https://openworks.mdanderson.org/sumexp21/1157/thumbnail.jp

Araujo, Jose

Arur, Swathi

Trimmer, Kenneth

English

Arur, Swathi, PhD

OpenWorks @ MD Anderson

Adapted from http://www.wormbook.org/chapters/www_meiosis/meiosis.htmlCrossover FormationMaturationFertilizationUterus1) Yokoo et al. Cell 2012; 149:75-872) Goudeau et al. Genetics 2021; 217: 43) Paix et al. Genetics 2015; 201:1:47-54Insertion of WrmScarlet11 into cosa-1 gene in C. elegans using CRISPRJose Araujo1,2, Kenny Trimmer1, Swathi Arur11University of Texas MD Anderson Cancer Center, Genetics Department2University of Texas MD Anderson Cancer Center, Faculty Diversity, Equity and Inclusion Department(A) A precise injection into the germline of C. elegans. Theinjection mixture is injected into the distal arm of the germline andwill disperse evenly in opposite directions, like shown with thearrows. (B) A photo of me performing precise and quick injectionsand adjusting the focal plane to distinguish the germline.(C) Asuccessful injection into the germline using a fluorescent label(blue) to visualize the result of an injection.- W- I- I Ihttp://www.wormbook.org/chapters/www_transformationmicroinjection/transformationmicroinjection.htmlA. B.C.(A) This worm displays a rolling phenotype (a helicallytwisted body). This worm is identifiable by its rollingmovement and the circle traces that it leaves behind. (B)This picture is showing a comparison between adumpy(left, short and wide) and a regular wildtype worm(right, long and skinny). (C) Worms cloned out from platescontaining dumpys and rollers were ran on the depicted gel.Worms marked with a star(*) appeared to beheterozygous/homozygous for wrmScarlett. The ladder islabeled with different band lengths and the length of aninsertion (I) and a wildtype worm (W) are also included.A BN23-13-23-33-43-53-63-73-83-93-10*3-113-123-133-143-163-173-183-24500600700800900100012001500400300200100N23-15*3-19*3-20*3-21*3-223-233-253-263-273-283-293-303-313-323-333-343-353-363-373-383-393-403-41100200300400500600700800900100012001500-W - I N23-423-433-443-453-463-473-483-493-503-513-52*3-533-543-553-563-573-583-593-603-613-623-633-64600700800900100012001500400300200100- W500Chttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1214229/http://www.faculty.ucr.edu/~mmaduro/oldnews.htmGel imaging of the second generation of worms (F2s) thatwere cloned from F1 worms that demonstrated awrmScarlet11 insertion. A total of 43 samples were screenedto detect a band length of 648 base pairs. The labels thatinclude a star (*) are the ones that showed promising bandlength due to bright white marks appearing in the 600-700bpladder length.Gel imaged from large scale PCR of worms that werepromising to have the wrmScarlet11 insertion. These wormswere picked out from plates of F2s that appeared to have theinsertion. The ones that have a star (*) label on them werethought to be homozygous for wrmScarlet11.Sequencing results from our 6 promising worms. In the insertionsite, there was a gap in the sequence traces; thus, demonstratingthe absence of nucleotides(left rectangle), and a nucleotide changewas also apparent in some sequences(right rectangle).A BShown above is the fluorescent protein wrmScarlet as viewed from the front(A) and top (B). It is made up of 11 beta strands and a fluorescent functionalgroup. In order to fluoresce, all the beta strands must be present in a betabarrel shape. However, beta strand 11 (rainbow) can be on a different proteinand still form the beta barrel with 1-102. Therefore, we can tag COSA-1 withthe 11th strand in an animal that expresses wrmScarlet1-10 constitutively,localizing fluorescence to COSA-1, and therefore to crossover sites. Toinsert the 11th beta strand into the cosa-1 gene, I used CRISPR .Background/IntroductionThe desired insertion of wormScarlet11 (red) into the cosa-1 gene (green) isshown above. The primers used for genotyping and sequencing are shownby the arrows and the expected band lengths are written above the primers.CRISPR-Cas9 can locate a target DNA sequence using a guide RNA and cut it.This induces the cell to repair its DNA. There are two types of DNA repair:NEHJ and HDR. While NEHJ just sticks the broken ends of DNA backtogether, HDR looks for donor