EXAMENSARBETE INOM BIOTEKNIK, AVANCERAD NIVÅ, 30 HP STOCKHOLM, SVERIGE 2017

Temporal regulation of neural stem cells during cortex development

AMICA JOHANSSON

KTH SKOLAN FÖR BIOTEKNOLOGI !

Temporal regulation of neural stem cells during cortex development

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Thesis for master level degree

Amica Johansson KTH Royal Institute of Technology

Abstract(

The emergence and evolution of the cerebral cortex in humans has been critical for the development of complex cognitive, perceptual and emotional abilities. In mammals, the cortex is a multi-layered structure organized in six layers of unique neuronal subpopulations; each with distinct nerve fiber organization and biological function. Interestingly, neurons of the distinct cortical layers are generated from the same stem cell population in a very predictive temporal order during cortex formation in the developing embryo. This fascinating developmental process exemplifies how the competence of a specified stem cell population changes over time. However, the processes and key regulators behind the temporally controlled layer formation of the cortex are poorly understood. This project aimed to investigate the competence of early and late cortical stem cell populations and to investigate the necessity of gene regulating enhancer regions to genes that are known to be characteristically expressed by early and late neuronal subpopulations. By studying data from Sox2 ChIP-sequencing and DNase-sequencing on early and late cortical stem cells, interesting enhancer regions to the genes Fezf2 (mainly expressed by neurons developed early in corticogenesis) and Nfix (mainly expressed by neurons developed late in corticogenesis) were selected for investigation. By utilizing the CRISPR/Cas9 system, the selected enhancer regions to Fezf2 and Nfix were knocked out, respectively. gRNAs were designed to target the enhancers and co-expressed together with Cas9 endonuclease in the PX333 plasmid. The enhancer knockout-system was first tested in vitro on P19 cell lines and successful genome editing was confirmed by using PCR followed by gel electrophoresis. The CRISPR/Cas9 constructs were subsequently tested in vivo on embryonic mouse cortices by in utero electroporation. By using immunofluorescence and confocal microscopy for detection, it was possible to show that the in vivo Fezf2- enhancer knockout resulted in a significant decrease of Fezf2 gene expression compared to the control. Furthermore, the Fezf2- enhancer knockout led to a significant reduce in expression of the late- cortical layer marker Ctip2 as well as an increase in expression of the early layer marker Satb2. Unfortunately, the staining results for the Nfix-enhancer knockout were unsatisfactory due to uninformative immunofluorescence staining. ! !

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! 2! Sammanfattning(

Uppkomsten och evolutionen av hjärnbarken hos människor har varit avgörande för utvecklingen av komplexa kognitiva, emotionella och perceptuella förmågor. I däggdjur är hjärnbarken en flerskiktsstruktur organiserad i sex lager av unika subpopulationer av neuroner; var och ett med distinkt biologisk funktion och organisation av nervfibrer. Utmärkande är att neuroner tillhörande de olika skikten utvecklas från samma stamcellspopulation i en förutsägbar temporärt kontrollerad ordning under den embryonala utvecklingen. Denna fascinerande utvecklingsprocess åskådliggör hur kompetensen hos en specifik stamcellspopulation förändras över tid. Dock är bakgrundsprocesserna och de regulatoriska elementen bakom den temporärt kontrollerade utvecklingen av hjärnbarken dåligt förstådda. Syftet med detta forskningsprojekt var att utforska nödvändigheten av genreglerande enhancer-regioner till gener som är kända för att vara karaktäristiskt uttryckta i neuroner som bildas tidigt respektive sent under hjärnans utveckling. Genom att analysera data från Sox2 ChIP- sekvensering och DNase-sekvensering, utfört på tidiga och sena neurala stamceller i hjärnbarken, valdes intressanta enhancer-regioner ut till generna Fezf2 (karaktäristikt uttryckt av tidigt utvecklade neuroner) och Nfix (karaktäristiskt uttryckt av sent utvecklade neuroner). Betydelsen av de två utvalda enhancer-elementen för genuttryck studerades genom att klyva ut respektive korresponderande DNA sekvens med CRISPR/Cas9. gRNA-oligonukleotider designades mot enhancer-sekvenserna och uttrycktes tillsammans med Cas9 i plasmidvektorn PX333. Strategin för enhancer-klyvning testades först in vitro på P19 cellinjer där lyckad CRISPR-redigering kunde bekräftas med PCR följt av gel- elektrofores. Därefter undersöktes CRISPR-konstrukten in vivo på musembryon genom in utero elektroporering av hjärnbarken. För att undersöka hur avlägsnandet av enhancer-regionerna påverkade uttrycket av Fezf2 och Nfix, respektive, användes immunfluorescens och konfokalmikroskopi för detektion. CRISPR/Cas9- inducerad knockout av enhancer-regionen till Fezf2 resulterade i signifikant minskning i genuttryck av Fezf2 jämfört med kontrollproverna. Dessutom sågs även en kraftig reducering i genuttrycket av Ctip2 (karaktäristiskt uttryckt av neuroner i de nedre hjärnbarkslagren) samt en kraftig ökning i genuttrycket av Satb2 (karaktäristiskt uttryckt av neuroner i de övre lagren). Resultaten från avlägsnandet av Nfix-enhancern in vivo var tyvärr svåra att tyda på grund av svåravlästa immunofluorescence-resultat.

! 3! Table(of(contents((

1.! Introduction and aims (5) 1.1. The cerebral cortex is a multi-layered structure (5) 1.2. Neuronal differentiation and layer specification is temporally regulated (6) 1.3. Sox2 binding to genes in NPCs used to identify gene regulating enhancers (7) 1.4. CRISPR/Cas9 as strategy for enhancer knockout (8)

2.! Materials and methods (10) 2.1. Identification of enhancers to target (10) 2.2. Design of gRNAs to generate enhancer knockouts (11) 2.3. Cloning of gRNAs into PX333 plasmid (12) 2.3.1. Cleavage reaction (12) 2.3.2. Annealing and ligation reaction (13) 2.3.3. Transformation (14) 2.3.4. Sanger sequencing to confirm successful cloning (14) 2.4. PCR optimization (14) 2.5. Transfection of P19 cells with PX333 CRISPR constructs (14) 2.6. PCR and gel electrophoresis to confirm CRISPR genome editing (15) 2.7. Cloning both enhancer-targeting gRNAs into the same plasmid (17) 2.8. In utero electroporation of mouse embryos with CRISPR plasmids (18) 2.9. Immunofluorescence and confocal microscopy analysis (18)

3.! Results (20) 3.1. Identification of enhancer regions to target (20) 3.2. PX333 plasmid linearization (23) 3.3. Cloning results (23) 3.4. Sanger sequencing results (single gRNA inserts) (24) 3.5. PCR optimization (27) 3.6. PCR results after transfection with combinations of single gRNA plasmids (27) 3.7. Sanger sequencing results (single gRNA inserts) (28) 3.8. PCR results after transfection (PX333 with double gRNA inserts (29) 3.9. In vivo results after in utero electroporation (30)

4.! Discussion (35)

5.! Bibliography (37) !

! 4! 1.!Introduction(and(aims(

Ever since the identification and staining of the neuron by Ramón y Cajal and Camillo Golgi over a century ago, the field of neuroscience has come a long way and our understanding of the complex human brain is continuously improving. New insights about how pluripotent embryonic stem cells are regulated to generate the diverse pool of neurons – the electrically excitable cells that constitute the nervous system - will greatly contribute to a better understanding about how the nervous system functions to transmit, integrate and process information throughout the entire body to coordinate movement, thought and record sensations. Understanding how the healthy nervous system is developed is further important for understanding the mechanisms of neurodegenerative disorders and diseases related to the nervous system. The aim of this research project is to study the temporal regulation of cortex development in the developing embryo. The focus is shed on investigating the competence of early and late neural progenitor cells (NPCs) during corticogenesis and aims to investigate regulatory DNA elements of the complex corticogenesis process, in which six different neuronal subpopulations are formed at different time points from the exact same stem cell population.

1.1.! The&cerebral&cortex&is&a&multi3layered&structure&& & The human brain is one of the most complex organs in the body. It is the main core of the CNS in humans and can be regarded as the control center of the entire body. The mature CNS consists of the nerves in the spinal cord and the brain and comprises approximately 100 billion neurons that are central for information processing, a sum which can be compared to the amount of stars in our galaxy.1 The cerebral cortex is the folded outermost layer that surrounds the brain hemispheres, as illustrated in Figure 1. It takes up about three quarters of the brain volume and is built up by a large diversity of cells; containing neurons of hundreds of different cell types as well as a wide variety of different glia cells. The cortex operates as a basis for cognitive as well as sophisticated perceptual functions such as consciousness, reasoning and thought. Interestingly, the mature human cortex contains unique subpopulations of neurons organized in six different layers. Each of the six layers encompasses unique neuronal density, size and shape as well as a distinct nerve fiber organization, as illustrated in Figure 1. Thus, neurons in the different layers further show different biological function.1, 2

Figure!1.!

Figure 1. Basic overview of the cerebral cortex organization in the human brain. The picture to the right shows an overview of the localization and neuronal organization of the six different cortical layers, from upper layers at the top to the deeper layers at the bottom.3

1 http://www.human-memory.net/brain_neurons.html 2 https://academic.oup.com/cercor/article/13/6/607/360931/Neuronal-Migration-in-the-Developing-Cerebral 3 http://vanat.cvm.umn.edu/brain18/images/optNeocortexLayers.jpg

! 5! 1.2.! Neuronal&differentiation&and&layer&specification is&temporally®ulated& & Interestingly, during embryonic development, the six different neuronal layers of the cortex are generated in a characteristic temporal order from the same population of stem cells that are located in the ventricular zone of the brain, as illustrated in Figure 2 below. In this temporally regulated process of neurogenesis, the deep layer neurons are generated first and the upper layer neurons are generated last. Accordingly, later developed neurons must migrate past the deep layer neurons in order to make up the upper cortical layers. The generation of differentiated neurons primarily takes place during embryonic development.2, 4 Neurons are developed from self-renewing progenitor cells located in the ventricular zone (VZ) of the CNS. The process begins with the initiation of embryonic stem cells to begin specializing into multipotent neural progenitor cells. During this developmental process, the stem cells must exit the cell cycle and begin migrating out from the VZ to reach the marginal zone (which will make up the six cortical layers) of the brain and begin to express neuronal markers.4, 5

The formation of the multi-layered mammalian cortex is thus a fascinating example illustrating how the competence of a defined stem cell population changes over time during development. One interesting question in research concerns how the generation of these various subtypes of neurons are developed from the same stem cell population, and how this process is temporally regulated during corticogenesis in the developing brain.

