Posts Tagged ‘Tay-Sachs’

Prime Editing as a New CRISPR Tool to Enhance Precision and Versatility


Reporter: Stephen J. Williams, PhD


CRISPR has become a powerful molecular for the editing of genomes tool in research, drug discovery, and the clinic

(see posts and ebook on this site below)


however, as discussed on this site

(see posts below)

there have been many instances of off-target effects where genes, other than the selected target, are edited out.  This ‘off-target’ issue has hampered much of the utility of CRISPR in gene-therapy and CART therapy

see posts


However, an article in Science by Jon Cohen explains a Nature paper’s finding of a new tool in the CRISPR arsenal called prime editing, meant to increase CRISPR specificity and precision editing capabilities.


By Jon Cohen | Oct 25th, 2019

Prime editing promises to be a cut above CRISPR Jon Cohen CRISPR, an extraordinarily powerful genome-editing tool invented in 2012, can still be clumsy. … Prime editing steers around shortcomings of both techniques by heavily modifying the Cas9 protein and the guide RNA. … ” Prime editing “well may become the way that disease-causing mutations are repaired,” he says.

Science Vol. 366, No. 6464; DOI: 10.1126/science.366.6464.406

The effort, led by Drs. David Liu and Andrew Anzalone at the Broad Institute (Cambridge, MA), relies on the modification of the Cas9 protein and guide RNA, so that there is only a nick in a single strand of the double helix.  The canonical Cas9 cuts both strands of DNA, and so relies on an efficient gap repair activity of the cell.  The second part, a new type of guide RNA called a pegRNA, contains an RNA template for a new DNA sequence to be added at the target location.  This pegRNA-directed synthesis of the new template requires the attachment of a reverse transcriptase enzymes to the Cas9.  So far Liu and his colleagues have tested the technology on over 175 human and rodent cell lines with great success.  In addition, they had also corrected mutations which cause Tay Sachs disease, which previous CRISPR systems could not do.  Liu claims that this technology could correct over 89% of pathogenic variants in human diseases.

A company Prime Medicine has been formed out of this effort.

Source: https://science.sciencemag.org/content/366/6464/406.abstract


Read an article on Dr. Liu, prime editing, and the companies that Dr. Liu has initiated including Editas Medicine, Beam Therapeutics, and Prime Medicine at https://www.statnews.com/2019/11/06/questions-david-liu-crispr-prime-editing-answers/

(interview by StatNews  SHARON BEGLEY @sxbegle)

As was announced, prime editing for human therapeutics will be jointly developed by both Prime Medicine and Beam Therapeutics, each focusing on different types of edits and distinct disease targets, which will help avoid redundancy and allow us to cover more disease territory overall. The companies will also share knowledge in prime editing as well as in accompanying technologies, such as delivery and manufacturing.

Reader of StatNews.: Can you please compare the pros and cons of prime editing versus base editing?

The first difference between base editing and prime editing is that base editing has been widely used for the past 3 1/2 years in organisms ranging from bacteria to plants to mice to primates. Addgene tells me that the DNA blueprints for base editors from our laboratory have been distributed more than 7,500 times to more than 1,000 researchers around the world, and more than 100 research papers from many different laboratories have been published using base editors to achieve desired gene edits for a wide variety of applications. While we are very excited about prime editing, it’s brand-new and there has only been one paper published thus far. So there’s much to do before we can know if prime editing will prove to be as general and robust as base editing has proven to be.

We directly compared prime editors and base editors in our study, and found that current base editors can offer higher editing efficiency and fewer indel byproducts than prime editors, while prime editors offer more targeting flexibility and greater editing precision. So when the desired edit is a transition point mutation (C to T, T to C, A to G, or G to A), and the target base is well-positioned for base editing (that is, a PAM sequence exists approximately 15 bases from the target site), then base editing can result in higher editing efficiencies and fewer byproducts. When the target base is not well-positioned for base editing, or when other “bystander” C or A bases are nearby that must not be edited, then prime editing offers major advantages since it does not require a precisely positioned PAM sequence and is a true “search-and-replace” editing capability, with no possibility of unwanted bystander editing at neighboring bases.

