Genome editing: modified CRISPR/Cas9 corrects genetic mutation causing Alzheimer’s disease or cancer without cleaving DNA
Genetic mutations account for a significant fraction of human disorders. Genetic analyses have shown that inherited mutations may predispose individuals to developing various illnesses. These include the mutant alleles of RB (retinoblastoma), p53 (Li-Fraumeni syndrome), APC (familial adenomatous polyposis), BRCA1/BRCA2 (breast cancer), VHL (von Hippel–Lindau disease) and other tumor suppressor genes (Knudson, 2002; Lee et al., 1994). Mutations leading to other types of disorders (ex. Bloom syndrome, xeroderma pigmentosum, hemophilia, cystic fibrosis and Duchenne muscular dystrophy) have also been identified. Increasingly, mutations affecting neural pathways, resulting in mental disorders affecting memory (ex. APP, APOEe4 for Alzheimer’s disease), behavior (ex. MAOA for aggression, autism), etc. are being uncovered.
Genetic mutations can also interfere with therapy. For the anticancer chemotherapeutic Taxol, specific mutations in the b-tubulin subunit gene confer resistance (Giannakakou et al., 1997). For tyrosine kinase inhibitors, the point mutation T790M in epidermal growth factor (EGF) receptor represents a common mechanism of resistance (Yun et al., 2008). In chronic myeloid leukemia, resistance to the targeted drug Gleevec could occur through specific point mutations in BCR/ABL tyrosine kinase. Consequently, researchers have strived to develop a means through which the predisposing mutation could be corrected to preempt the disorder or to restore therapeutic efficacy.
Initially, genome editing was attempted using zinc-finger nuclease (ZFN). ZFN is comprised of a DNA binding domain formed by zinc fingers (from transcription factors) and the DNA cleavage domain of bacterial restriction endonuclease Fok-1 (Gaj et al. 2013). Recognition of specific sequence by its DNA binding domain enables site-specific double strand cleavage. This property could be exploited to abolish a dominantly acting mutant allele if subsequently repaired by error-prone nonhomologous end joining (NHEJ) pathway. Alternatively, it may allow gene editing if repaired via homologous recombination (HR) by supplying a template DNA. A similar principle underlies genome editing by TALEN (transcription activator-like effector nucleases), which represents the DNA recognition motif of TAL effector (secreted bacterial protein) fused to Fok-1’s cleavage domain. The main caveat is that both require custom protein engineering for target sequence.
Clustered regularly interspaced short palindromic repeats (CRISPR) represent a region in bacterial genome where the double stranded DNA fragments of previously infected virus’ genome are captured and inserted at the ‘spacer’ loci by Cas1 and Cas2. The RNA transcribed from the spacer is processed into crRNA, which serves as guide RNA to direct Cas9 endonuclease to target DNA. Upon identifying a foreign DNA complementary to the spacer, Cas9 (also requires trans-activating tracrRNA) would cleave it to compromise the invading agent. To harness this bacterial immunity function for genome editing, tracrRNA and crRNA have been fused into a single guide RNA (sgRNA) molecule (Deltcheva et al, 2011). The CRISPR gene editing system can be directed to cleave cellular genome at any location by modifying guide RNA.
Nevertheless, CRISPR genome editing poses problems associated with mutagenesis at off-target sites as it can be inherited in the case of germ line. To resolve, investigators (Harvard University) modified Cas9 to edit genome without cleaving DNA. Catalytically inactive Cas9 was tethered to rAPOBEC1 cytidine deaminase, which was termed ‘based editor’ (Komor et al., 2016). It deaminated cytidine (C) to uridine, which converted to thymidine (T) following replication. The base editor (3rd generation) was able to correct mutation in apolipoprotein variant APOE4 (associated with Alzheimer’s disease) or p53 (linked to cancer) by causing C to T conversion. Using a similar strategy, another base editor containing TadA (E. coli tRNA adenine deaminase; converts adenine to inosine in tRNAArg) was used to deaminate adenine, driving A to G conversion in target DNA (Gaudelli et al., 2017). Subsequent analyses, however, revealed that cytosine base editor alters single nucleotides at a number of off-target sites in the genome (Zuo et al., 2019) whereas both cytosine and adenine base editors mutate numerous coding or non-coding RNAs in the transcriptome (Grunwald et al., 2019), suggesting that further improvements are necessary.
As an alternative approach, recent works have shown that the genome editing efficacy of CRISPR-Cas system can be improved by modifying sgRNA, i.e. by incorporating chemically modified nucleosides in the synthesis of sgRNA (Hendel et al. in 2015). Bio-Synthesis, Inc. specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA). It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides. To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade. Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA. The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance. It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe). More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.
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