Figure!2!

Figure 2. Neuronal cell type specification is temporally regulated in the developing cortex. Neurons of the six different cortical layers are generated at different time points from the same stem cell population located in the ventricular zone (VZ). The first neurons of the deep layers are born on embryonic development day 11 (E11).5

In order to investigate the temporal control and regulation of neuronal cell type specification, it is interesting to study the regulation of gene expression, which differs between the different types of neurons in the upper vs the deep layers. Interestingly, some genes that demonstrate layer- as well as neuronal subtype-specificity have been identified 4. These discoveries have made it possible to more properly investigate the underlying mechanisms behind the characterization of the distinct subtypes of neurons. Thus, to further investigate cortex development in this manner, it would be of great interest to study regulatory elements of identified genes that are characteristic for the different subpopulations, respectively. Discovering regulatory elements that are critical for gene expression of upper vs. deep layer genes would greatly increase our understanding about how the process of corticogenesis is regulated.

4 http://www.nature.com/neuro/journal/v6/n11/full/nn1131.html 5 http://www.nature.com/nrn/journal/v8/n6/full/nrn2151.html

! 6! In this project, it was decided to investigate the regulatory elements of gene expression of two layer specific genes; one that is expressed early and one that is expressed late in cortex development. The first gene to investigate in this project was Fezf2 (Family Zink Finger 2), which is characteristically expressed by deep layer neurons.6 Fezf2 is thus mainly expressed early in cortical development. It has been shown that inactivation of Fezf2 disturbs the the specification of deep layer neurons as well as the formation of corticospinal axons.6 Since it is known that Fezf2 is a clear marker for deep layer neurons, it was of great interest to study and define regulatory elements of Fezf2. The second gene decided to investigate was the upper layer gene Nfix (Nuclear factor one X). This gene is characteristically expressed later in cortex development by neurons of the upper cortical layers.7

1.3.! Sox2&binding&to&genes&in&NPCs&used&to&identify&gene®ulating&enhancers& & A regulatory enhancer region can be defined as a sequence of DNA that can be bound by activating transcription factors to increase the likelihood of transcription of a certain gene8. The aim with this project is to study the importance of gene regulating enhancer regions for neuronal cell type specification in the developing cortex. More specifically, the main aim in this project was to investigate the necessity of gene regulating enhancer regions to genes that are characteristically expressed by early and late neuronal subpopulations. In order to identify critical enhancer regions to the selected genes of interest (Fezf2 and Nfix), transcription factor binding was studied. Transcription factors can be described as proteins that control the rate of transcription, and regulates gene expression by communicating and controlling the transcription process, in which a DNA encoding gene is converted to mRNA9. These regulatory proteins have DNA-binding domains and are thus are able to bind to specific sequences of DNA in regulatory regions, such as to enhancer regions.8, 9

Interestingly, transcription factors belonging to the SOX family of transcription factors have been shown to serve an important key role in the regulation of neural lineage formation. Characteristic for the proteins belonging to this large family is the presence of a so called high-mobility group-box, HMG-box, important for DNA binding. SOX proteins have been highly conserved throughout evolution.10 One example is the transcription factor Sox2, which shows as much as 98% sequence similarity between the human and mouse protein. SOX proteins are affecting gene regulation by affecting the structure of the chromatin, but also through interaction with partner proteins that affects the activity and also the main target gene selection.10, 11

Sox2 is a well known key regulator of neurogenesis and in the regulation of stem cell characteristics. It has been shown that loss of Sox2 in the mouse brain leads to premature differentiation of NPCs, and overexpression of Sox2 instead keeps the NPCs in a slowly dividing state, characteristic for stem cells.10, 12Interestingly, it has been shown that Sox2 pre-binds to many neuronal genes in NPCs at different time points during corticogenesis. It has further been shown that the NPC genes bound by Sox2 are later bound and activated by other transcription factor proteins in differentiating neurons. One can see it as Sox2 is marking these NPC genes which subsequently become destined to be activated by other proteins, and thereby also commit to a certain neural lineage. The genes in NPCs that are bound by Sox2 are not expressed until the NPC has developed into a mature neuron and Sox2 is at that stage no longer present.13There are thus reasons to state that Sox2 binding to NPC genes is important for the temporal regulation of neuronal subpopulation formation. However, exactly how this

6 Molyneaux et.al., Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47, 817–831 (2005) 7 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776400/ 8 https://www.nature.com/scitable/definition/enhancer-163 9 https://www.nature.com/scitable/definition/transcription-factor-general-transcription-factor-167 10 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106650/ 11 http://www.scielo.br/pdf/zool/v26n1/a17v26n1 12 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106650/ 13 H. Kondoh et. al., Sox2: Biology and Role in Development and Disease, page 149

! 7! process is regulated is unknown. Thus, the strategy to investigate the competence of early and late neuronal subpopulations in this project was to study Sox2 transcription factor binding of enhancer regions to genes characteristic for upper vs deep layer neurons. By studying Sox2 ChIP-sequencing data as well as DNase sequencing data performed on early and late cortical stem cells, Sox2 binding sites as well as chromatin accessibility on Fezf2 and Nfix genes could be identified. In this way, interesting enhancer regions to both genes were selected, respectively.

Notably, a critical enhancer region to the Fezf2 deep layer gene identified on the Sox2 ChIP- sequencing data has already been discovered by N. Sestan et. al, published in Nature 2012. By generating a knockout mouse in which the whole enhancer region, called E4, was removed, the researchers were able to show a loss of Fezf2 expression and corticospinal axons in E4-/- mice. They could further show that Fezf2 is important for the formation of deep layer neurons.14According to these findings, it was of great interest to identify and knock out the same enhancer, as a control. If similar results could be generated by knocking out E4, the designed knock-out system in this project would give a higher credibility to the results generated when knocking out the selected enhancer region for Nfix, as well as other regulatory regions in the future.

In order to study the necessity of the selected enhancers in cortical patterning, the strategy was to knockout the enhancer regions to which Sox2 binds by using the CRISPR/Cas9 system; to first design, then clone and confirm guide RNAs (gRNAs), test the system on cell lines and subsequently deliver the CRISPR constructs to the cortices of mouse embryos by in utero electroporation. By knocking out the selected enhancers to Fezf2 and Nfix, the expectation was to see a significant reduce in gene expression of both genes, respectively. Since it is known that Fezf2 and Nfix are characteristically expressed by deep layer neurons and upper layer neurons, respectively, is was further expected to see that neurons of the layers were these genes usually are highly expressed cannot form properly.

1.4.! CRISPR/Cas9&as&strategy&for&enhancer&knockout& & Techniques that are capable of generating targeted and highly specific genome alteration are important for revealing physiological patterns and mechanisms in the body as well as for understanding pathological systems of disease. One technique that has revolutionized the research community in the recent years is the CRISPR/Cas9 system. This system of Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, has been developed for proper programming and targeting of nucleases to enable precise and highly specific editing of endogenous DNA in a wide range of different biological systems.15In this project, the CRISPR technique will be used in order to delete the enhancer regions to the two genes Fezf2 and Nfix, respectively.

The CRISPR/Cas system is a prokaryotic adaptive immune system originally discovered in E.coli bacteria. This system is built up by segments of DNA containing short palindromic repetitive sequences that are identical to each other. Interestingly, each repetition is interspaced by so called spacer DNA, as shown in Figure 3 below. The spacer DNA sequences are all unique and represent exogenous DNA from earlier exposures to foreign DNA, e.g. from virus infections. Accordingly, this prokaryotic immune system allows for rapid recognition and response to repetitive infections by previously encountered infectious agents. Upon a secondary infection, the information from the associated spacer DNA sequence will be used to form so called guideRNA (gRNA). This gRNA will serve to guide the Cas nuclease to disrupt the inserted nucleic acid from the infectious agent. Together, the repetitive sequences along with the unique spacer DNA sequences make up the so called CRISPR array, or crRNA array. Each crRNA segment will thus contain a repeat sequence together with a 20-

14 hhttp://www.nature.com.focus.lib.kth.se/nature/journal/v486/n7401/full/nature11094.html 15 https://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html

! 8! nucleotide gRNA sequence. Next to the CRISPR region are cas genes localized. These represent helicases needed for DNA unwinding of the target sequence as well as the Cas-nuclease for DNA cleavage.15, 16, 17

Figure!3!

Figure 3: A: Schematic overview of the CRISPR/Cas adaptive immune system in bacteria. The three white segments to the left represent Cas genes and the blue, red and green segments correspond to spacer DNA from previous exposures to foreign DNA. Upon virus infection, the viral DNA is identified by the bacteria and inserted in the crRNA array region as a unique spacer DNA. B: Upon re-infection with the same type of virus, the bacteria can easily recognize the exogenous DNA and activates a directed immune response towards the virus by co-expressing the cas nucleases along with the gRNA sequence to disrupt the viral DNA.

The targeted genomic cleavage requires two main elements; the Cas9 endonuclease as well as small gRNAs. For the system to function, the Cas9 nuclease must be guided to the genomic sequence of interest by the gRNA through homologous base pairing with the target sequence. It is thus possible to design the 20 nt long gRNAs to target any desired location in the genome. Cas9 then allows for genome editing by generating a targeted double strand break, DSB, in the target DNA sequence. The CRISPR/Cas9 system can be designed in a plasmid in which the gRNAs are co-expressed together with Cas9 in a plasmid. Upon induced DSB in the genomic DNA, the cell will either repair the DNA through the less frequent DNA repair pathway homology directed repair (HDR) or non-homologous 18 end joining (NHEJ).