Of course, for classes of mutations other than the four types of point mutations that base editors can make, such as insertions, deletions, and the eight other kinds of point mutations, to our knowledge prime editing is currently the only approach that can make these mutations in human cells without requiring double-stranded DNA cuts or separate DNA templates.

Nucleases (such as the zinc-finger nucleases, TALE nucleases, and the original CRISPR-Cas9), base editors, and prime editors each have complementary strengths and weaknesses, just as scissors, pencils, and word processors each have unique and useful roles. All three classes of editing agents already have or will have roles in basic research and in applications such as human therapeutics and agriculture.

Nature Paper on Prime Editing CRISPR

Search-and-replace genome editing without double-strand breaks or donor DNA (6)


Andrew V. Anzalone,  Peyton B. Randolph, Jessie R. Davis, Alexander A. Sousa,

Luke W. Koblan, Jonathan M. Levy, Peter J. Chen, Christopher Wilson,

Gregory A. Newby, Aditya Raguram & David R. Liu


Nature volume 576, pages149–157(2019)



Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2,3,4,5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.



From Anzolone et al. Nature 2019 Figure 1.

Prime editing strategy

Cas9 targets DNA using a guide RNA containing a spacer sequence that hybridizes to the target DNA site. We envisioned the generation of guide RNAs that both specify the DNA target and contain new genetic information that replaces target DNA nucleotides. To transfer information from these engineered guide RNAs to target DNA, we proposed that genomic DNA, nicked at the target site to expose a 3′-hydroxyl group, could be used to prime the reverse transcription of an edit-encoding extension on the engineered guide RNA (the pegRNA) directly into the target site (Fig. 1b, cSupplementary Discussion).

These initial steps result in a branched intermediate with two redundant single-stranded DNA flaps: a 5′ flap that contains the unedited DNA sequence and a 3′ flap that contains the edited sequence copied from the pegRNA (Fig. 1c). Although hybridization of the perfectly complementary 5′ flap to the unedited strand is likely to be thermodynamically favoured, 5′ flaps are the preferred substrate for structure-specific endonucleases such as FEN122, which excises 5′ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. The redundant unedited DNA may also be removed by 5′ exonucleases such as EXO123.

  • The authors reasoned that preferential 5′ flap excision and 3′ flap ligation could drive the incorporation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand (Fig. 1c).
  • DNA repair to resolve the heteroduplex by copying the information in the edited strand to the complementary strand would permanently install the edit (Fig. 1c).
  • They had hypothesized that nicking the non-edited DNA strand might bias DNA repair to preferentially replace the non-edited strand.


