2) and demonstrate the location of the mutation. Adhikari 12 Results: Trial #1 In Table #1 below, the target sequence used for Trial #1 is listed, as well as the designed forward and reverse primers for that specific target site. The highlighted portion of the forward primer matches the target sequence. The initial DNA concentrations and restriction enzyme digest of the plasmid data are not included for Trial #1 since it ultimately did not succeed in the progeny of the injected worms. Table#1: Target sequence and primers in cbr-unc-119 gene for Trial #1 unc-119 sgRNA #1 GGAAGTGCTAAAACGTCGTT GGAAGTGCTAAAACGTCGTTGTTTTAGAGCTAGAAATAGCAAG Forward Primer (sgRNA#1) (GL1047) AAACATTTAGATTTGCAATTCAATTATAT Reverse Primer (sgRNA#1) (GL1048) Table #1: Forward and reverse primer for Q5 site directed mutagenesis of pU6::cel-unc-119 sgRNA plasmid to substitute cbr-unc-119 sgRNA #1 sequence. Below, Table #2 illustrates the mutation results for Trial #1. Initially, 14 AF16 worms were injected with the injection mix, and 50 worms showing GFP expression were plated. However, there were no worms in the F2 generation that illustrated the unc-119 mutation, thus resulting in a 0% efficiency of genetic engineering. Table #2: Mutation Results for Trial #1 Targeted Gene cbr-unc-119 P0 14 worms F1 50 worms (GFP) F2 0 Efficiency 0% Adhikari 13 Trial #2 In Table #3 below, the target sequence used for Trial #2 is listed, as well as the designed forward primer and same reverse primer from Trial #1 Table #3: Target sequence and primers in cbr-unc-119 gene for Trial #2 unc-119 sgRNA #2 GGGAAGGTCGCCGAGCCGGG GGGAAGGTCGCCGAGCCGGGGTTTTAGAGCTAGAAATAGCAAG Forward Primer (sgRNA#2) (GL1079) AAACATTTAGATTTGCAATTCAATTATAT Reverse Primer (sgRNA#2) (GL1048) Table #3: Forward and reverse primer for Q5 site directed mutagenesis of pU6::cel-unc-119 sgRNA plasmid to substitute cbr-unc-119 sgRNA #2 sequence. Image 1 below shows the restriction digest of the mutated pUC57 plasmids containing the inserted unc-119 sgRNA #2 target site. Three colonies (labeled A-C) were either double digested with HindIII + EcoRI or only digested with Acl1. The control for this experiment was the pUC57 plasmid containing lin-10 insertion. Bands for the double digest of the mutated plasmid appeared at 3.1kb for all of the colonies. For the Acl1 digest, unc-119 sgRNA #2 C gave a band at 2.6kb. Image 1: Restriction Digest of pUC57::pU6::cbr-unc-119 sgRNA #2 Plasmid DNA Adhikari 14 Below, Table #4 illustrates the initial and final concentrations of the plasmids containing the inserted unc-119 sgRNA #2 target site for. DNA Spectrophotometer was used to achieve these readings. Table #4: Initial and Final Plasmid DNA Concentrations, Trial #2 Initial Reading (ng/µl) Final Reading (ng/µl) unc-119 sgRNA #2 A 168 248 unc-119 sgRNA #2 B 184 268 unc-119 sgRNA #2 C 172 N/A* *Since unc-119 sgRNA #2 C provided a faulty digest for Acl1, it was discarded and thus not used later on. Below, Table #5 illustrates the mutation results of Trial #2. Initially, 16 AF16 worms were microinjected, and 54 F1 worms that illustrated GFP expression were plated. Additionally, 84 worms that did not express GFP but resided on plates that contained GFP expressing worms were plated.. 6 plates of the F2 generation illustrated visible phenotypic unc-119 mutations. Additionally, both F1 plates with 1 worm ended up producing mutant F2 progeny. Two out of six F2 candidates produced unhealthy eggs and only two out of six candidates were sequenced (See Supplemental Table #2). Table #5: Mutation Results for Trial #2 Targeted Gene cbr-unc-119 P0 16 worms F1 54 worms* (GFP) 84 worms (non-GFP) F2 6 plates** 0 Efficiency 11-19% 0% *Cloned 2 worms per plate, but two plates had 1 worm/plate (see Supplemental Table #1). **Cloned 1 worm per plate. Adhikari 15 Trial #2: Alignment Data Below, Figure 1 shows the alignment between the wt DNA and F2-8-10-1 candidate for the unc-119 gene. The inserted sequence takes place 4 bp upstream of the PAM site and introduces a frameshift that leads to a premature stop codon.The inserted sequence is 16bp long the sequence itself matches part of the sgRNA target site. The insertion takes place immediately after a base pair mutation (cytosine to thymine). There were a few nucleotides that were able to be corrected by peak analysis of the DNA sequencing sheet. The key for the figure is shown below the figure. Figure 1: wt (AF16) and unc-10-1 DNA sequence alignment of unc-119 gene CLUSTAL 2.1 multiple