The strategy in this project was to induce two double strand breaks on each end of the selected enhancer regions to the Fezf2 and Nfix genes, respectively, and thereby knockout the enhancer regions for both genes, as illustrated in Figure 4. The ultimate goal was to test the designed enhancer targeting CRISPR constructs in mouse cortex and then study if gene expression was altered. However, before exposing mice to the CRISPR constructs by in utero electroporation, the system had to be tested and confirmed successful on P19 cell lines.

! ! ! ! ! ! ! !

16 https://www.nature.com/nature/journal/v526/n7571/full/nature15386.html 17 http://science.sciencemag.org/content 18 https://www.nature.com/articles/srep37895

! 9! ! Figure!4!

Figure 4: Basic illustration of enhancer knockout strategy by using the CRISPR/Cas9 system. By using CRISPR/Cas9, it is possible to generate targeted double strand breaks (DSBs) in the genomic DNA. This system was thus used in order to generate the enhancer deletions for enhancers to Fezf2 and Nfix. 2.!Materials(and(methods(

2.1.! Identification&of&enhancers&to&target&

The strategy in this project was to use the CRISPR/Cas9 system to knock out enhancers to genes that are known to be characteristic for upper vs. deep-layer neuronal subpopulations, in order to study their necessity in corticogenesis. As mentioned in the introduction section, the deep layer gene Fezf2 (expressed by neurons of the deep layers V and VI) was chosen, as well as the upper-layer gene Nfix (mainly expressed by later born neurons of the upper cortical layers). Figure 5 shows an overview of the project workflow.

Figure!5!

Identification!of! in vitro:& Cloning!of!gRNAs! enhancers!to! Design of gRNAs Transfection!of! into!vectors knock!out P19!cells

Immunostaining In vivo:&in&utero& Confirm to!study!gene! electroporation!of! CRISPR/Cas9! expression mice editing!with!PCR

Figure 5. Basic overview of the project workflow

In order to start the project, enhancer regions which indicated to be important for gene expression of Fezf2 and Nfix had to be identified, respectively. As mentioned in the introduction section, Sox2 binding sites were studied to find interesting enhancer regions to knock out for both genes, respectively. This was possible since Sox2 ChIP-sequencing as well as DNase-sequencing on early and late cortical stem cells had been performed by D. Hagey. et. al. prior to this project.

As described in the introduction section, an enhancer region, E4, to the deep layer gene Fezf2 had already been discovered and proven to be important for Fezf2 expression and deep layer formation14. It was decided to search for the same enhancer region in the Sox2 ChIP-sequencing -and the DNase- sequencing data, and knock the same region out as a positive control. Since Fezf2 is mainly expressed by deep layer neurons and thus early in cortex development, it was desired to find high Sox2 binding

! 10! to the enhancer in NPCs early in cortex development, rather than later. A figure showing the selected enhancer region to knock out for the Fezf2 gene is shown in Figure 6 and Figure 7 in the results section. To identify a critical enhancer region for Nfix (which mainly is expressed by upper layer neurons and thus later in cortex development), the aim was to find an enhancer region in which Sox2 binds to the NPCs late in corticogenesis. A figure showing the selected enhancer region to knock out for the Nfix gene is shown in Figure 8 and Figure 9 in the results section.

2.2.! Design&of&gRNAs&to&generate&enhancer&knockouts&

When the sequences of DNA to knock out for both genes were selected, the CRISPR/Cas9 system could be designed to generate the targeted sequence deletions. A basic overview of the knockout strategy can be seen in Figure 4 in the introduction section. Targeted double strand breaks can be generated by co-expressing the guide RNA (gRNA) and the Cas9 endonuclease in the same plasmid. The idea in this project was to target both ends of the enhancers with the CRISPR/Cas9 constructs in order to knock it out. To increase the likelihood of obtaining successful CRISPR/Cas9 knockout editing, three gRNAs were designed to guide the Cas9 to each end of the enhancer, for both the Fezf2 enhancer and the Nfix enhancer. As illustrated in Figure 10; gRNA 1, gRNA 2 and gRNA 3 were designed to guide the Cas9 nuclease to the 3’-end of the enhancer and gRNA 4, gRNA 5 and gRNA 6 were designed to guide Cas 9 to the 5’-end. In this way, different combinations of 3’-end gRNAs and 5-’ end gRNAs could be tested to find which pair of gRNAs that yields the most efficient enhancer deletion. Thus, for each of the two enhancers, 9 different gRNA combinations could be tested on P19 cell lines.

Figure!10!

Figure 10. Illustration showing how the gRNAs were designed in order to generate the enhancer deletion. Three gRNAs were designed to each end of the enhancer in order to compare the enhancer knockout efficiency between different combinations of 3’-end and 5’-end enhancers.

In order to clone the oligonucleotide gRNAs into the PX333 vector, they need to be carefully designed in order to be properly inserted. The role of the gRNAs is to direct the Cas9 endonuclease to a targeted position in the genomic sequence. Thus, when designing the gRNAs, it is important to consider that these need to match a target genome sequence of 20 nucleotides in the genome, often called the protospacer sequence. Furthermore, the target genomic sequence must be followed by a three- nucleotide long protospacer adjacent motif (PAM) sequence, NGG, which is necessary for DNA digestion by Cas9.

To design the gRNAs, the position of the enhancer regions for the two genes to knock out were identified from the Sox2 ChIP-sequencing data, respectively. By typing in the coordinates of the genomic target position in the UCSC genome browser for mouse (NCBI37/mm9 Assembly, July 2007), the raw bases of the region to knock out was obtained. Subsequently, the CRISPR-design tool on the website https://benchling.com/crispr was used to generate suggestions of gRNA oligonucleotides aimed to target the region of interest. The different gRNA suggestions from Benchling demonstrated different efficiency scores describing the gRNA activity at the target site. Higher scores indicate a higher likelihood of the gRNA being active. Thus, three gRNAs with high efficiency scores aimed to target the 3’-end of the enhancer and three gRNAs targeting the 5’-end of the enhancer were selected.

! 11!

After the six gRNA sequences were chosen for each enhancer, they had to be further designed in order to be properly inserted into one of the restriction sites of the PX333 plasmid. The idea was to cleave the plasmid with the HF-BbsI restriction enzyme. The recognition site of HF-BbsI consists of the non- palindromic sequence “GAAGAC” and upon plasmid cleavage, the two sticky-end overhangs “CACC” and “GTTT” are left, see Figure 11.

Figure!11!

Figure 11. Illustration of the cleavage sites of the BbsI restriction enzyme

Thus, the gRNAs need to be designed to match the overhangs. Furthermore, the genomic target sequence must begin with a “G” nucleotide in order to optimize transcription driven by the human U6 promoter. Each gRNA was designed and ordered as a pair of homologous forward-and reverse strand DNA according to the two last columns in Table 1 and Table 2 for the Fezf2 enhancer and the Nfix enhancer, respectively.

Table 1. gRNA design for the six different gRNAs designed to target the enhancer for Fezf2. For each gRNA, one forward and one reverse strand were designed and ordered according to the two far-right columns. The red letters correspond to the nucleotides added to match the plasmid overhangs. The green “G” and “C” were added to optimize U6 driven transcription.

Table 2. gRNA design for the six different gRNAs designed to target the enhancer for Nfix. For each gRNA, one forward and one reverse strand were designed and ordered according to the two far-right columns. The red letters correspond to the nucleotides added to match the plasmid overhangs. The green “G” and “C” were added to optimize U6 driven transcription.

2.3.! Cloning&of&gRNAs&into&PX333&plasmid&

To start the cloning, the PX333 plasmid was cultivated on Cb-agar plates at 37°C overnight. After this, single colonies were picked from the plates and the plasmid DNA was purified by using the Qiagen Miniprep kit, according to the manufacturer’s recommendations.

2.3.1.! Cleavage&reaction& For insertion of the single gRNA oligonucleotides into different PX333 plasmids, the plasmid was initially cleaved with the restriction enzyme HF-BbsI according to the following protocol:

! 12! ! 2 ng PX333 plasmid DNA 4 µl NE2 buffer 2 µl HF-BbsI restriction enzyme H2O up to 40 µl

The reaction mixture was incubated for 20 minutes at 37°C. In order to test if the linearization reaction was successful, a small volume from the cleavage reaction mixture was run on gel electrophoresis (0.6% agarose, 60 V) along with a size reference ladder (1 kb+ DNA ladder, ThermoFisher) and a sample of un-cleaved vector as control. Results of vector linearization are shown in Figure 12 in the results section. The size of the PX333 plasmid is 8990 bp and a satisfactory cleavage result would show a clear band corresponding to the size of the linearized PX333 vector. When proper linearization was confirmed, the rest of the cleavage reaction was run on the gel and the linearized plasmid DNA was then purified from the agarose gel by using the Qiagen Gel Extraction Kit, according to the manufacturers recommendations. Since the cleavage site for the BbsI restriction enzyme is non- palindromic, the ends of the plasmid cannot ligate themselves together and thus, a de-phosphorylation reaction was not needed to be performed.

2.3.2.! Annealing&and&ligation&reaction& The ordered gRNA insert oligonucleotides arrive as single stranded DNA. Thus, an annealing reaction had to be performed for each homologous pair of forward and reverse gRNA sequence. Since three gRNAs for each end of the enhancer were designed, six annealing reactions in total were performed for each enhancer to target. The following annealing protocol was used:

1 µl gRNA forward 1 µl gRNA reverse H20 up to 10 µl

The mixture was heated to 95°C for 5 minutes in a PCR thermocycler. In order for the oligonucleotides to anneal, the double stranded gRNA oligonucleotides were then left in room temperature for an hour. In order to then ligate the annealed gRNA oligonucleotide sequences into the linearized PX333 plasmid, the following ligation protocol was used:

100 ng linearized PX333 vector 2 µl of 1:250 diluted annealing reaction mixture 4 µl 10X DNA ligation buffer 1 µl T4 ligase H2O up to 40 µl

The ligation mixture was incubated at room temperature for one hour.

