Materials and Methods

“Designer babies?!” ACRISPR‐based learning module for undergraduates built around theCCR5gene

2.1. Intended audience and learning time

This work is intended for sophomore‐ and junior‐level undergraduate Biology students enrolled in a Genetics or Biotechnology course, but can be integrated and modified for Introductory Biology course or upper‐level Molecular Biology course. The main activity was implemented during a 2‐hour 45‐minute laboratory period. The activity can be adapted to fit two traditional 90‐minute class/lecture periods. Subsequent post‐activity student work where students created a research proposal was carried out over the next two laboratory periods and during students' own time.

2.1.1. Prerequisite student knowledge

Student should have prior exposure to basic molecular genetics principles that include DNA/RNA structure, codons, reading frames, mutation. However, this lesson can be adapted to introduce and apply the concepts of codons/mutations.

2.2. Materials

Each student will require a computer/laptop with access to the internet and the software program, SnapGene Viewer (full version, A free 30‐day trial to SnapGene Viewer can be downloaded by each student with their email address. Alternative molecular biology software could be used with modifications to the below resources.

The student handouts and accompanying instructor guide/key are provided in Supporting Information.

2.3. Instructions for faculty and students

2.3.1. Comparative analysis of CCR5 gene and modified allele variants on paper

The activity utilizes the gene encoding human CCR5; the intended target gene used in Dr. He's study. CCR5 gene encodes for a chemokine GPCR in T cells, and was targeted because of the known role of the CCR5 as a co‐receptor in HIV infection of humans. 28 CCR5 delta 32 (Δ32) is a naturally occurring allele lacking 32 nucleotides that correspond to a sequence that normally codes for part of the co‐receptors second extracellular loop. 28 Removing these 32 nucleotides results in a premature stop codon due to frameshift, and the resultant truncated protein product can no longer be exocytosed to the cell membrane. 29 Individuals homozygous for the CCR5 Δ32 variant are resistant to HIV infection, as CCR5 is required for membrane fusion during HIV infection. The HIV life cycle and the key molecular components of a HIV infection cycle in immune T cell can be introduced to students using this animation by Janet Iwasa. 30

Dr. He's goal of editing human embryos was to create a similar non‐functional HIV‐resistant CCR5 variants using CRISPR‐cas technology. 31 However, the results of these experiments have purportedly generated three new CCR5 allelic variants named after the twin girls, Lulu and Nana, with Lulu being heterozygotic for a novel CCR5 variant (the Lulu CCR5 allele) and wild‐type allele, and Nana being heterozygotic at the CCR5 locus (Nana CCR5 alleles 1 and 2 respectively). 4 , 31 He also claimed that a third child has been genetically modified in a similar manner.

To begin their comparative analysis of the different alleles, students were given a worksheet (Supporting Information) that contains the partial coding DNA sequences of an unmodified CCR5 allele, the HIV resistant allele Δ32 CCR5, and three novel alleles generated via CRISPR‐cas9‐based modification. They were tasked to determine the nucleotide differences between them and the effect on the reading frame, codon and amino acid (Figure ​(Figure2a,2a, example of student work; Table ​Table2,2, summary of CRISPR alleles). Students worked on their own initially and then in a group to compare answers.


Student work aligning unmodified CCR5 allele to HIV resistant Δ32 and CRISPR‐cas modified alleles (a) on paper, and (b) using computer software program SnapGene. B) shows all alleles aligned to the unmodified allele. Panel (c) shows an expanded alignment view with the change in reading frame and early stop codon (red asterisks). Expanded alignment views for other modified alleles are shown in Figure S1


