New genome editing technologies:

New genome editing technologies work well in Xenopus. CRISPR/Cas and TALEN/ZFN editing technologies function by inducing site-specific DNA strand breaks anywhere in the Xenopus genome. Mutations are induced by inefficient, error-prone non-homologous end joining (NHEJ). In addition, site-specific DNA breaks promote precise knock-in, homologous recombination (HR) gene editing (Gaj et al (2013), Menke (2013)).

For developing your own custom CRISPR/TALEN transgenic frogs, research housing and facilities are available at the National Xenopus Resource (NXR) at Woods Hole, MA. Please contact the NXR at: Xenopus@mbl.edu

Design Tools:

CRISPR/Cas:

CRISPR/Cas (clustered regularly interspaced short palindromic repeats-associated/CRISPR associated) systems are taken from host-defense mechanisms of the bacterium Streptococcus pyogenes (Barrangou (2012), Bilkard & Marraffini (2013)). CRISPR RNA (crRNAs) homologous target sequences are followed by a tri-nucleotide protospacer adjacent motif (PAM) - NGG. crRNAs basepair with transactivating CRISPR RNA (tracrRNA) which together bind Cas9 endonuclease enzyme. This complex binds DNA in a sequence-specific manner. Genomic sequences are corrected to maintain genomic stability by binding and cleaving inserted foreign DNA.

For use in animal systems like Xenopus (Mashimo (2014), Sampson & Weiss (2014)), the RNA components of the bacterial CRISPR/Cas proofreading machinery are combined in a single construct: synthetic guide RNA (sgRNA). sgRNA constructs contain a ~20 nucleotide homologous sequence for gene targeting placed adjacent to a NGG PAM followed by tracrRNA scaffold. Co-injection of this construct with mRNA encoding Cas9 in early fertilized eggs directs site-specific DNA binding and cleavage. A structure of the DNA/RNA/Cas9 complex has recently been released. Mutations are introduced by DNA strand breaks followed by NHEJ and/or HR. In designing sgRNA, a BLAST should be run on the targeting sequence against the genome to prevent off-target binding as sequences with up to 5 base mismatches can still mediate unwanted hybridization.

Xenbase literature search for CRISPR

TALENs/ZFNs:

Utilizing nucleotide specific binding abilities of transcription activator-like effector nucleases (TALENs) and zinc-finger endonucleases (ZFNs), researchers can custom design DNA binding proteins for site-specific targeting (Sun & Zhao (2013)). TALEs and ZFs are coupled with the cleavage domain of FokI endonuclease to target double stranded DNA breaks. Like CRISPRs, TALENs and ZFNs cause mutations by inefficient DNA repair (NHEJ). TALE repeats bind individual nucleotides, whereas ZFs bind DNA triplets. For this reason, ZFN specificity is trickier to engineer compared to TALENs.

Xenbase literature search for TALEN

Select reviews of CRISPR, TALEN, and ZFN technologies:

• Nakade (2014)
• Mashimo (2014)
• Sampson & Weiss (2014)
• Bilkard & Marraffini (2013)
• Gaj et al (2013)
• Barrangou (2012)
• Menke (2013)
• Sun & Zhao (2013)