! 13! 2.3.3.! Transformation& After the gRNA inserts had been ligated into the PX333 vector, the circularized plasmid containing the inserts were transformed into competent bacteria (One Shot TOP10 Chemically Competent E. Coli, Thermofisher) for amplification of the cloned vector. For transformation of the plasmid DNA to bacteria, 4 µl of the ligation mixture and the control sample was carefully added to 50 µl of competent cells, respectively. The tubes were incubated for 20 minutes on ice and then heat shocked for 30 seconds at 42C. After this, 250 µl of SOC medium was added to each of the two tubes. The tubes were incubated for 1 hour and the transformed cells were then added to Cb-agarplates. The agarplates were subsequently incubated at 37°C overnight. The PX333 plasmid contains a gene for ampicillin resistance. Therefore, only bacteria that are transformed with the circularized PX333 plasmid should be able to grow on Cb-agarplates. Furthermore, bacteria are only able to amplify closed, ligated plasmids. Accordingly, colonies appearing on the Cb-agarplates after transformation should guarantee proper circularization of the PX333 plasmids containing the gRNA inserts. The cloning results after transformation can be seen in Figure 13 and Figure 14 in the results section. However, in order to properly confirm that the plasmid contains the inserted oligonucleotides, the plasmid DNA from the bacterial colonies were purified and sent for Sanger sequencing.

2.3.4.!Sanger&Sequencing&to&confirm&successful&cloning& Before exposing the P19 cells to the CRISPR/Cas9 designed PX333 plasmids, it is desirable to confirm that the different gRNAs are properly inserted into the plasmids, respectively. Thus, the newly transformed PX333 plasmids designed to target the Fezf2 enhancer as well as the Nfix enhancer were purified from the bacteria culture with the Qiagen Miniprep kit according to the manufacturers recommendations and sent for Sanger sequencing at GATC-biotech. The obtained sequencing results were aligned against the original PX333 plasmid sequence by using the bioinformatics tool CLC Sequence Viewer 7 (Qiagen Bioinformatics) in order to see whether the gRNA oligonucleotides were properly inserted or not. The sequencing results for Fezf2- and Nfix targeting plasmids can be seen in Figure 15 and Figure 16 in the result section, respectively.

2.4.! PCR&optimization& PCR followed by gel electrophoresis was used detecting CRISPR/Cas9 induced enhancer knockouts on genomic DNA. It is therefore important to have a well optimized PCR protocol and well functioning primers for both enhancer regions; the Fezf2 enhancer as well as the Nfix enhancer region). Thus, PCR primers were designed to cover the whole enhancer region for both enhancers, respectively. Thereafter, different protocols containing different amounts of PCR reagents and combinations of forward and reverse PCR primers were tested in order to generate clear bands on the gel showing the amplified DNA fragment. In Figure 17 in the results section, the most successful PCR optimization results for Fezf2 and Nfix are illustrated, respectively.

2.5.! Transfection&of&P19&cells&with&PX333&CRISPR&constructs& After confirming that the gRNA oligonucleotides were properly inserted into the plasmids, the CRISPR/Cas9 constructs were tested on P19 cell lines cultivated in the lab. It was desired to confirm successful enhancer knockouts in vitro before performing the in vivo studies and exposing mice to in utero electroporation.

Lipid based transfection, e.g. lipofection, was used in order to introduce the plasmids with the different designed CRISPR/Cas9 systems to the cells. Lipofectamine was utilized to facilitate the introduction

! 14! of DNA. The cationic lipid molecules in Lipofectamine will form complexes with the anionic DNA molecules and process enables the negatively charged molecules to overcome the electrostatic repulsion with the negatively charged plasma membrane and thus, the cationic liposomes containing the transfection payload DNA are able to fuse with the membrane and enter the cells.

Prior to transfection, a 24-well transfection plate containing 50000 cells/well was prepared and incubated over night to let the cells grow. For transfection, the three plasmids containing 3’-end targeting gRNAs (gRNA 1, gRNA 2 and gRNA 3) were initially combined with the three plasmids containing 5’-end targeting gRNAs (gRNA 4, gRNA 5 and gRNA 6), respectively. Thus, nine combinations of gRNAs in duplicates were prepared according to the structure in Figure 18 below. Control samples were also made in which two gRNAs that targets the same end of the enhancer were mixed.

Figure!18! gRNA 1 gRNA 1 gRNA 1 gRNA 1 gRNA 1 gRNA 1 gRNA 4 gRNA 4 gRNA 5 gRNA 5 gRNA 6 gRNA 6 gRNA 2 gRNA 2 gRNA 2 gRNA 2 gRNA 2 gRNA 2 gRNA 4 gRNA 4 gRNA 5 gRNA 5 gRNA 6 gRNA 6 gRNA 3 gRNA 3 gRNA 3 gRNA 3 gRNA 3 gRNA 3 gRNA 4 gRNA 4 gRNA 5 gRNA 5 gRNA 6 gRNA 6 gRNA 1 gRNA 1 gRNA 4 gRNA 4 No plasmid No plasmid Control Control Control Control added, Control added, Control Figure 18 Organization of the transfection plate made for each enhancer knockout. gRNAs targeting one side of the enhancer were mixed with gRNAs targeting the other side of it. Control samples were also prepared by mixing two 3’-end targeting gRNAs together as well as by mixing two 5’-end targeting gRNAs together.

The Lipofectamine based transfection mixture was prepared according to:

0.5µl of plasmid DNA (PX333 + gRNA) from a 5’-end gRNA vector was mixed with 0.5µl from a 3’- end gRNA vector in Eppendorf tubes according to the structure in Figure 18. 1 µl +plus reagent together with 23 µl (-) medium was added to each tube and the mixture was incubated for 15 minutes at room temperature. Subsequently, 1 µl of Lipofectamine together with 24 µl (-) medium was added to each tube and the samples were left at room temperature for 15 minutes. After that, the 24-well plate containing P19 cells cultivated overnight was removed from the 37°C incubator and the medium was removed from the wells, leaving cells bound to the bottom of the wells. Now, 200 µl of (-) medium was added to the lipofectamine based mixtures containing the CRISPR/Cas9 constructs as well as the control sample, and the 250 µl mixtures were then added to the transfection plate wells according to the prepared structure. The plates were incubated for three hours and then, 1 µl of (+)-medium was added to each well. The transfection plates were then incubated over night.

After exposing the P19 cells to the CRISPR constructs, it was desired to study if the transfection resulted in enhancer knockouts on the genomic DNA of the cells. Thus, the cells were lysed and the genomic DNA was purified in order to check for genome editing.

2.6.! PCR&and&gel&electrophoresis&to&confirm&CRISPR&genome&editing& In order to confirm CRISPR/Cas9 genome editing on the cell lines, PCR followed by gel electrophoresis was performed on the purified genomic DNA after transfection. A successful enhancer knockout with CRISPR/Cas9 would show two bands on the gel; one band of unaltered genomic DNA since CRISPR/Cas9 is not 100% efficient, and also a smaller sized band of the same region but with the enhancer region knocked out. The length of the wildtype region of interest (the distance between

! 15! the designed PCR primers) for both genes correspond to 650 bp for Fezf2 and 800 bp for Nfix. On the gel, the band for the un-cleaved DNA was thus expected to have the length 650 bp and 800 bp for the two different genes after PCR, respectively. Since different combinations of gRNAs were tested, the sizes of the edited DNA fragments would differ slightly. The expected sizes of the CRISPR/Cas9 edited fragments was calculated for each combination of gRNAs, as demonstrated in Table 3 for the Fezf2 enhancer and in Table 4 for the Nfix enhancer. The size of the knocked-out enhancer would equal the length between each combination of gRNAs. The column to the right in both figures (in blue) correspond to the expected sizes of the genome edited fragments that are expected to be seen on the gel.

Fezf2& & gRNA&combination& Size&of&knocked3out&fragment&[bp]& Size&of®ion&with&knockout&[bp]& gRNA!1!&!gRNA!4! 331! 319! gRNA!1!&!gRNA!5! 322! 328! gRNA!1!&!gRNA!6! 313! 337! gRNA!2!&!gRNA!4! 358! 292! gRNA!2!&!gRNA!5! 349! 301! gRNA!2!&!gRNA!6! 340! 310! gRNA!3!&!gRNA!4! 365! 285! gRNA!3!&!gRNA!5! 356! 294! gRNA!3!&!gRNA!6! 347! 303! Table 3. Calculation showing the length of the knocked out fragment that the different combination of gRNAs are designed to yield. The column to the right represents the expected size of the genome edited fragments that should generate a band on the gel after PCR. The sizes of DNA regions with knockouts (in blue) were calculated by subtracting the length between the PCR primers (650 bp for the Fezf2 enhancer) with the size of the knocked out enhancer.

Nfix& & gRNA&combination& Size&of&knocked3out&fragment&[bp]& Size&of®ion&with&knockout&[bp]& gRNA!1!&!gRNA!4! 392! 408! gRNA!1!&!gRNA!5! 410! 390! gRNA!1!&!gRNA!6! 457! 343! gRNA!2!&!gRNA!4! 335! 465! gRNA!2!&!gRNA!5! 353! 447! gRNA!2!&!gRNA!6! 400! 400! gRNA!3!&!gRNA!4! 330! 470! gRNA!3!&!gRNA!5! 348! 452! gRNA!3!&!gRNA!6! 395! 405! Table 4. Calculation showing the length of the knocked out fragment that the different combination of gRNAs are designed to yield. The column to the right represents the expected size of the genome edited fragments that should generate a band on the gel after PCR. The sizes of DNA regions with knockouts (in blue) were calculated by subtracting the length between the PCR primers (800 bp for the Nfix2 enhancer) with the size of the knocked out enhancer.

