Chemically Modified Nucleic Acids for CRISPR-Cas
Chemically modified nucleic acids allow for the enhancement of genome editing efficiency using CRISPR-Cas system type II. Recently scientists showed that the CRISPR Cas system type II can be used with artificially modified nucleic acids that were synthetically incorporated into CRISPR guide RNA (gRNA).
Recently, Hendel et al. in 2015 reported the use of chemically modified and protected nucleoside phosphoramidites for the synthesis of single guide RNAs (sgRNAs) to enhance genome editing efficiency in human primary T cells, CD34+ hematopoietic stem and progenitor cells. The researchers argued that co-delivery of chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system. According to Hendel et al. this approach is a simple and effective way for the development of new genome editing methods. This technique is thought to potentially accelerate the development of a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
Hendel et al. used three different modifications for the synthesis of sgRNAs. The modified nucleosides 2’-O-methyl (M), 2’-O-methyl-3’-phosphorothioate (MS), and 2’-O-methyl-3’-thiophosphonoacetate (MSP) were incorporated as protected nucleoside phosphoramidites. However, other modified nucleosides, such as bridged nucleic acids, can be used as well. The modified nucleic acids were incorporated at the 5′ and 3′ terminal positions of the synthetic sgRNAs.
Figure 1: Structures of chemical modifications that can be incorporated into RNAs, in this case into sgRNAs. Structures of RNA dimers and modified RNA chimeras are shown.
Many scientists now use the CRISPR-Cas system for targeted gene editing. Because of its ease of use CRISPR-Cas appears to have become the gene editing tool of choice. The increasing number of papers investigating the CRISPR-Cas systems and their use illustrate this nicely. Figure 1 shows the number of published papers as a result of a Pubmed search.
Figure 2: Numbers of published CRISPR-Cas papers in Pubmed.
The CRISPR (clustered regularly interspaced short palindromic repeat) loci together with a diverse cassette of CRISPR-associated (Cas) genes provide a sophisticated adaptive immune system to bacteria and archaea. The pre-crRNA is encoded in the CRISPR locus. This locus consists of repeat and spacer sequences. In some cases, the repeat sequences fold into stem-loop structures. The spacer sequences originate from previously cell attacking invader DNA. CRISPR locus transcription starts from a leader region yielding the pre-crRNA. The pre-crRNA is processed to generate crRNAs. Each crRNA is specific for one invader. (Li, et al., 2015).
Figure 2: The CRISPR locus. The CRISPR locus encodes the pre-crRNA that consists of repeat and spacer sequences. Some of the repeat sequences fold into stem-loop structures. Spacer sequences are derived from invader DNA from previous cell attacks. CRISPR locus transcription starts from the leader region (black arrow) generating pre-crRNA. The pre-crRNA is subsequently processed into crRNAs, and each crRNA is specific for one invader. (Maier et al., 2012).
The CRISPR Cas base immune defense proceeds in three stages:
Invading nucleic acid of the invading element enters the cell. This is immediately recognized as a foreign element. A piece of the invader DNA (the protospacer) is selected and integrated into the CRISPR locus as a new spacer. The protospacer as part of the invading DNA sequence is called a spacer after integration into the CRISPR locus. Selection of a new spacer depends on the presence of a specific neighboring sequence, the protospacer adjacent motif (PAM). This has been shown to be the case for CRISPR-Cas systems type I and type II.
The CRISPR locus is expressed. A pre-crRNA is generated and subsequently processed to short crRNAs. Each crRNA is specific for a single invader sequence.
Cas proteins together with crRNA recognize the invader during the defense reaction. The spacer sequence of the crRNA form base pairs with the invader sequence from which it was derived and hybridizes with it. This makes the defense sequence specific.
CRISPR-Cas immune systems are classified into three main types and eleven or more subtypes. All CRISPR-Cas systems operate through three stages: acquisition, CRISPR RNA (crRNA) biogenesis, and target interference. CRISPR-Cas based genome editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage. The Cas endonuclease facilitates the cleavage. CRISPR-derived RNAs (crRNAs) together with Cas proteins capture and degrade invading genetic materials in prokaryotes.