The effect of CCR5 mutant alleles on reading frame and protein structure

AlleleNucleotide change in coding sequenceEffect on reading frame (RF)Effect on amino acidsProtein lengthProtein structureUnmodified _CCR5_Normal (RF +1)3527 TM domains with extracellular N′ and intracellular C′Delta 32 (Δ32) _CCR5_Deletion of 32 nucleotides (911..942)Frameshift (RF +3) leading to early stop codonNew amino acids on C terminus (31 different amino acids from AA185)215Lacks 3 TM domains, altered C′ protein sequence after TM 3Lulu allele (Delta 15)Deletion of 15 nucleotides (900..914)In‐frame deletionDeletion of 5 amino acids from AA181 in extracellular loop 2347Extracellular loop 2 missing 5 AA

Nana allele 1

(Delta 4)

Deletion of 4 nucleotides (913..916)Frameshift leading to early stop codon (RF +2)9 different amino acids on C terminus from AA186194Lacks 3 TM domains, altered C′ protein sequence after TM 3Nana allele 2 (Plus 1)Addition of 1 nucleotide at bp 911Frameshift (RF +3) leading to early stop codon42 different amino acids on C terminus from AA185(31 of the amino acids are the same as Δ32)226Lacks 3 TM domains, altered C′ protein sequence after TM 3Open in a separate window

2.3.2. Comparative analysis of CCR5 gene and modified allele variants on computer

Students were guided to obtain the full reference coding sequence of CCR5 from NCBI (Accession number NM_000579). They created a “new sequence” in the DNA analysis software SnapGene. Students were tasked to create “new features” that represent the three exons to show where intron/exon boundaries would exist in genomic DNA. Using the open reading frame feature, the students determine the translated protein sequence and normal length of protein. They were given the three other new allele variants (Δ32, Lulu, Nana allele 1 and Nana allele 2) that had been created by the instructor based on reported CRISPR modified sequences. 32 Students were guided to align these sequences together and determine the nucleotide, reading frame, amino acid sequence and protein length changes that were created. The use of the full version SnapGene is key in visualizing the alignment. Although NCBI and free DNA softwares ApE and Benchling enables alignment of sequences together, the alignment in Snapgene is presented in an effective scheme that enables students to still visualize the double‐stranded DNA molecule with polarity of each DNA strain, the resulting translation, and the features labeled in one window (Figure 2b,c, Figure S1). Students compared their analysis on SnapGene to their worksheet. Using SnapGene allows students to determine whether premature stop codons are formed further downstream from the nucleotide change.

2.3.3. Predicting the effect of the mutations on protein structure

The nucleotide sequence alignment allows students to visualize the effect on the linear primary sequence of the protein, but it is important that students move beyond the linear protein sequence and establish the effects of changes in the primary sequence to the tertiary structure. Students were shown published tertiary structures of the CCR5 protein 33 , 34 where the unmodified protein consists of seven transmembrane helices, characteristic of all G protein‐coupled receptors. Additionally, the web‐based 3D structure viewer iCn3D can be used by students to visualize the tertiary structure of the protein. The students were tasked to discuss and predict how the new alleles affect protein structure and draw the various predicted protein structures out on paper. Students of the Advanced Genetics class utilized the software BioRender (, a free biology drawing software to create their schematics. Examples of student work are shown in Figure 3a,b.


Student work depicting protein structure of CCR5 and predicted structures of its various modified versions (a) on paper and (b) using Biorender. Nucleotide sequences that were inserted or deleted are indicated, and nucleotides shared between Δ32 and Nana+1 alleles are highlighted

2.3.4. CRISPR‐cas9 editing design and strategy of CCR5 gene

Students were guided to design the strategy to program the cas9 nuclease to target and cut CCR5 gene at the appropriate region in order to create a loss‐of‐function CCR5 protein. Specifically, students identified and designed 3 key components: (a) the cas9 nuclear target sequence in CCR5, (b) the protospacer adjacent motif (PAM) sequence in CCR5 gene utilized by cas9, and (c) the complementary guide RNA that is guided to the target sequence by cas9 (Figure ​(Figure44).