The gel pictures after PCR, performed to confirm CRISPR editing on the cell lines are shown in Figure 19 for the Fezf2 enhancer and for the Nfix enhancer, respectively in the results section. Based on the results shown on the gels, the combination that yielded the most effective CRISPR enhancer knockout was selected. Thus, the ratio between the edited and non-edited DNA was considered, and it was desired to obtain a high level of CRISPR edited DNA.

! 16!

2.7.! Cloning&both&enhancer3targeting&gRNAs&into&the&same&plasmid& After selecting the combination of gRNAs that yielded the most efficient enhancer knockout for both Fezf2 and Nfix respectively, it was decided to clone the best combination of the 3-’ and 5’- gRNA oligonucleotides into the same plasmid. This was possible since the PX333 plasmid contains two different restriction sites for the enzymes BbsI and BsaI. Conveniently, these two enzymes leave the same sticky-end overhangs and thus, the gRNAs could be designed in the same way as earlier in order to be properly inserted. The restriction site for BsaI, similar to BbsI, is shown in Figure 20. For Fezf2, the combination of gRNA 1 and gRNA 5 was chosen. Thus, the PX333 plasmid containing gRNA 1 was linearized with the BsaI enzyme and gRNA 3 was inserted in the same manner as earlier. Thereafter, the double gRNA-plasmid was transformed to competent bacteria, purified and sent for Sanger sequencing according to the same method as earlier described. The sequencing results for the double-insert plasmid (targeting the Fezf2 enhancer) is shown in Figure 21 in the results section. Similarly for the Nfix enhancer, gRNA 3 and gRNA 5 was selected and cloned into the same PX333 plasmid. Sequencing results for the double-insert plasmid (targeting the Nfix enhancer) is shown in Figure 22 in the results section. In both cases, the double-insert plasmid was aligned to the original PX333 sequence, to study if the oligonucleotides were inserted or not. In this way, each plasmid was designed to cause two double strand break at each end of the two enhancers, respectively.

Figure!20!

Figure 20. Restriction site for the enzyme BsaI used to clone in the second gRNA into the PX333 plasmid.

A basic overview of the double gRNA-insert plasmids for Fezf2 and Nfix, respectively, are shown in Figure 23 below.

Figure!23!

Figure 23. Basic illustration of PX333 plasmids containing double gRNA inserts. The left figure shows an overview of the Fezf2 enhancer targeting plasmid containing the most efficient gRNA combination for CRISPR editing (gRNA 1 and gRNA 5). The figure to the right shows an overview of the Nfix-enhancer targeting plasmid containing the most efficient gRNA combination for CRISPR editing (gRNA 3 and gRNA 5).

P19 cells were then transfected the CRISPR plasmids containing the double gRNA inserts in order to confirm proper CRISPR editing. PCR and gel electrophoresis were once again used to confirm CRISPR editing. For both Fezf2 and Nfix, the gel electrophoresis results from this are shown in Figure 24 in the results section, respectively.

! 17! 2.8.! In&utero&electroporation&of&mouse&embryos&with&CRISPR&plasmids&& After the enhancer targeting CRISPR plasmids were generated and confirmed on cell lines for both genes, they were to be introduced to mice by in utero electroporation. A basic overview of the electroporation procedure is illustrated in Figure 25. In order to study temporal pattering and NPC cell commitment properly, the CRISPR constructs were electroporated to the cortices of mouse embryos at day 11 (E11) after fertilization, which corresponds to the approximate time when the first neurons are born in the mouse brain. The idea was to knock out the enhancers to both Fezf2 and Nfix, respectively, in the NPC cells as early in cortex development as possible to properly be able to follow the developmental process of these when the enhancers are removed. The cortices of six E11 embryos of one pregnant mouse were injected with the Fezf2 enhancer-targeting plasmids and the cortices of E11 embryos of another pregnant mouse were injected with the Nfix enhancer-targeting plasmids. GFP was co-injected to the cortices to enable confirmation of successfully electroporated regions in the brain when later analyzing the results. Furthermore, in order to increase the efficiency of the CRISPR/Cas9 system, additional Cas9 mRNA was co-injected into the cortices. The embryos of one pregnant mouse were used as negative controls – these were only electroporated with GFP and were not exposed to the enhancer-knockout CRISPR plasmids. Unfortunately, as presented in 3.9 in the result section, the first electroporation attempt was unsuccessful since the embryos did not survive post-electroporation. It was hypothesized that the E11 embryos were too young and unstable to survive the electroporation and thus, a similar attempt was performed again but on mouse embryos at day 12 (E12) after fertilization.

Figure!25!

Figure 25. Overview of the electroporation procedure, in which the enhancer targeting CRISPR plasmids were introduced into embryonic mouse cortices. Initially, the uterus with the embryos of a pregnant mouse were exposed. The plasmid DNA was then injected along with PBS and GFP as a marker into the cortex of the embryo. The injected mouse cortices are then electroporated at 50 V by placing electrodes on each side of the brain.19

2.9.! Immunofluorescence&and&confocalµscopy&analysis&&

After electroporation of the E12 mouse embryos, they were left growing inside the uterus until day 18, the time when the six cortical layers are fully developed in mice. The treated mice were subsequently sacrificed and the embryonic mouse cortices were fixated by using 4% paraformaldehyde in PBS (pH 7.4) for about 15 minutes in order to preserve the tissue and prevent autolysis. The brain tissues were sectioned in thin brain sections and portioned out on glass coverslips to then be stored in -20C over night.

In order to study if gene expression of Fezf2 and Nfix was affected by the in vivo enhancer knockouts, respectively, immunofluorescence staining of the brain sections was used for detection. The mouse cortices treated with the Fezf2-enhancer targeting CRISPR constructs were stained with primary

19 https://www.nature.com/articles/ncomms1961

! 18! antibodies against Fezf2, and the ones exposed to the Nfix-enhancer targeting CRISPR constructs were stained with primary antibodies against Nfix. Furthermore, it was desired to investigate if the enhancer knockout affected neuronal development and cortical layer formation in other ways. It was therefore decided to stain for two other characteristic layer markers; Ctip2 (mainly expressed by deep layer neurons) and Satb2 (mainly expressed by upper layer neurons). Accordingly, the cortices treated with the Fezf2 enhancer targeting CRISPR constructs were separately also dubble-stained with antibodies against both Ctip2 and Satb2. The cortices treated with the Nfix-targeting CRISPR constructs were also separately stained for Ctip2 and Satb2. In order for proper analysis, the brain tissues from the control mice (mouse embryos electroporated with only GFP and no CRISPR constructs) were stained in the same manner as the CRISPR treated brain tissues. Thus, as illustrated in Table 5 below, eight glass coverslips with electroporated brain tissue were stained for in total.

Table 5. Immunostaining strategy of sectioned mouse brain tissue after in utero electroporation experiments. In order to study how the enhancer knockouts affected layer formation, immunostaining of cortical layer markers was used. Fezf2 and Ctip2 are characteristically expressed by deep layer neurons whereas Nfix and Satb2 are characteristically expressed by upper layer neurons.

To initiate the immunofluorescence staining, the glass coverslips with the brain sections were washed three times with PBS and subsequently incubated for 1 hour in blocking solution (22.52 mg/mL glycine in PBST (0.1% Tween 20 + PBS), 1% BSA) in order to prevent unspecific binding of the antibodies. Thereafter, the glass coverslips were incubated in diluted primary antibody (with 1% BSA in PBS) for two hours in room temperature. For proper detection of the double immunofluorescence staining where Ctip2 and Satb2 primary antibodies were combined (coverslip 2, 4, 6 and 8 in Table 5), primary antibodies from different species were used in order for the different secondary antibodies to selectively bind to the desired targets. The samples were then washed three times with PBS and incubated in diluted secondary antibody (with 1% BSA in PBS) for two hours in room temperature. Additionally, the DNA stain DAPI was added to each sample to be able to detect nuclei (and thus all cells in the tissue) when investigating the results of the immunofluorescence staining with confocal microscopy. Subsequently, the solution was discarded from the coverslips and the slides were washed three times in PBS. In order to analyze the results after the staining, confocal microscopy was used. Thus, a drop of mounting medium and thin mount coverslips were added to each glass slide before analyzing the samples under the microscope. Gene expression levels from the different markers could be studied and distinguished since the secondary antibodies used for the different primary antibodies contain different fluorophores. Results from confocal microscopy for Fezf2 expression, Ctip2 expression and Satb2 expression of the Fezf2 enhancer-knockout samples are presented in section 3.9, Figure26, Figure 27 and Figure 28 in the results section.

To strengthen the results from the immunofluorescence stainings after in vivo Fezf2 enhancer- knockout, cell counts were made on the confocal microscopy images to study and compare the intensity of gene expression in successfully electroporated cells. Figure 29 (3.9 in the results section)

! 19! shows a cell count of Fezf2 expression in electroporated cells compared to the Fezf2 expression in electroporated cells of the negative control. 50 electroporated cells were counted for the control and for the CRISPR-treated sample and the intensity of Fezf2 expression for each cell was noted by the tool ImageJ on the computer. The mean intensity of Fezf2 expression for the counted cells was then calculated for the treated sample and for the control. It is important to note that the background intensity may differ between the treated sample and the control sample, and that the noted values of Fezf2 expression intensity for the counted cells might differ because of this. Therefore, intensities of 20 cells in the background of both images (identified by the DAPI marker) were noted and a mean of the background intensity was calculated for both the treated sample and for the control. Likewise, the same procedure was done for the cell count of Ctip2 expression as well as for Satb2 expression, shown in Figure 30 and Figure 31 in the results section, respectively. However, 96 cells were counted for these two markers. The bars in Figure 29, Figure 30 and Figure 31 correspond to the mean intensity of Fezf2 background in electroporated cells divided by the mean of the background intensity of the image, for the CRISPR treated sample and for the control, respectively. 3.!Results(

3.1.! Identification&of&enhancer®ions&to&target& In order to find an interesting enhancer region to knock out for the Fezf2 gene, sequencing data illustrated in Figure 6 and Figure 7 were used. These figures illustrate Sox2 ChIP-sequencing data and DNase-sequencing data on E11-mouse embryos and E15-mouse embryos, respectively.

