the target protein on the matrix. Following the interaction of the primary antibody with the target protein, any unbound antibody is washed away, and the matrix is treated with a second antibody (secondary antibody) that is specific for the primary antibody. The secondary antibody is attached to an enzyme, such as alkaline phosphatase, that converts a colorless substrate to a colored or light-emitting (chemiluminescent) product that can readily identify positive interactions.
Figure 2.16 Screening of a genomic DNA library using an immunological assay. Transformed cells are plated onto solid agar medium under conditions that permit transformed but not nontransformed cells to grow. (1) From the discrete colonies formed on this master plate, a sample from each colony is transferred to a solid matrix such as a nylon membrane. (2) The cells on the matrix are lysed, and their proteins are bound to the matrix. (3) The matrix is treated with a primary antibody that binds only to the target protein. (4) Unbound primary antibody is washed away, and the matrix is treated with a secondary antibody that binds only to the primary antibody. (5) Any unbound secondary antibody is washed away, and a colorimetric (or chemiluminescent) reaction is carried out. The reaction can occur only if the secondary antibody, which is attached to an enzyme (E) that performs the reaction, is present. (6) A colony on the master plate that corresponds to a positive response on the matrix is identified. Cells from the positive colony on the master plate are subcultured because they may carry the plasmid–insert DNA construct that encodes the protein that binds the primary antibody.
Genome Engineering Using CRISPR Technology
Recently, researchers have designed strategies to insert, replace, or disrupt sequences at targeted sites in intact genomes in vivo. The method is based on a prokaryotic system that protects bacteria against invasion by foreign DNA such as bacteriophage genomic DNA and plasmids. This is a type of bacterial adaptive immune system that consists of genomic clustered regularly interspaced short palindromic repeats (CRISPR) containing fragments of foreign DNA molecules that the bacterium was previously exposed to and CRISPR-associated (Cas) proteins, including an endonuclease that cleaves homologous foreign DNA upon subsequent exposures. The CRISPR-Cas system has been adapted to introduce or replace genes in the genomes of a variety of organisms, both prokaryotes and eukaryotes, and also to edit genomes, that is, remove or alter targeted nucleotides (chapters 6 and 12).
In the natural bacterial CRISPR-Cas systems, short sequences (“protospacers”) from an invading DNA molecule are incorporated as “spacers” between repeat sequences in the CRISPR locus of the bacterial genome (Fig. 2.17A). Thus, the CRISPR locus contains an array of spacers separated by repeat sequences that are a record of past foreign DNA invasions from which the bacterium survived. When the bacterium is subsequently invaded by a virus or plasmid whose DNA contains a sequence that is homologous to a spacer sequence, the spacer DNA is transcribed, producing a CRISPR RNA (crRNA) molecule that binds to and guides the Cas endonuclease complex to the target sequence in the invading DNA, which is cleaved (Fig. 2.17B). Recognition of the target sequence on the invading DNA requires that it is adjacent to a short specific sequence known as a protospacer adjacent motif (PAM). For example, the Streptococcus pyogenes endonuclease Cas9 recognizes a target sequence that is complementary to a crRNA only if it is immediately upstream of the motif NGG (where N is any nucleotide). PAMs are also important for selection of protospacers during spacer acquisition. The PAM requirement prevents cleavage of the bacterium’s own genome at sequences that are complementary to the crRNA, including the site in the CRISPR array from which the crRNA was transcribed, which lacks a PAM.
Figure 2.17 Bacterial CRISPR-Cas system for protection against invading bacteriophage. (A) Fragments of bacteriophage DNA (protospacer) are incorporated into the host bacterial genome as spacers between repeat sequences (gray) in the CRISPR array. (B) On subsequent invasion, the spacer DNA is transcribed to produce CRISPR RNA (crRNA) that guides an endonuclease (Cas) to a sequence in the invading DNA that is homologous to the spacer sequence and is adjacent to a protospacer adjacent motif (PAM). The viral genome is cleaved. Adapted by permission from Macmillan Publishers Ltd. from Yosef and Qimron, Nature 519:166–167, 2015.
Because of its relative simplicity compared to systems in other bacteria, the CRISPR-Cas system from S. pyogenes has been adapted for use as a genome engineering tool. In the natural S. pyogenes system, two RNA molecules, crRNA and transactivating crRNA (tracrRNA), form a crRNA:tracrRNA hybrid that directs the Cas9 endonuclease to the target site. For ease of use in genome engineering, the two RNAs are combined into a single guide RNA (sgRNA) that is 80 to 100 nucleotides long. The sgRNA is designed to include a 20-nucleotide sequence that is complementary to the target site (which is located adjacent to a PAM), and the fused crRNA:tracrRNA sequence that forms a stem loop structure involved in endonuclease binding (Fig. 2.18A). Following binding to the target sequence, the endonuclease makes a double-stranded break in the target DNA (Fig. 2.18B). This damage activates the cellular systems for DNA repair either by homologous recombination, in which DNA sequences with sufficient similarity are exchanged, or nonhomologous end joining, in which sequences are deleted or inserted. The repair systems can be harnessed to disrupt, insert, or replace a DNA sequence at a targeted site.
Figure 2.18 CRISPR-Cas system for genome editing. (A) An 80- to 100- nucleotide long single guide RNA (sgRNA) is constructed that contains a 20-nucleotide guide sequence (orange) that is complementary to the target site. The secondary structure, stabilized by intramolecular base-pairing between regions of the fused crRNA and tracrRNA sequences, is required for binding to the Cas9 endonuclease. (B) The sgRNA guides Cas9 to the target sequence (blue) in the genome. Target recognition requires an adjacent PAM sequence (red) NGG and complementarity between the guide sequence and the target sequence. Cas9 makes a double-stranded break in the target DNA (arrows) which is repaired by homologous recombination or nonhomologous end joining. The repair systems generate deletions and insertions at the target site.
Insertion of a DNA sequence (donor sequence) into a target site requires introduction of the sequences for the sgRNA, the Cas9 endonuclease, and the donor DNA into a recipient cell. The sgRNA and Cas9 coding sequences may be introduced on a vector (Fig. 2.19A), or the sgRNA and Cas9 mRNA may be directly injected along with the donor DNA. When the genes are introduced on a vector, the promoters that drive expression of the sgRNA and endonuclease, and the coding sequence (e.g., codon usage) for the endonuclease are optimized for expression in the chosen host. The donor DNA sequence is flanked by sequences that are homologous to the target genomic site for insertion by homologous recombination (Fig. 2.19B). The vector is introduced into the recipient cell and following expression of the sgRNA and the endonuclease the recipient cell genome is cleaved at the target site. Activation of recombinases that mediate DNA repair results in recombination between homologous sequences on the vector and in the recipient genome, and thereby, insertion of the donor DNA into the genome at the target site (Fig. 2.19B).
Figure 2.19 Vector for production of sgRNA and Cas9 in host
