Nonviral Delivery of CRISPR/Cas Systems in mRNA Format

Abstract

From the battleground of bacteria and archaea against virus and plasmid DNA (pDNA), the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated (Cas) gene system gives an efficient and promising toolbox for genetic engineering. [1][2][3][4][5] Compared with other genome editing technologies, such as transcription activator-like effector nucleases (TALEN) and zinc-finger nucleases (ZFN), the CRISPR/Cas system lowers the bar of conducting genome editing experiments because it does not require redesign of nucleases to recognize distinct target gene. [6] With only one decade of development, the CRISPR/Cas systems have emerged as a revolutionary engineering tool for sequence-specific genome modifications and have brought significant advances in gene therapy, developmental biology, cancer research, etc. [7] Currently, dozens of CRISPR/Cas-based therapeutics have entered clinical trials, paving the way for precision medicine. [8][9][10] The CRISPR/Cas systems can be classified into two categories (class 1 and class 2) according to the number of Cas proteins involved. [11] The class 1 systems contain multiple Cas subunits, while class 2 systems only contain a single Cas effector protein. [11] The simplicity and flexibility of type II (CRISPR/Cas9) and V (CRISPR/Cas12) systems from class 2 make them more suitable for genetic engineering. [12] Both CRISPR/Cas9 and CRISPR/Cas12 utilize their own single-guide RNAs (sgRNAs) and protospacer adjacent motifs (PAM) to recognize and bind the target gene. [2,13,14] After the formation of the R-loop, the RNA-bounded Cas protein undergoes conformational rearrangement, and induces DNA double-strand break (DSB). [15,16] Eventually, the DNA repair system in eukaryotic cells is activated to repair the cleaved DNA primarily by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways (Figure 1). NHEJ is usually considered as a highly effective but error-prone mechanism due to the direct ligation of the broken DNA. The ligation process is easy to introduce random insertions or deletions (indels) at the target loci. [17] Thus, NHEJ repair is widely employed in treating diseases caused by the overexpression of abnormal proteins, for instance, transthyretin amyloidosis. [6] The HDR pathway uses homologous DNA templates to repair DNA lesions. [18] As a result, diseases caused by loss-of-function mutations, such as hemophilia, phenylketonuria, and X-linked retinitis pigmentosa, can benefit from CRISPR/Cas-mediated HDR. [10,19] In addition to NHEJ and HDR, alternative mechanisms for DSB repair including homology-mediated end joining (HMEJ), microhomology-mediated end joining (MMEJ), and homology-independent targeted integration (HITI) were also developed to broaden the scope of CRISPR/Cas system. [20][21][22][23] Although CRISPR/Cas systems hold great potential for therapeutic applications, there are two major challenges that need to be addressed. [24] The first challenge is to minimize off-target effects. [25] The second one i...

Intracellular Delivery of mRNA for Cell‐Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles

“…Thus, it is highly desirable to reduce the risk of off‐target editing by regulating the activity of CRISPR RNA delivery [81–83] . In contrast, delivering CRISPR RNA by using LNPs might provide cell‐selective targeting and enable genome editing in specific cell types, thus reducing the risk of off‐target effects [84] . To increase the specificity of CRISPR RNA delivery and reduce off‐target effects, recent studies have focused on the design of biodegradable LNPs with cell‐selective targeting.…”

“…[81][82][83] In contrast, delivering CRISPR RNA by using LNPs might provide cellselective targeting and enable genome editing in specific cell types, thus reducing the risk of off-target effects. [84] To increase the specificity of CRISPR RNA delivery and reduce off-target effects, recent studies have focused on the design of biodegradable LNPs with cell-selective targeting. For example, the introduction of disulfide bonds into the hydrophobic tail of ionizable lipids has been shown to promote endosomal escape and intracellular release of Cas9 mRNA, while also allowing for degradation under the action of intracellular glutathione.…”

A PEG-lipid-free COVID-19 mRNA vaccine triggers robust immune responses in mice

“…[1][2][3][4][5] As one of the most advanced delivery technologies, LNPs show broad applications ranging from genome editing to mRNA vaccines. [1][2][3][4][5][6][7][8][9][10] In recent years, major breakthroughs have been made in the field of mRNA vaccines as evidenced by the emergency use authorization of two LNP-formulated COVID-19 mRNA vaccines from Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273) at an unparalleled speed. 6,9,11,12 Both vaccines exclusively utilize PEGylated LNPs to deliver nucleoside-modified mRNA encoding the spike protein of SARS-CoV-2.…”

“…[15][16][17][18][19][20][21][22] LNPs confer mRNA resistance to nuclease degradation and facilitate its cellular uptake. [1][2][3][4][5][6][7][8][9][10] Typically, LNPs are comprised of four types of lipids including key lipids, phospholipids (helper lipids), cholesterol, and polyethylene glycol-conjugated lipids (PEG-lipids). 6,11 Among them, the key lipids are always ionizable or cationic lipids that drive self-assembly and facilitate intracellular delivery.…”

“…6,11 Among them, the key lipids are always ionizable or cationic lipids that drive self-assembly and facilitate intracellular delivery. 6,7,[9][10][11] The helper lipids and cholesterol contribute to stability, cellular uptake, endosomal escape, etc. 6,7,11 The remaining polyethylene glycol (PEG)-lipids play a critical role in preventing the aggregation and prolonging the half-life of LNPs.…”