Figure 3: Models of the CRISPR-Cas systems (2011). CRISPR-Cas systems act in three stages: adaptation, expression, expression, and interference. In type I and type II: Selection of proto-spacers in invading nucleic acid may depend on a protospacer-adjacent motif (PAM). How exactly this works, is presently intensively investigated. (Stage 1) It is thought that during the initial step Cas1 and Cas2 proteins incorporate the proto-spacers into the CRISPR locus by forming spacers. (Stage 2) During the expression stage, the CRISPR locus with the spacers is expressed. A long primary CRISPR transcript (pre-crRNA) is produced. Next, the CRISPR-associated complex for antiviral defense (Cascade) complex binds the pre-crRNA. Cas6e and Cas6f subunits cleave the pre-crRNA to produce crRNAs with a typical 8-nucleotide repeat fragment on the 5’ end and the remainder of the repeat fragment. In general, the repeat fragment forms a hairpin structure on the 3’ flank. Type II systems use a trans-encoded small RNA (tracerRNA) that pairs with the repeat fragment of the pre-crRNA. After binding, the Cas9 RNase III complex cleaves the RNA within the repeats. During maturation, spacers are cleaved at a fixed distance within the spacers. In type III systems, Cas6 is starting the processing of crRNA. In a the next step crRNA appears to be transferred to a different Cas complex called Cms in subtype III A and Cmr in subtype III B. In subtype III B, the 3’ end of crRNA is trimmed further. (Stage 3) The invading nucleic acid is cleaved during the interference step. Type I systems: crRNA guides the Cascade complex to targets that contain complementary DNA. The Cas3 subunit is thought to cleave the invading DNA. In addition, the PAM most likely is important for target recognition. Type II systems: Ca9 is loaded with crRNA and is thought to target directly invading DNA. This process requires PAM. Type III systems: A chromosomal CRISPR locus and an invading DNA fragment. Subtype III A systems target DNA and subtype III B systems target RNA. Base pairing to the 5’ repeat fragment of the mature crRNA results in no interference and no base pairing results in interference. (Makarova et al. 2011).
Type I and II bacterial CRISPR-Cas9 system
The selection of proto-spacers in invading nucleic acid in type I and type II CRISPR-Cas systems probably depend on a proto-spacer-adjacent motif (PAM). The type II bacterial CRISPR-Cas9 system consists of an RNA-guided nuclease (Cas9) and a short guide RNA (gRNA). The type II bacterial CRISPR-Cas9 system generates site-specific DNA breaks. DNA breaks are repaired by endogenous cellular mechanisms.
This mechanism can have several results:
(1) Mutagenic non-homologous end-joining (NHEJ),
(2) Creation of insertions or deletions (indels) at the site of the break, and
(3) Precise change of a genomic sequence through homologous recombination (HR).
The guide RNA is composed of two RNAs, CRISPR RNA (crRNA) and trans-activating crRNA. The trans-activating crRNA (tracrRNA) can be combined in a chimeric, single guide RNA (sgRNA). The sgRNAs are typically 100 nucleotides (nt) long. Twenty nucleotides at the 5’-end hybridize to a target DNA sequence by Watson-Crick base pairing. This hybridization reaction guides the Cas endonuclease to cleave the target genomic DNA. The remaining double-stranded structure at the 3’ side is critical for Cas9 recognition.
Figure 4: Model of crRNA processing and interference in type I systems. The pre-crRNA is processed by Cas5 or Cas6. DNA target interference requires Cas3 in addition to Cascade and crRNA (Rath et al. 2015).
Type II CRISPR-Cas is considered to be the minimal CRISPR-Cas system. The system includes the CRISPR repeat-spacer array and four or three cas genes. However, additional bacterial factors, such as tracrRNA and RNase III contribute to its function. SgRNA can be delivered into cells as synthetic RNA or by using a DNA vector expressing the sgRNA. Synthetic sgRNA can be generated using in vitro transcription (IVT).
Type III-B CRISPR-Cas system
The type III-B CRISPR-Cas system contains six Cas proteins (Cmr1 to Cmr6) and a crRNA that form an RNA silencing complex.
Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E. et al., 2015 Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotech advance online publication: doi: 10.1038/nbt.3290
Yingjun Li, Saifu Pan, Yan Zhang, Min Ren, Mingxia Feng, Nan Peng, Lanming Chen, Yun Xiang Liang, and Qunxin She; Harnessing Type I and Type III CRISPR-Cas systems for genome editing Nucl. Acids Res. first published online October 13, 2015 doi:10.1093/nar/gkv1044
Maier L-K, Fischer S, Stoll B, et al. The immune system of halophilic archaea. Mobile Genetic Elements. 2012;2(5):228-232. doi:10.4161/mge.22530
Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV; Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011; 9(6):467-477. doi:10.1038/nrmicro2577.
Meghdad Rahdar, Moira A. McMahon, Thazha P. Prakash, Eric E. Swayze, C. Frank Bennett, and Don W. Cleveland; Synthetic CRISPR RNA-Cas9–guided genome editing in human cells. http://www.pnas.org/content/early/2015/11/18/1520883112. Modified nucleic acids including bridged nucleic acids were incorporated into a designed chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), to replace the natural guide RNA in the CRISPR Cas9 nuclease system for genome editing.
Devashish Rath, Lina Amlinger, Archana Rath, Magnus Lundgren; Review: The CRISPR-Cas immune system: Biology, mechanisms and applications. Biochimie, Volume 117, October 2015, Pages 119–128;
Paul B.G. van Erp, Ryan N. Jackson, Joshua Carter, Sarah M. Golden, Scott Bailey, and Blake Wiedenheft; Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. Nucleic Acids Research, 2015, Vol. 43, No. 17 8381–8391. doi: 10.1093/nar/gkv793.