DNA that is similar around the cut region anduses it as template for repair. By making a repair template that includeswrmScarlet11 along with some homologous sequence to the cosa-1 gene, HDRuses it as donor DNAand inserts wrmScarlet 11 into cosa-1, as shown below.https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biologyThe C. elegans germline is a U-shaped tissue with mitosis occurring distalfrom the uterus and meiosis proceeding as cells move proximally, as seen inthe drawing above. Meiosis encompasses a number of different stages:pairing of chromosomes, crossover formation, chromosome remodeling, andgerm cell maturation. During crossover formation, COSA-1licencescrossovers between paired chromosomes1. To visualize crossovers in vivo, Iattempted to insert part of a fluorescent protein into cosa-1.WA comparison of a hermaphrodite (top, slim tail) and a male(middle, blunt tail with fan) found in my cloned-out plates.ConclusionsFuture Directions • We will determine why the insertion was not as weexpected.• We will reinject with corrected injection mix and screen to generate the correct insertion.• I got worms with insertions in cosa-1• A frame shift occurred due to the deletion of twonucleotides resulting in loss of function in cosa-1• Loss of function in cosa-1 led to having more males• Some isolates had a nucleotide change resulting in a missense mutationcosa-1cosa-1WS11506 base pairs(bp)648 base pairs(bp)BeforeAfterFirst, into the distal arm of the germline, I injected amixture containing CRISPR-Cas9 protein and a guideRNA and repair template for cosa-1. As part of acoCRISPR strategy, the injection mixture alsocontained a guide RNA and repair template for dpy-10which would produce a mutation which results inrollers when heterozygous and dumpy rollers whenhomozygous3. I incubated the injected worms andwaited for progeny (F1).Progeny from F1 (F2s) were cloned out, incubatedand then genotyped. Four F2 worms contained asingle white mark between the 600bp and 700bpsuggesting that they were homozygous.N23-523-21-3*3-21-43-21-8500600700800900100012001500400300200100W I3-10-2*I looked at the four homozygous lines, and I sawmore males than wildtype. This phenotype hintedat a loss of function in cosa-1. Since cosa-1 isrequired for crossovers, and crossovers hold thepaired chromosomes together for the first division,an absence of cosa-1 might result in chromosomenondisjunction1. This may in turn result in someprogeny receiving one X chromosome, resulting inmales. This suggested that the insertion may not beas I had planned, so I prepared to sequence the 4homozygous lines.I obtained the results from the worms that weresequenced. In 3 of them a missense mutationoccurred. However, in all of them I saw the deletionof two nucleotides. This would result in a frameshift, changing the amino acid sequence andconfirming that we had produced a loss of function.From each homozygous line of worms, I amplifiedthe DNA in preparation for sequencing. Since most ofthe lines had multiple DNA bands and we were onlyinterested in the 648bp band, the PCR product of eachline was run on a gel, the correct band was cut out,and the DNA was purified before sending it forsequencing.ResultsProgeny(F1) that were rollers and dumpys had beenCRISPR-edited for dpy-10 in the mother, so they werecloned into individual plates to determine whether theywere also CRISPR-edited for cosa-1. Then I incubatedthe F1s and waited for more progeny. I genotyped theF1s and found that six lines had potential insertions.Progeny of these F1s were followed up.N25006007008009001000120015004003002001005004003002001006007008009001000120015003-10-13-10-2*3-10-33-10-43-10-53-10-63-10-73-10-83-10-93-10-103-19-13-19-23-19-33-19-43-19-53-19-63-19-73-19-83-19-93-19-103-20-13-20-23-20-3N23-20-43-20-53-20-63-20-73-20-83-20-93-20-103-21-13-21-23-21-3*3-21-4*3-21-53-21-63-21-73-21-8*3-21-93-21-103-52 W I IReferencesAcknowledgementsThank you to my mentor Kenny for helpful discussions

Insertion of wrmScarlet11 into cosa-1 gene in C. elegans using CRISPR

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1157&context=sumexp21

Insertion of wrmScarlet11 into cosa-1 gene in C. elegans using CRISPR

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