Figure!6!

Figure 6. Sox2 ChIP-sequencing data (green) and DNase-sequencing data (blue) studied to find enhancers for the Fezf2 gene. The illustrated region is located in the Fezf2 gene on chromosome 14. The first ChIP-sequencing row as well as the first DNase-sequencing row corresponds to sequencing done on 11 days old mouse embryos (E11) and the second ChIP- sequencing row as well as the second DNase-sequencing row corresponds to sequencing done on 15 days old mouse embryos.

!! ! !

! 20! Figure!7!

Figure 7. Zoom in-picture of the selected region thought to affect Fezf2 gene expression to knock out by using CRISPR/Cas9. The figure is illustrating the Sox2 ChIP-sequencing data in green and DNase-sequencing data in blue. The illustrated region is located in the Fezf2 gene on chromosome 14. The first ChIP-sequencing row as well as the first DNase- sequencing row corresponds to sequencing done on 11 days old mouse embryos (E11) and the second ChIP-sequencing row as well as the second DNase-sequencing row corresponds to sequencing done on 15 days old mouse embryos.

When studying the Sox2 ChIP-sequencing data and DNase-sequencing data on E11-mouse embryos and E15-mouse embryos in Figure 6 and Figure 7, it is possible to clearly find a region of DNA that indicate to be a critical enhancer region to the deep layer gene Fezf2. In this region, Sox2 binding is much higher in E11 NPCs (early in cortex development) than in E15 NPCs (late in cortex development). This identified enhancer region have been studied before and it has been confirmed that it is a critical enhancer for Fezf2 expression and for the development of cortical layer V and VI. Thus, it was important to find this region in the sequencing data, as a control. It was further decided to knock this enhancer region out.

In order to find an interesting enhancer region to knock out for the gene Nfix, Sox2 ChIP-sequencing data and DNase-sequencing data on E11-mouse embryos and E15-mouse embryos were studied.The selected enhancer region for the gene Nfix is shown in Figure 8 and Figure 9 below.

! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 21! Figure!8!

Figure 8. Sox2 ChIP-sequencing data (green) and DNase-sequencing data(blue) studied to find critical enhancers for the Nfix gene. The illustrated region is located in the Nfix gene on chromosome 8. The first ChIP-sequencing row as well as the first DNase-sequencing row corresponds to sequencing done on 11 days old mouse embryos (E11) and the second ChIP-sequencing row as well as the second DNase-sequencing row corresponds to sequencing done on 15 days old mouse embryos. The red arrow in the figure points at the enhancer region which was selected to be knocked out.

Figure!9!

Figure 9. Zoom in-picture of the selected Nfix- enhancer region thought to affect gene expression. This region was decided to knock out by using CRISPR/Cas9. The figure is illustrating the Sox2 ChIP-sequencing data in green and DNase-sequencing data in blue. The illustrated region is located in the Nfix gene on chromosome 8. The first ChIP-sequencing row as well as the first DNase-sequencing row corresponds to sequencing done on 11 days old mouse embryos (E11) and the second ChIP-sequencing row as well as the second DNase-sequencing row corresponds to sequencing done on 15 days old mouse embryos.

By studying the Sox2 ChIP-sequencing data in Figure 8 and 9, a clear peak can be seen in E15 NPCs which is not present in E11 NPCs. Thus, Sox2 binding to this particular enhancer region is much higher later in cortex development (E15) than in early cortex development (E11) and therefore, this enhancer

! 22! can be described as a new enhancer in E15 NPCs. Nfix is known to be expressed by neurons of the upper layers that are generated later in cortex development. Thus, this enhancer region is of particular interest and it was selected to be knocked out.

3.2.! PX333&plasmid&linearization&

The linearization of the PX333 plasmid with the restriction enzyme HF-BbsI was successful, and can be studied in Figure 12 below. The first three wells on the gel contain, in order from left: 1 kb+ size reference ladder, un-cleaved plasmid DNA and cleaved DNA.

Figure!12!

Figure 12: The first three wells contain, in order: 1kb+ reference ladder, un-cleaved plasmid DNA, cleaved plasmid DNA (with HF-BbsI)

When comparing the size of the cleaved PX333-plasmid with the 1kb+ size reference ladder, a size of 9000 bp was confirmed, which is the size of the PX333 plasmid. Furthermore, the un-cleaved plasmid (the sample closest to the ladder in Figure 12) did not give rise to a clean, linearized band. Thus, the linearization of the PX333 plasmid was successful.

3.3.! Cloning&results&& & The transformation results for the plasmids containing the six different inserted Fezf2-targeting gRNAs (gRNA 1, gRNA 2, gRNA 3, gRNA 4, gRNA 5 and gRNA 6, respectively) were successful. All six Cb-agarplates did contain colonies after the overnight incubation. Furthermore, the control plate which was treated without gRNA insert during plasmid ligation did not show any colonies, as desired. This result strongly indicates that all six gRNA inserts were properly inserted into PX333 plasmids, respectively. Figure 13 is showing the successful cloning results for one of the transformed gRNA plasmids (PX333 with gRNA 1) compared to the negative control plate.

! ! ! ! ! ! ! ! !

! 23! Figure!13!

Figure 13. Illustration of results after transformation of PX333 plasmids containing inserted Fezf2 gRNAs to Top10 competent bacteria. The picture to the left corresponds to transformation of PX333 plasmid containing Fezf2 gRNA 1 and the picture to the right corresponds to the control plate in which the bacteria were not exposed to any gRNA.

As for Fezf2, the transformation results for the plasmids containing the six different inserted Nfix- targeting gRNAs (gRNA 1, gRNA 2, gRNA 3, gRNA 4, gRNA 5 and gRNA 6, respectively) were successful. All six Cb-agarplates did contain colonies after incubation overnight, except from the control plate which did not contain any colonies. This can be illustrated in Figure 14 were the transformed PX333 plasmid containing gRNA 1 is shown and compared with the negative control plate.

Figure!14!

Figure 14. Illustration of results after transformation of PX333 plasmids containing inserted Nfix gRNAs to Top10 competent bacteria. The picture to the left corresponds to transformation of PX333 plasmid containing Nfix gRNA 1 and the picture to the right corresponds to the control plate in which the bacteria were not exposed to any gRNA

3.4.! Sanger&sequencing&results&(single&gRNA&inserts)&& The cloning of the six different single gRNA oligonucleotides into different plasmids was successful for both the Fezf2- and the Nfix targeting constructs, illustrated in Figure 15 and Figure 16 below. The sequencing results (of the transformed PX333 with gRNA inserts) were all aligned against the original PX333 sequence separately, in order to confirm that the gRNAs were properly inserted. For alignment, the bioinformatics tool CLC Sequence Viewer 7 (Qiagen Bioinformatics) was used.

! ! ! ! ! ! !

! 24! Figure!15!

Figure 15: Sequencing results illustrating that the six different gRNAs designed to target the Fezf2 enhancer were properly inserted. The sequencing results (the upper row of DNA sequence) were aligned against the original PX333 DNA sequence (the lower row of DNA sequence, labeled PX333).

! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 25! ! Figure!16!!

Figure 16: Sequencing results illustrating that the six different gRNAs designed to target the Nfix enhancer were properly inserted. The sequencing results (the upper row of DNA sequence) were aligned against the original PX333 DNA sequence (the lower row of DNA sequence, labeled PX333).

As desired, all six different gRNA inserts were properly inserted as 20-nt gRNA sequences in six different plasmids for both of the enhancers to knockout. Thus, for each enhancer, three gRNAs with the aim of targeting the 5’ end of the enhancer and three with the aim of targeting the 3’ end of the enhancer was successfully generated.

! 26! 3.5.! PCR&optimization&& ! The results from the PCR optimization for Fezf2 and Nfix, respectively, are illustrated in Figure 17.

Figure!17!

& Figure 17. PCR optimization PCRs on genomic DNA and annealing temperature gradients (55C-75C). Left picture: PCR optimization on PCR primers designed to cover the Fezf2 enhancer region. The arrow points at the selected optimal reaction. Right picture: PCR optimization on PCR primers designed to cover the Nfix enhancer region. The arrow points at the selected optimal reaction.

The arrows in Figure 17 points at the most optimal PCR reaction for the two enhancers, respectively. For Fezf2, the PCR optimization experiments led to the conclusion that the PCR reaction is most efficient at annealing temperature 65°C. This is since the electrophoresis gel band at 65°C have the highest intensity. Furthermore, the amplified region have the desired size covering the whole enhancer region; around 720 bp. For Nfix, the most efficient annealing temperature was not as clear to decide as Fezf2 since most of the annealing temperatures tested led to intense gel bands. However, after carefully studying the intensity of the bands, it was decided to select 55°C as the annealing temperature for the Nfix enhancer PCR. Furthermore, the size of the amplified region had the desired size; around 800 bp.

3.6.! PCR&results&after&transfection&with&combinations&of&single&gRNA&plasmids&

Figure 19 shows the results after exposing P19 cells to the different enhancer-targeting combinations of CRISPR-plasmids. gRNA 1, gRNA 2 and gRNA 3 were designed to target the 5’-end of the enhancer, and gRNA 4, gRNA 5 and gRNA 6 were designed to target the 3’-end, for both the Fezf2 and Nfix- enhancers, respectively.

Figure!19!