Necessary components for CRISPR‐cas9‐based genetic modifications. (a) cas9 and gRNA form a complex, then gRNA base pairs with complementary sequence in CCR5 gene with PAM aiding in bringing cas9 nuclease in, resulting in cas9 double‐stranded cleavage 3 nucleotides from PAM sequence. (b) A comparison of endogenous DNA repair mechanisms, non‐homologous end joining (NHEJ) and homology directed repair (HDR), which utilizes a repair template containing the desired nucleotide change. Nana alleles Δ4 and +1 are shown as outcomes of NHEJ, and HIV resistant Δ32 is shown as an outcome of HDR with the use of an appropriate repair template

Using the reference unmodified CCR5 gene, students utilized the “features” tool on SnapGene to identify and annotate the cas9 specific PAM sequence (5′NGG3′) chosen by Dr. He that was presented in his oral presentation at the Second International Summit on Human Genome Editing meeting (Start at: 1:17:57). 8 The guanine dinucleotides of the PAM sequence that is on the non‐target strand of the gRNA interacts with arginine amino acids of cas9 to assist the unwinding of double‐stranded DNA for subsequence nuclease activity (as reviewed in Chen and Doudna 3 ). Next, students were guided to label the target sequence of the cas9 nuclease, which are the 20 nucleotides upstream of the 5′TGG3′ PAM sequence (Figure ​(Figure5a).5a). The cas9 nuclease is brought to the target sequence by the single guide RNA, which is composed of an RNA sequence complementary to the target strand and an 80‐mer universal scaffold region that aids in cas9 binding. To model formation of gRNA‐cas9‐target DNA complexes, students were tasked to hand draw how all these components interact, labeling important components and sites including their PAM sequence, the location of the cas9 cut site, the annealing of the gRNA to its specific target nucleotide sequence. We intentionally had students draw this out based on our prior teaching experiences where assessment revealed students found it challenging to conceptualize the action and polarity of gRNA and cas9 complex with two complementary DNA strands (target and non‐target strands) of the target gene. 19


Key components utilized by CRISPR‐cas9 system. View in SnapGene of (b) PAM sequence (green) and gRNA‐cas9 target sequence (light green) are annotated in unmodified CCR5 allele in top half. Potential PAM sequences are highlighted in yellow. Bottom half shows the alignment with Δ32 allelic variant to allow visualization of which PAM sequence is most appropriate. (b) An example HDR repair template lacking the 32 nucleotides missing in the Δ32 allelic version, and containing two homology arms. The blue line indicates where the 32 nucleotides in Δ32 allelic version has been deleted. (c) Student work showing a schematic of gRNA and cas9 complexing with target sequence in optix gene

Students were prompted to discuss why this specific PAM associated with the target sequence was chosen of all possible PAM sequences of (5′NGG3′) as a quick “Find” of “GG” highlights in yellow all the GGs present in the sequence (yellow boxes, Figure ​Figure5a),5a), and students observe that there are many potential PAM sequences present throughout the gene. The discussion led the class to conclude that this specific 5′NGG3′ was chosen of many potential 5′NGG3′ because of the necessity for cas9 cut the CCR5 genomic DNA in close proximity to the physical location of the desired nucleotide change that could potentially give rise to Δ32 allele variant.

2.3.5. Interpreting DNA repair mechanisms that created Nana and Lulu alleles; designing a homology directed repair (HDR) repair template

