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  • CRISPR-Cas9 Genome Editing

    20160617 Lab Animal Pathology Rounds

    Han Tan ( TwiCer: @ekhtn

  • Why Genome Editing?

    Accelerate basic research Generate mulGple alleles Generate mulGple gene mutaGons

    Analysis of linked genes Analysis of lethal genes

    Disease modeling Gene Therapy Replace defecGve genes Fix specific cell-types

    Agriculture Non-transgenic approaches to improve crops

    Genome engineering of plants and animals

    Biotechnology Ecological control of vectors that transmit diseases

    SyntheGc biology

  • Engineering POLLED Phenotype in Dairy Cows


    confirmed the homozygous introgression of PC into RCI-001, RCI-002 (named Spotigy) and RCI-003 (named Buri), and heterozygous introgression into RCI-004 (RCI, Recombinetics, numbered in order of birth), whereas progenitor cells (2120) were negative for the PC allele (Fig. 1a,b). Two healthy, homozygous polled animals, Spotigy and Buri (Fig. 1c,d and Table 1), which are now more than 10 months old, were phenotypically polled.

    To evaluate off-target effects in two distinct edited lines, we sequenced the genomes of RCI-001 and RCI-002, derived from clones HP14-B4 and HP7-P4-A1 respectively, along with those of their progenitor cell lines, 2122 and 2120 respectively (Table 1). We did not identify

    costly but is also painful for the animals and has come under scrutiny owing to public concerns about farm animal welfare3,4. Numerous animal advocacy groups have campaigned for mandated anesthesia during dehorning or complete cessation of dehorning as a management practice. In response to this sentiment, retailers such as Wal-Mart, Starbucks, Nestle and Kroger prioritize dehorning in their animal welfare policies5. The increased use of polled Holstein sires has been impeded by their lower estimated breeding values (a genetic merit score) for milk production, likely a result of genomic drag due to POLLED introgression from non-dairy animals. Even if producers ignore the substantial value difference of $252 per lactation cycle between horned and polled animals, it would still take >20 years of classic breeding to reach a frequency of 50% polled animals6. Further attempts to increase the frequency of polled animals in dairy herds by cross-breeding with other polled breeds, such as Angus, is not feasible because the total genetic merit for milk production would suffer losses, and the genetic admixture would take many generations to unwind7.

    Identification of the genetic cause of hornlessness in cattle has been the subject of intensive genetic and genomic research, culminating in the nomination of two different candidate neomutations on cattle chromosome 1 that are predicted to have arisen 500 1,000 years ago8: a complex allele of Friesian origin (PF), an 80,128 base pair (bp) duplication (19093521989480 bp)9, and a second, simple allele of Celtic origin (PC) corresponding to a duplication of 212 bp (chromosome 1 positions 17058341706045) in place of a 10-bp deletion (17060511706060).

    We report the use of genome editing using transcription activator-like effector nucleases (TALENs) to introgress the putative PC POLLED allele into the genome of bovine embryo fibroblasts to try and produce a genotype identical to what is

    achievable using natural mating, but without the attendant genetic drag and admixture7. In our previous studies, we used TALEN-stimulated homology-dependent repair to produce four cell lines either homozygous or heterozygous for the PC allele10 (Table 1). Each of the four lines were cloned by somatic cell nuclear transfer11, and full-term pregnancies were established for three of the four lines. In total, five live calves were produced, representing two different dairy genetic backgrounds, 2122 and 2120 (Table 1). When calves were born, a board-certified veterinarian inspected each of the live-born calves visually and by palpation for horn buds, and observed none, which suggested polledness. Analysis of the polled genotype using diagnostic PCR10

    Table 1 Animal production statistics

    Cell line Parental cells Genotype SCNT rep Blast rate (%)Embryos/ recipients

    Pregnant at day 40

    Pregnant at day 90 Liveborn Alive at 60 d

    HP14-B6 2122 Homozygous POLLED 1 27/64 (42) 9/9 6 0 0 0

    HP14-B4 2122 Homozygous POLLED 1 3/15 (20) 1/1 1 1 1a 0

    HP7-P4-A1 2120 Homozygous POLLED 2 25/82 (30) 9/9 2 2 2 2b

    HP-24.8 2120 Heterozygous POLLED 3 35/151 (23) 7/7 5 2 2a 0

    Summary 70/295(24%)

    26/26 14/26(54%)




    aRCI-001 (HP14-B6), RCI-004 and RCI-005 (HP-24.8). Consistent with known cloning inefficiencies, these animals were not viable and were humanely euthanized within 24 h of birth11. bSpotigy (RCI-002) and Buri (RCI-003). SCNT rep, somatic cell nuclear transfer replicates.