sequence alignment GGCACCCTCTAATTACCATTTTCCTGTCACACCACACTCTTTTCCTTCTTCCACTTCTTT 60 ---------------------------NNNNNNNNNNTNTTTTCNTTCTTCN-CTTCTTT 32 * ***** ****** ******* CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA 120 CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA 89 ******* ******** * * ************************************* CCCG----------------GCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACT 164 CCCTCGGCGACCTTCCCGTCGCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACT 149 *** **************************************** AGAGAAACCGCTCAACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGT 224 AGAGAAACCGCTCAACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGT 209 ************************************************************ TGAACTTTTCCATATTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATC- 281 TGAACTTTTCCATATTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATCA 267 ********************************************************* Key for Figure 1 & 2: Yellow highlight = PAM Sequence Green Highlight = sgRNA #2 Target Site Red Text = Corrected Nucleotides Blue Text = Inserted Sequence wt unc-10-1 wt unc-10-1 wt unc-10-1 wt unc-10-1 wt unc-10-1 Adhikari 16 Figure 2: wt (AF16) and unc-13-2 DNA sequence alignment of unc-119 gene CLUSTAL 2.1 multiple sequence alignment GGCACCCTCTAATTACCATTTTCCTGTCACACCACACTCTTTTCCTTCTTCCACTTCTTT -----------------------------NNNNNNNNTCTTTTCCTTCTTNN-CTTCTTT ************* ******* wt CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA ******* ******** * * ************************************* CCCG--GCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACTAGAGAAACCGCTCA CCCAAAGCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACTAGAGAAACCGCTCA *** ****************************************************** ACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGTTGAACTTTTCCATA ACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGTTGAACTTTTCCATA ************************************************************ TTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATC- 281 TTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATCA 251 ******************************************* 60 30 wt unc-13-2 120 87 wt unc-13-2 178 147 wt unc-13-2 238 207 wt unc-13-2 wt unc-13-2 Above, Figure 2 shows the alignment between the wt DNA and F2-13-2-1 candidate for the unc-119 gene. The inserted sequence takes place 4 bp upstream of the PAM site and introduces a frameshift mutation that leads to a premature stop codon. The inserted sequence is a two base pair insertion. The insertion takes place immediately after a base pair mutation (cytosine to adenine). There were a few nucleotides that were able to be corrected by peak analysis of the DNA sequencing sheet. Figure 2 follows the same key as Figure 1 above. Adhikari 17 Discussion Trial #1 vs Trial #2 Efficiencies Looking at the results, Trial #1 proved to be unsuccessful due to a faulty target sgRNA of choice. None of the F2 progeny illustrated any mutations, and the 0% efficiency may be in part due to inability of CRISPR/Cas9 to recognize the target sequence on the gene. The problem was not due to a poor injection, since GFP expression was found in the F1 generation of worms (Table #2). None of the specific data (e.g. DNA concentrations, restriction digest) from this experiment was included in the results section since it was a faulty Trial. By changing the target sequence and having it end with a “GG” motif for Trial #2, there was success in generating the cbr-unc-119 mutation in six candidates in the F2 generation (Table #5). Only the forward primer was modified, since the location of the reverse primer was far enough downstream from the target. The efficiency had a range of 11-19% since we are unsure of whether both original F1 worms (on plates containing two F1 worms) gave rise to F2 unc-119 candidates. Although the efficiency for Trial #2 seems low, it is comparable to the efficiency of genetic engineering with CRISPR/Cas9 found in previous studies.2 Restriction Digest The restriction digest shown in Image 1illustrates the three transformed bacterial colonies that had the unc-119 sgRNA #2 inserted into the pUC57 plasmid. Looking at the Acl1 digest, unc-119 sgRNA #3 was unable to properly be digested by Acl1, and gave a band of 2.6kb when the digest was supposed to cut after 3.1kb. This may have been due to mutation in the plasmid, or contamination of the enzyme. As a result, unc-119 sgRNA #2 C was