Identification of the likely CCR5 PAM sequence and cas9 cut site was followed by discussion of the outcomes for the CCR5 gene after cas9 cleavage, and how different endogenous repair mechanisms are used to achieve different research goals. Animation are shown to illustrate the two processes. 35 , 36 The process of non‐homologous end joining (NHEJ) is used after cas9 makes the double stranded cut to produce indels and potential loss‐of‐function alleles due to frameshift mutations. Homology Directed Repair (HDR) is typically utilized when a specific nucleotide change is desired. HDR requires the addition of a repair template with homology arms that enables the cell to utilize HDR to incorporate a desired nucleotide sequence after the cas9‐mediated dsDNA cut. As a class, we discussed the features that the HDR must contain: (a) the desired nucleotide change, (b) regions of DNA that were similar to the original allele (homology arms), and (c) a PAM sequence that was altered such that the repair template would not be cut by cas9. Students were guided to create an HDR template in SnapGene (Figure ​(Figure5b).5b). Students were then asked to work in groups to discuss which DNA repair strategy was likely utilized by Dr. He to produce the three new variant alleles found in Nana and Lulu. Most students report that NHEJ occurred in the embryos to create the three new variants and that a repair template was likely not used as the HIV resistant Δ32 allele was not found in Lulu and Nana.

2.3.6. Putting knowledge into practice: CRISPR‐cas9 gene editing strategy with butterfly wing patterning gene optix and human oncogenic Ras gene

Students worked on two follow‐up hypothetical scenarios to practice the CRISPR design and strategy of sgRNA and target sequence. The first scenario was that students were investigating the function of the gene optix in Lepidoptera butterflies, and were tasked to design a CRISPR‐cas9 strategy to create a loss‐of‐function optix gene. The students were shown published results of a CRISPR‐cas9 strategy, where optix CRISPR‐ed butterflies displayed color and patterning defects. 37 An example of student work is shown in Figure ​Figure5c.5c. The second scenario was that they had cultured cancer cells growing in lab with Ras gene that has the oncogenic mutation that causes abnormal cell proliferation. 38 , 39 The students' goal was to design a strategy to take this mutant Ras and genetically modify it to wild‐type Ras. They were given the coding sequence for the Ras gene in SnapGene with the oncogenic mutation causing 12th amino acid Glycine to Valine change (G12V). Students were asked to discuss in pairs what they would predict the phenotypic change would be after successful gene edit. The work was submitted to be checked by the instructor.

2.3.7. Bioethics discussion on implications of CRISPR‐cas9 technology and CCR5 editing in human society

Following the exercise, we engaged the students in a robust bioethical discussion on the bioethical implications of CRISPR‐cas9 technology and the modification of the CCR5 gene. Questions that were posed to students for discussion are included in the Tables S2 and S3. One topic was the discussion of the novel and Δ32 mutations on CCR5's normal function in human health and disease. Whether Dr. He's novel CCR5 mutations in the twins actually confer HIV protection or has an effect on normal immune function has not been published yet. We asked students whether Lulu and Nana should be monitored by a specialized group of doctors and researchers for unintended consequences of their genetic edits since they have novel modified alleles that have not been studied before. Since Dr. He's made his claim, studies show that humans who are carriers for the Δ32 CCR5 allele have a faster recovery from strokes. 40 Furthermore, CCR5 plays a role in brain cognition, as mice that lack CCR5 have improved memory. 41

Article Title“Designer babies?!” ACRISPR‐based learning module for undergraduates built around theCCR5gene


CRISPR‐cas technology is being incorporated into undergraduate biology curriculum through lab experiences to immerse students in modern technology that is rapidly changing the landscape of science, medicine and agriculture. We developed and implemented an educational module that introduces students to CRISPR‐cas technology in a Genetic course and an Advanced Genetics course. Our primary teaching objective was to immerse students in the design, strategy, conceptual modeling, and application of CRISPR‐cas technology using the current research claim of the modification of theCCR5gene in twin girls. This also allowed us to engage students in an open conversation about the bioethical implications of heritable germline and non‐heritable somatic genomic editing. We assessed student‐learning outcomes and conclude that this learning module is an effective strategy for teaching undergraduates the fundamentals and application of CRISPR‐cas gene editing technology and can be adapted to other genes and diseases that are currently being treated with CRISPR‐cas technology.

Login or Signup to leave a comment
Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact or check out our support page.