    Figure 1 Phenotypic and genotypic confirmation of POLLED introgression in Spotigy and Buri. (a,b) Diagnostic PCRs for the Pc allele using primer pairs btHP-F1 + btHP-R2 (a) and HP1748-F1 + HP1594_ 1748-R1 (b) (Supplementary Methods), respectively, confirmed homozygous introgression in RCI-001, Spotigy (RCI-002) and Buri (RCI-003), and heterozygous introgression in RCI-004 relative to the donor cell line that is negative. The identity of PCR products was confirmed by Sanger sequencing. The positive control (p1748) was a plasmid containing the Pc allele10. (c) Photograph of Spotigy at 2 months of age, so named after the black spots where horn buds would have developed. (d) Photograph of Buri (left) and Spotigy at 2 months of age.

    900 3,0002,5002,000















    a b

    c d

    Size (bp)

    Size (bp)


    (Carlson et al., Nature Biotech. 2016)

    *Instant introgression directly into elite breeds bypasses tradiGonal breeding

  • Outline

    Genome EdiGng

    The CRISPR-Cas9 System

    The many flavors of Cas9

    Genome EdiGng in the lab and beyond

  • What is Genome Editing?

    Targeted modificaGon to DNA


    Using customized sequence-specific nucleases

  • Zinc Finger Nucleases (ZFNs)

    (Baltes et al., Trends in Biotech. 2015)

  • Transcription Activator-Like Effector Nucleases (TALENs)

    (Baltes et al., Trends in Biotech. 2015)

    N- -C

    Repeat Variable Diresidue

  • Cas9





  • Comparisons


    DNA recogni=on MulGmeric protein DNA interacGon

    Protein DNA interacGon

    RNA DNA Watson-Crick base-pairing

    DNA cleavage Coupling to non-specific nuclease FokI

    Coupling to non-specific nuclease FokI

    Innate to Cas9

    Requirements Two large protein constructs

    Two large protein constructs

    Simple 20nt change to construct

    Targe=ng Poor Good Good

    Feasibility Difficult Difficult Easy


    as of 6/16/16

  • Outline

    Genome EdiGng

    The CRISPR-Cas9 System

    The many flavors of Cas9

    CRISPR-Cas9 in the lab and beyond

  • Discovery of CRISPR-Cas

    CRISPR = Clustered Regularly Interspersed Short Palindromic Repeats [DNA repeats]

    Cas = CRISPR associated [Protein coding sequences]

    Discovered in 1987 from the analysis of E. coli genomes (Ishino et al., J. Bacteriol. 1987)

    Is important for adapGve immunity in bacteria and archaea

  • Type II CRISPR-Cas

    activity, whereasmutating both domains (dCas9;Asp10 Ala, His840 Ala) results in an RNA-guidedDNAbinding protein (64, 65). DNA targetrecognition requires both base pairing to thecrRNA sequence and the presence of a short se-quence (PAM) adjacent to the targeted sequencein the DNA (64, 65) (Fig. 2).The dual tracrRNA:crRNA was then engineered

    as a single guide RNA (sgRNA) that retains twocritical features: the 20-nucleotide sequence atthe 5 end of the sgRNA that determines theDNAtarget site by Watson-Crick base pairing, and thedouble-stranded structure at the 3 side of theguide sequence that binds to Cas9 (64) (Fig. 2).This created a simple two-component system inwhich changes to the guide sequence (20 nucleo-tides in the native RNA) of the sgRNA can beused to program CRISPR-Cas9 to target any DNAsequence of interest as long as it is adjacent toa PAM (64). In contrast to ZFNs and TALENs,which require substantial protein engineering foreach DNA target site to be modified, the CRISPR-Cas9 system requires only a change in the guideRNA sequence. For this reason, the CRISPR-Cas9

    technology using the S. pyogenes system has beenrapidly and widely adopted by the scientific com-munity to target, edit, or modify the genomes of avast array of cells and organisms. Phylogeneticstudies (6971) as well as in vitro and in vivoexperiments (64, 71, 72) show that naturallyoccurring Cas9 orthologs use distinct tracrRNA:crRNA transcripts as guides, defined by thespecificity to the dual-RNA structures (6971) (Fig.3). The reported collection of Cas9 orthologs con-stitutes a large source of CRISPR-Cas9 systems formultiplex gene targeting, and several orthologousCRISPR-Cas9 systems have already been appliedsuccessfully for genome editing in human cells[Neisseria meningitidis (73, 74), S. thermophilus(73, 75), and Treponema denticola (73)].Although the CRISPR acronym has attracted

    media attention and is widely used in the scien-tific and


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