Figure 19. Results from transfection of P19 cells with the different combinations of CRISPR-plasmids containing 3’- enhancer targeting gRNAs with CRISPR-plasmids containing 5’-enhancer targeting gRNAs, for the Fezf2 and Nfix- enhancers. Right picture: The arrows point at the most efficient Fezf2-enhancer (E4) knockout, generated by gRNA 1

! 27! together with gRNA 5. The well to the far right is the negative control (only gRNA 1)l. Left picture: The arrows point at the most efficient Nfix-enhancer knockout with CRISPR, generated by gRNA 3 together with gRNA 5. The band to the left corresponds to the negative control (only gRNA 1).

The bands on the gel pictures in Figure 19 correspond to the different combinations of 3’-targeting plasmids and 5’-targeting plasmids. It was expected that all gRNA combinations would generate enhancer knockouts, but with variable efficiencies. For the Fezf2 enhancer knockout, it is clear that the 3rd band from the ladder shows the most efficient CRISPR- enhancer knockout reaction, as clarified by the blue arrow in Figure 19. The smaller sized band corresponding to the fragment with enhancer- knockout (398 bp) is clearly the most intense among the different combinations of gRNAs. The genomic un-altered wild type band for Fezf2 is 720 bp. Thus, the most efficient enhancer-knockout combination was a result of the combination of gRNA 1 & gRNA 5. For the Nfix enhancer, the best gRNA combination was not as easy to decide. The smaller sized bands corresponding to the CRISPR edited fragments were much weaker than for the Fezf2 enhancer. However, the intensities of the different bands were carefully compared and the arrows point at the selected combination that produced the best ratio between altered and non-altered DNA for the Nfix enhancer – and thus the sample with as much edited DNA as possible. The blue arrow in the left image of Figure 19 points at the most efficient combination of gRNAs for Nfix-enhancer knockout, gRNA 3 and gRNA 5. The unaltered genomic DNA for the Nfix-region was 800 bp and the fragment with enhancer-knockout (sample 9 from the reference ladder) corresponds to 460 bp. As desired, the result for the control samples (for both the Fezf2 enhancer knockout and for the Nfix enhancer knockout) did show one band corresponding to the unaltered genomic DNA, and no band for CRISPRed DNA with enhancer knockout.

3.7.! Sanger&sequencing&results&(double&gRNA&inserts)&&

Figure 21 and Figure 22 demonstrates the results from the Sanger sequencing of the cloned PX333 plasmids containing two gRNAs designed to target each end of the enhancer for Fezf2 and Nfix, respectively. In both images, the sequencing results (upper row of DNA sequence) were against the original PX333 plasmid sequence.

Figure!21!!

Figure Q. Sequencing results from Sanger sequencing of plasmids with double gRNA inserts, designed to target the Fezf2-enhancer.

Figure 21 shows successful sequencing results for the cloned PX333 plasmid aimed to target and knockout the Fezf2 enhancer. As desired, gRNA 5 was properly cloned into the second restriction site of the PX333 plasmid which contained gRNA 1.

! 28! Figure!22!

Figure 22. Sequencing results from Sanger sequencing of plasmids with double gRNA inserts, designed to target the Nfix-enhancer.

Figure 22 shows successful sequencing results for the cloned PX333 plasmid aimed to target and knockout the Nfix enhancer. As desired, gRNA 5 was properly cloned in the PX333 plasmid which contained gRNA 3.

3.8.! PCR&results&after&transfection&(PX333&with&double&gRNA&inserts)&

The in vitro enhancer knockout results after transfection of P19 cells with the double gRNA-insert plasmids for both the Fezf2 enhancer and the Nfix enhancer are shown in Figure 24 below.

Figure!24!

Figure 24. In vitro enhancer knockout results. P19 cells were exposed to CRISPR plasmids containing the most efficient combination of gRNAs to knock out the Fezf2 enhancer and the Nfix enhancer, respectively. The green arrows point at the band corresponding to DNA with enhancer knockout.

The enhancer-knockout results after transfection of P19 cells with the CRISPR plasmids were successful for both the Fezf2 enhancer and for the Nfix enhancer. When comparing to the controls in both cases, it is clear that the CRISPR-plasmid resulted in enhancer-knockout in both cases. However, the intensity of the gel bands shown in Figure 24 were much weaker than the gel bands in Figure 19 (the in vitro results after testing different combinations of gRNAs). A contributing factor to this could be varying efficiency during the transfection of cells, and accuracy during preparation of PCR mixtures. However, the interesting part is to study in the results is the ratio between un-CRISPRed

! 29! DNA and CRISPRed DNA. According to this, it is clear that the enhancer-knockout was efficient and resulted in genome editing.

3.9.! In&vivo&results&after&in&utero&electroporation&&

The electroporation of E11 mouse embryos was unsuccessful since none of the embryos did survive. It was therefore not possible to study cortex development in these. It was believed that the E11 embryos were weak and eletroporated too early in development. The following attempt done on E12 embryos was more successful; all electroporated embryos did survive the electroporation and was sacrificed at E18.

The immunofluorescence staining results after in utero electroporation of E12 mice with the Fezf2 enhancer-targeting CRISPR constructs are shown in Figure 26, Figure 27 and Figure 28.

Figure 26 shows the affect on Fezf2 expression after exposure to the Fezf2-enhancer knockout CRISPR agents performed by electroporation in E12 embryonic mouse cortex. The green areas (co- electroporated GFP) correspond to successfully electroporated regions in the brain. The red colored areas show Fezf2 expression.

Figure!26!

Figure 26. Confocal microscopy image of sectioned E18- mouse cortex after in utero electroporation on mice with Fezf2 enhancer targeting constructs, stained for Fezf2 expression. Brain sections were stained with immunofluorescence by using a primary antibody against Fezf2 and a secondary antibody for detection. Green areas correspond to the GFP marker, showing successfully electroporated areas in the cortex. Red colored areas shows Fezf2 expression. Yellow areas illustrate electroporated areas which still show Fezf2 expression. The control sample was electroporated with GFP and not with the Fezf2-enhancer targeting CRISPR constructs.

Electroporation was successful for both the control and for the Fezf2 enhancer CRISPR-treated brain tissue. This is possible to state since several green areas can be seen in both images in Figure 26. Yellow areas in the images correspond to an overlap in GFP and Fezf2 expression - electroporated cells showing Fezf2 expression. By comparing the control and the CRISPR treated sample, it is possible to see that there are less yellow areas in the CRISPR treated sample. Thus, the majority of cells that were exposed to the enhancer-knockout plasmids seem to have lost expression of Fezf2. By

! 30! studying Figure 26, one can thus claim that removal of the enhancer region (E4) seem to cause a reduce in Fezf2 expression, as expected. Thus, the enhancer region (E4) indicates to be important for expression of Fezf2.

Figure 27 and Figure 28 illustrate how the markers Ctip2 (deep layer marker) and Satb2 (upper layer marker) were affected by the Fezf2 enhancer knockout in vivo, respectively. Since Ctip2 and Satb2 were double- stained on the same glass coverslip, the same cortex region is illustrated in both Figure 27 and Figure 28.

Figure!27!

Figure 27. Confocal microscopy image of sectioned E18- mouse cortex after in utero electroporation on mice with Fezf2 enhancer targeting constructs, stained for Ctip2 expression. Brain sections were stained with immunofluorescence by using a primary antibody against Ctip2 and a secondary antibody for detection. Green areas correspond to the GFP marker, showing successfully electroporated areas in the cortex. Dark blue dye shows Ctip2 expression. Light blue areas illustrate electroporated ares which still show Ctip2 expression. The control sample was electroporated with GFP and not with the Fezf2-enhancer targeting CRISPR constructs.

The Fezf2 enhancer- CRISPR treated sample in Figure 27 shows no overlap between GFP and Ctip2 compared to the control, which show many regions of overlap (light blue). Thus, cells that are electroporated and exposed to the CRISPR enhancer-knockout agents do not seem to express the deep layer marker Ctip2 whereas several of the electroporated areas in the control still show expression of Ctip2. This strongly indicates that removal of the enhancer region (E4) to Fezf2 is critical for expression of Ctip2. Since Ctip2 is a known marker for deep layer neurons of layer V, there are strong reasons to believe that the removed enhancer region to Fezf2 is critical for proper development of deep layer neurons of layer V. These findings are in line with previous research done on the E4 enhancer for Fezf214 and strongly suggest that the designed CRISPR/Cas9 system for in vivo enhancer- knockouts is effective and efficient.

! 31! Figure!28!

Figure 28. Confocal microscopy image of sectioned E18- mouse cortex after in utero electroporation on mice with Fezf2 enhancer targeting constructs, stained for Satb2 expression. Brain sections were stained with immunofluorescence by using a primary antibody against Satb2 and a secondary antibody for detection. Green areas correspond to the GFP marker, showing successfully electroporated areas in the cortex. Red dye shows Satb2 expression. Yellow areas illustrate electroporated ares which still show Satb2 expression. The control sample was electroporated with GFP and not with the Fezf2-enhancer targeting CRISPR constructs.

The Fezf2 enhancer- CRISPR treated sample in Figure 28 shows many areas of overlap (yellow) between GFP and the upper layer marker Satb2. Yellow areas are also seen for the control. This indicates that the Fezf2 enhancer-knockout did not lead to a reduced expression of the upper layer marker Satb2. According to Figure 28, the knocked out enhancer is not critical for expression of Satb2.

Gene expression was further investigated by performing a cell count on the confocal microscopy images to study and compare the intensity of gene expression in successfully electroporated cells. Figure 29 shows a cell count of Fezf2 expression in electroporated cells compared to the Fezf2 expression in electroporated cells of the negative control. Figure 30 and Figure 31 below illustrate cell counts of Ctip2- and Satb2 expression levels in electroporated cells exposed to the Fezf2 enhancer- targeting CRISPR plasmids, compared to Fezf2 expression in the control, respectively. The height of the bars in Figure 29, Figure 30 and Figure 31 correspond to the mean intensities of Fezf2 expression in electroporated cells divided by the mean of background intensity in the images, respectively.

! 32!

Figure!29!