discarded from that point on and was not vacufuged (Table #4). The digest was also unsuccessful in the control, as the lin-10 Adhikari 18 plasmid was supposed to provide a band at 3.1kb but instead cut at 2.6kb. This is likely due to the old age of the plasmid. The HindIII and EcoR1 double digest, however, was successful for all three of the plasmids, and illustrates that the mutagenesis was successful for both A and B. Trial #2 Additional Analysis Looking at Table #5, we can conclude that the unc-119 mutation in C. briggsae is linked to GFP expression in F1 generation worms, since none of the non-GFP worms gave rise to mutant F2s. All of the candidates for the unc-119 mutation arose from the same original parental worm. This means that CRISPR/Cas9 mechanism may have only been successful in one of the injected worms (see Supplemental Figure #1). Additionally, two of the mutant candidates were unable to give rise to an F3 generation and had eggs that did not undergo proper gastrulation. This may have been due to an additional mutation caused by the CRISPR/Cas9 mechanism. Sequencing Analysis Two of the six candidates were able to be successfully sequenced. Figure 1 shows the sequencing data for F2-10-1. The inserted sequence is found in the sgRNA site, and the insertion takes place 4bp upstream of the PAM sequence. F2-13-2 also had an insertion 4bp upstream of the PAM sequences, illustrating that this recognition for the target site is highly conserved in C. briggsae. Both of the insertions led to a premature stop codon. The non-mutant F2s may have also had an altered DNA sequence that may have included silent mutations instead. In summary, different targeted modifications of gene sequences in C. briggsae were acquired through the CRISPR/Cas9 system of genetic engineering. An sgRNA site was chosen in the unc-119 gene that led to different insertions causing loss of function for the mutants. Adhikari 19 Pitfalls and Future Experimentation The timeframe of this project spanned over three and a half months, when it could have easily been completed in two. If it was known that an sgRNA site ending with a GG motif increases the efficiency of CRISPR/Cas9, this project could have been expanded to multiple genes of interest instead of just cbr-unc-119. Other barriers to completing the experiments on time included various places of contamination (on the agar plates, as well as with some of the reagents) as well as learning and reviewing some of the basic experimental techniques that I was more unfamiliar with (e.g. gel extraction, restriction digest). Additionally, it took the unc-119 mutants a longer amount of time to progress through their life cycle compared to the AF16 worms, which made things a little bit slower as well. For future experimentation, I would investigate other genes that are similar in function between C. elegans and C. briggsae and involved with developmental pathways such as Wnt and Ras. I would also further investigate the F2 candidates that produced sick eggs, and see whether CRISPR/Cas9 was able to introduce side effect mutations as well as the expected insertion to these worms. By expanding this effort towards other genes shared by C. elegans and C. briggsae, the evolutionary divergence between these two species can be better understood and mapped more accurately. Future studies can also focus on genes that are conserved to humans, to see if similar effects of genetic modifications can be applied to these same pathway genes found in humans. Adhikari 20 Supplementary Tables Supplementary Table #1: F1 Plates (GFP Expression) F1 (GFP) Plate Notes F1 (GFP) Plate F1-1-1 F1-8-1 F1-1-2 F1-8-2 F1-1-3 F1-8-3 F1-2-1 F1-8-4 F1-7-1 F1-8-5 F1-7-2 F1-8-6 F1-7-3 1 sick worm F1-8-7 F1-7-4 F1-8-8 F1-7-5 F1-8-9 F1-7-6 F1-8-10 F1-7-7 F1-8-11 F1-7-8 F1-8-12 F1-7-9 F1-8-13 F1-14-1 F1-14-2 Notes 1 sick worm 1 worm 1 worm The table above shows a more detailed description of the identity of each of the F1 GFP plates. Each plate had 2 worms each, except for F1-8-11 and F1-8-13 Two of the plates had 1 sick worm each. The highlighted plates are the ones that produced mutant F2s. Additionally, it is important to note that GFP-expressing worms were only found from 5 of the 14 parents (P1,P2,P7,P8,P14), and each of the mutant candidates arose from the same injected parental worm (P8). Supplementary Table #2 below shows the identity of each of the picked non-GFP expressing worms. Adhikari 21 Supplementary Table #2: F1 Plates (Non-GFP Expression) Notes Notes F1 (non-GFP) F1 (non-GFP) Plate Plate F1-1-1 F1-8-1 F1-1-2 F1-8-2 F1-1-3 F1-8-3 F1-1-4 F1-8-4 F1-1-5 F1-8-5 F1-2-1 F1-8-6 F1-2-2 F1-8-7 F1-2-3 F1-8-8 F1-2-4 F1-8-9 F1-2-5 F1-8-10 F1-7-1 F1-8-11 F1-7-2 F1-8-12 F1-7-3 F1-14-1 F1-7-4 F1-14-2 F1-7-5 F1-14-3 F1-7-6 F1-14-4 F1-7-7 F1-14-5 F1-7-8 F1-14-6 F1-7-9 F1-14-7 F1-7-10 F1-14-8 F1-7-11 F1-14-9 Supplementary Table #3 below shows each of the F2 candidates that were picked (1 per plate). Candidates from F1-8-7 and F1-8-8 produced sick eggs and were unable to produce any offspring. Candidates F2-8-10-1 and F2-13-2 were sequenced. Supplementary Table #3: F2 Candidates F2 (unc-119) Plates Notes F2 (unc-119) Plates F2-8-1-1 F2-8-8-4 F2-8-1-2 F2-8-10-1 F2-8-1-3 F2-8-10-2 F2-8-1-4 F2-8-10-3 F2-8-7-1 Sick Eggs F2-8-10-4 F2-8-7-2 Sick Eggs F2-8-11-1 F2-8-8-1 Sick Eggs F2-8-11-2 F2-8-8-2 Sick Eggs F2-8-13-1 F2-8-8-3 Sick Eggs F2-8-13-2 Notes Sick Eggs Sequenced Sequenced Adhikari 22 References: 1. Barrangou, Rodolphe, et al. "CRISPR provides acquired resistance against viruses in prokaryotes." Science 315.5819 (2007): 1709-1712. 2. Friedland, Ari E., et al. "Heritable genome editing in C. elegans via a CRISPR-Cas9 system." Nature methods 10.8 (2013): 741-743. 3. Maduro, Morris, and David Pilgrim. "Conservation of function and expression of unc-119 from two Caenorhabditis species despite divergence of non-coding DNA." Gene 183.1 (1996): 77-85. 4. Maduro, Morris F., et al. "The UNC-119 family of neural proteins is functionally conserved between humans, Drosophila and C. elegans." Journal of neurogenetics 13.4 (2000): 191-212. 5. Miller, Jeffrey C., et al. "An improved zinc-finger nuclease architecture for highly specific genome editing." Nature biotechnology 25.7 (2007): 778-785. 6. Sander, Jeffry D., et al. "Selection-free zinc-finger-nuclease engineering by contextdependent assembly (CoDA)." Nature methods 8.1 (2011): 67-69. 7. Christian, Michelle, et al. "Targeting DNA double-strand breaks with TAL effector nucleases." Genetics 186.2 (2010): 757-761. 8. Gaj, Thomas, Charles A. Gersbach, and Carlos F. Barbas. "ZFN, TALEN, and CRISPR/Casbased methods for genome engineering." Trends in biotechnology31.7 (2013): 397-405. 9. Ran, F. Ann, et al. "Genome engineering using the CRISPR-Cas9 system."Nature protocols 8.11 (2013): 2281-2308. 10. Dong, L. H., et al. "Plasmid pUC57-BT11 construction and application of genetic modified maize line BT11." Acta Metrologica Sinica 32.6 (2011): 1-5. 11. Tzur, Yonatan B., et al. "Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR–Cas9 system." Genetics 195.3 (2013): 1181-1185. 12. Farboud, Behnom, and Barbara J. Meyer. "Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design." Genetics 199.4 (2015): 959-971. Adhikari 23 13. Zhao, Zhongying, et al. "New tools for investigating the comparative biology of Caenorhabditis briggsae and C. elegans." Genetics 184.3 (2010): 853-863. 14. Sander, Jeffry D., et al. "Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool." Nucleic acids research 35.suppl 2 (2007): W599-W605. 15. Sherry, Stephen T., et al. "dbSNP: the NCBI database of genetic variation."Nucleic acids research 29.1 (2001): 308-311. 16. Altschul, Stephen F., et al. "Basic local alignment search tool." Journal of molecular biology 215.3 (1990): 403-410. 17. Waaijers, Selma, et al. "CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans." Genetics (2013): genetics-113. 18. Walquist, Mari, Elisabeth Kjeldsen Buvang, and M. Raafat El‐Gewely. "Mutagenesis: Site‐Specific." eLS. 19. Wilson, Kate. "Preparation of genomic DNA from bacteria." Current protocols in molecular biology (1987): 2-4. 20. Friedland, Ari E., et al. "Heritable genome editing in C. elegans via a CRISPR-Cas9 system." Nature methods 10.8 (2013): 741-743. 21. Mello, Craig C., et al. "Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences." The EMBO journal10.12 (1991): 3959. 22. Liu, Leo X., et al. "High-throughput isolation of Caenorhabditis elegans deletion mutants." Genome research 9.9 (1999): 859-867. 23. Barnes, Wayne M. "PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates." Proceedings of the National Academy of Sciences 91.6 (1994): 2216-2220. 24. Vogelstein, Bert, and David Gillespie. "Preparative and analytical purification of DNA from agarose." Proceedings of the National Academy of Sciences 76.2 (1979): 615-619. 25. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. (2011) Molecular systems biology 7 :539