Figure 29. Cell count of successfully electroporated cells (treated with Fezf2 enhancer targeting plasmids) expressing Fezf2. Gene expression of electroporated cells was analyzed by using the tool ImageJ on the confocal microscopy images. 50 electroporated cells were counted for both the treated samples and for the control, and the intensity of Fezf2 expression for each cell was noted. The bars in the figure were generated by calculating the mean intensity of the 50 cells, respectively and dividing this number by the mean intensity of the background in the image. Error bars were generated by calculating the standard deviation for the treated and for the control samples. A T-test gave the value 0,00329804, demonstrating that the results are significant. &

The cell count for Fezf2-expression of the electroporated cells show that Fezf2 expression is significantly reduced in the Fezf2 enhancer-knockout treated samples compared to the control. A statistical t-test was performed on the gene expression intensity data, to compare the difference between the results from the CRISPR treated sample and the control. The t-test gave the value 0,00329804 which shows that the result is significant. This highly suggests that the Fezf2 enhancer (E4) is critical for Fezf2 expression of deep layer neurons, as the confocal microscopy image in Figure 26 also suggest.

Figure!30!

! 33! Figure 30. Cell count of successfully electroporated cells (treated with Fezf2 enhancer targeting plasmids) expressing Ctip2. Ctip2 gene expression of electroporated tissue was analyzed by using the tool ImageJ on the confocal microscopy images. 96 electroporated cells were counted for both the treated samples and for the control, and the intensity of Ctip2 expression for each cell was noted. The bars in the figure were generated by calculating the mean intensity of the 96 cells, respectively and then dividing this number by the mean intensity of the background in the image.

Figure 30 shows that Ctip2 expression is significantly reduced in electroporated cells treated with the Fezf2 enhancer-knockout constructs compared to the control. The Ctip2 expression cell count strengthens the results showing that the Fezf2 enhancer (E4) is important for expression of the deep layer marker Ctip2, as also suggested in Figure 27.

Figure!31!

Figure 31. Cell count of successfully electroporated cells (treated with Fezf2 enhancer targeting plasmids) expressing Satb2. Satb2 gene expression of electroporated tissue was analyzed by using the tool ImageJ on the confocal microscopy images. 96 electroporated cells were counted for both the treated samples and for the control, and the intensity of Satb2 expression for each cell was noted. The bars in the figure were generated by calculating the mean intensity of the 96 cells, respectively and then dividing this number by the mean intensity of the background in the image.

In Figure 31, a clear increase in Satb2 expression is identified for cells treated with the Fezf2-enhancer knockout agents during electroporation, compared to the control. These results suggest that the enhancer to Fezf2 (E4) is not critical for Satb2 expression. However, removal of this enhancer seems to highly affect the gene expression level of the upper cortical layer gene Satb2 indirectly.

Unfortunately, the immunofluorescence results for the brain tissue exposed to the Nfix- enhancer knockout plasmids were not satisfactory. It was possible to see green areas corresponding to GFP during confocal microscopy, which indicate that electroporation was successful. However, the primary antibody against Nfix did not gave proper staining results – the stainings were uninformative. Due to the time limit of this project, there was not enough time to order a new primary antibody against Nfix and thus, new Nfix- stainings will be performed after this project.

! 34! 4.!Discussion(

The aim of this project was to investigate the necessity of gene regulating enhancer regions in cortical layer formation during development. By knocking out enhancers to one gene that is mainly expressed by deep layer neurons (Fezf2) and to one gene typically expressed by upper layer neurons (Nfix), the objective was to study how each enhancer removal affected gene expression and layer formation in the developing cortex, respectively.

The in vitro results of the CRISPR/Cas9 induced enhancer-knockouts were successful. After using PCR followed by gel electrophoresis as detection system for genome editing, the bands on the gel did clearly show that enhancer knockout did occur for both the Fezf2 enhancer and for the Nfix enhancer in separate experiments. It was expected to see two bands on the gel; one band corresponding to un- edited wild type DNA and one smaller sized band which corresponds to DNA with the enhancer region removed.

It is important to consider that many factors may affect the appearance of the in vitro results, and the intensity of the gel bands after PCR. One significant contributing factor to varying intensities of the results after gel electrophoresis is transfection efficiency, when exposing the cell lines to the CRISPR constructs. A poor transfection efficiency will give rise to less intense bands on the gel. For example, factors related to the preparation of transfection plates such as the amount of cells in each well, passage number and quality of the cells as well as the incubation time after transfection may affect the efficiency of transfection efficiency and thus also CRISPR editing. One way to improve the problem with transfection efficiency and differing intensities of the gel bands could be to perform replicate transfection experiments on different transfection plates. This would increase the robustness of the results. Nevertheless, in this project, PCR and gel electrophoresis was a convenient and proper method to use for detection since it was only desired to confirm that the CRISPR editing did work; to compare which pair of gRNA resulted in the best enhancer knockout for each enhancer respectively, and to confirm that transfection with the double gRNA-insert plasmid further also resulted in CRISPR editing.

The reason why two bands are seen in the in vitro results is that the efficiency of CRISPR/Cas9 is limited and not all cells exposed to the CRISPR plasmids are edited. The efficiency of CRISPR/Cas9 is limited by the DNA repair pathway “nonhomologous end joining, NHEJ”, a pathway of DNA repair, competing with the other DNA-repair pathway “homology directed repair, HDR”. Because of this, it is important to consider that the efficiency of the CRISPR/Cas9- system in vivo also will have an effect of the final results generated after in utero electroporation of embryonic mouse cortices. Both NHEJ and HDR are active in the majority of all cell types. However, the more frequent NHEJ pathway is active throughout the whole cell cycle whereas the HDR pathway only occurs during the S- and G2 phases. The NHEJ pathway of DNA repair is error prone and thus, insertions and deletions are frequently introduced in the DNA. This pathway is therefore often used for introducing random mutations. However, the system does not ensure precise targeted genome engineering by HDR- mediated insertion of exogenous DNA-sequences.17, 18 As an attempt to increase the efficiency of the CRISPR/Cas9 system, additional Cas9 mRNA was co-injected during in utero electroporation. However, it is possible that the efficiency can be increased further by increasing the concentration of injected CRISPR-plasmids and to co-inject an even higher concentration of Cas9 mRNA.

After knocking out the enhancers to Fezf2 and Nfix, respectively, it was interesting to study if the gene expression of these two genes were affected. It was also interesting to study if the enhancer-knockouts did lead to a shift in cortical layering. The Fezf2 enhancer-knockout results after in utero electroporation of E12 mouse cortices were interesting and satisfactory. In this project, the Fezf2-

! 35! enhancer knockout was performed as a positive control, to show that the designed knockout system with CRISPR/Cas9 is effective and reliable. According to the in vivo results in section 3.9, the Fezf2- enhancer knockout caused a significant reduce in Fezf2 expression, as desired. This result is in line with previous research stating that this particular enhancer region is critical for Fezf2 expression and also for proper development of deep layer neurons of layer V and VI. One important question to consider is if the CRISPR-treated neurons still will be developed, but without expression of Fezf2. When immunostaining the Fezf2-enhancer knockout –samples for the deep layer marker Ctip2, a clear reduce in Ctip2 expression was found; almost 90% of the electroporated cells did loose Ctip2 expression compared to the control. This clearly indicates that the development of deep layer neurons is affected by the enhancer deletion. Furthermore, the Fezf2-enhancer knockout resulted in a significant increase in expression of the upper-cortical layer marker Satb2 in cells that were successfully electroporated. These results indicate that the enhancer-knockout caused a fate-switch in the cells during development; that more upper layer neurons were generated compared to deep layer neurons. This further suggests that the gene Fezf2 is essential for proper cortical layering, and that the enhancer region E4 is critical for Fezf2 expression, as earlier research also suggest. However, it is important to consider that the cell counts of gene expression in electroporated cells presented in Figure 29, Figure 30 and Figure 31 are results of 50 cells and around 100 cells, respectively. In order to strengthen the results statistically, more cells would have to be included in the cell count of the immunofluorescence images. However, the results provide a proper indication of how the enhancer- knockout affected gene expression of Fezf2, Ctip2 and Satb2.

It was desired to generate the in vitro enhancer-knockout as early as possible during cortex development, in order to properly be able to study the affect on cell fate decision and layer formation. The electroporation was made at E12, which is the day after the first neurons are born in the mouse brain. Since the deep layer neurons are generated first during corticogenesis, it is important to consider how electroporation at this time point affected the results. If the first neurons are born at E11 and the enhancer knockout occurs in the NPC state, it is important to question if some neurons are “missed” when exposing the mouse cortices to the CRISPR/Cas9 reaction the day after. The in vivo results suggested that the Fezf2-enhancer knockout causes a fate switch - the deep layer marker Ctip2 was significantly reduced and the upper layer marker Satb2 was highly increased. One possibility is that this fate switch is affected when the enhancer knockout occurs in the NPC state. However, the CRISPR/Cas9 reaction can also happen in post-mitotic neurons, which is important to consider.

Unfortunately, the immunofluorescence stainings of the tissue with Nfix-enhancer knockouts were not as satisfactory. The electroporation was successful, indicated by identified green areas of GFP expression. However, the primary antibody for Nfix did not generate properly comprehensible results. It was thus hard to get an indication about how the gene expression of Nfix, Ctip2 and Satb2 was affected by the enhancer knockout. Due to the time limitation of this project, there was not enough time to order a new antibody and re-do the stainings. However, this experiment will be followed up in the future. Since Nfix mainly is expressed by upper layer neurons, it would be very interesting to see how enhancer knockout in E12 mice affected the gene expression. In this case, no neurons of the upper layers had begun to form when the CRISPR/Cas9 plasmids were was injected into the embryonic mice cortices.

There are still many questions regarding the evolution of our complex brain. Specification and development of the cortical layers in the brain highly rely on the precise regulation of location, timing as well as on levels of gene expression. Hopefully, the field of neuroscience will continue to increase and generate new insight about how the cortex is developed, to better understand the mechanisms behind our fascinating cognitive-, perceptual- and emotional abilities.

! 36! 5.!Bibliography((

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