  • The authors evaluated the eukaryotic cell DNA repair outcomes of 3′ flaps produced by pegRNA-programmed reverse transcription in vitro, and performed in vitro prime editing on reporter plasmids, then transformed the reaction products into yeast cells (Extended Data Fig. 2).
  • Reporter plasmids encoding EGFP and mCherry separated by a linker containing an in-frame stop codon, +1 frameshift, or −1 frameshift were constructed and when plasmids were edited in vitro with Cas9 nickase, RT, and 3′-extended pegRNAs encoding a transversion that corrects the premature stop codon, 37% of yeast transformants expressed both GFP and mCherry (Fig. 1f, Extended Data Fig. 2).
  • They fused a variant of M—MLV-RT (reverse transcriptase) to Cas9 with an extended linker and this M-MLV RT fused to the C terminus of Cas9(H840A) nickase was designated as PE1. This strategy allowed the authors to generate a cell line containing all the required components of the primer editing system. They constructed 19 variants of PE1 containing a variety of RT mutations to evaluate their editing efficiency in human cells
  • Generated a pentamutant RT incorporated into PE1 (Cas9(H840A)–M-MLV RT(D200N/L603W/T330P/T306K/W313F)) is hereafter referred to as prime editor 2 (PE2).  These were more thermostable versions of RT with higher efficiency.
  • Optimized the guide (pegRNA) using a series of permutations and  recommend starting with about 10–16 nt and testing shorter and longer RT templates during pegRNA optimization.
  • In the previous attempts (PE1 and PE2 systems), mismatch repair resolves the heteroduplex to give either edited or non-edited products. So they next developed an optimal editing system (PE3) to produce optimal nickase activity and found nicks positioned 3′ of the edit about 40–90 bp from the pegRNA-induced nick generally increased editing efficiency (averaging 41%) without excess indel formation (6.8% average indels for the sgRNA with the highest editing efficiency) (Fig. 3b).
  • The cell line used to finalize and validate the system was predominantly HEK293T immortalized cell line
  • Together, their findings establish that PE3 systems improve editing efficiencies about threefold compared with PE2, albeit with a higher range of indels than PE2. When it is possible to nick the non-edited strand with an sgRNA that requires editing before nicking, the PE3b system offers PE3-like editing levels while greatly reducing indel formation.
  • Off Target Effects: Strikingly, PE3 or PE2 with the same 16 pegRNAs containing these four target spacers resulted in detectable off-target editing at only 3 out of 16 off-target sites, with only 1 of 16 showing an off-target editing efficiency of 1% or more (Extended Data Fig. 6h). Average off-target prime editing for pegRNAs targeting HEK3HEK4EMX1, and FANCFat the top four known Cas9 off-target sites for each protospacer was <0.1%, <2.2 ± 5.2%, <0.1%, and <0.13 ± 0.11%, respectively (Extended Data Fig. 6h).
  • The PE3 system was very efficient at editing the most common mutation that causes Tay-Sachs disease, a 4-bp insertion in HEXA(HEXA1278+TATC).


  1. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res44, D862–D868 (2016).
  2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816–821 (2012).
  3. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science339, 819–823 (2013).


  1. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science339, 823–826 (2013).
  2. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.  Biotechnol. 36, 765–771 (2018).
  3. Anzalone, A.V., Randolph, P.B., Davis, J.R. et al.Search-and-replace genome editing without double-strand breaks or donor DNA. Nature576, 149–157 (2019). https://doi.org/10.1038/s41586-019-1711-4

Read Full Post »

Reproductive Genetic Testing

Reporter and Curator: Sudipta Saha, Ph.D.

Reproductive genetics, a field of medical genetics integrated with reproductive medicine, assisted reproduction, and developmental genetics, involves a wide array of genetic tests that are conducted with the intent of informing individuals about the possible outcomes of current or future pregnancies. The tests themselves can include the analysis of chromosomes, DNA, RNA, genes, and/or gene products to determine whether an alteration is present that is causing or is likely to cause a specific disease or condition.

Types of Tests

In general, reproductive genetic testing involves the following categories of tests:

Carrier testing is performed to determine whether an individual carries one copy of an altered gene for a particular recessive disease. The term recessive refers to diseases that will occur only if both copies of a gene that an individual receives have a disease-associated mutation; thus, each child born to two carriers of a mutation in the same gene has a 25 percent risk of being affected with the disorder. Examples of carrier tests include those for

Couples are likely to have carrier tests if they are at higher risk of having a child with a specific disorder because of their racial or ethnic heritage or family history. Carrier testing is often done in the context of family planning and reproductive health.

Preimplantation diagnosis is used following in vitro fertilization to diagnose a genetic disease or condition in a preimplantation embryo. Preimplantation genetic diagnosis is essentially an alternative to prenatal diagnosis, as it allows prenatal testing to occur months earlier than conventional tests such as amniocentesis on week 18th of pregnancy, even before a pregnancy begins. Doctors can test a single cell from an eight-cell embryo that is just days old to determine, among other things, whether it is a male or female. This can provide crucial information for genetic diseases that afflict just one sex. Preimplantation genetic diagnosis has been applied to patients carrying chromosomal rearrangements, such as translocations, in which it has been proven to decrease the number of spontaneous abortions and prevent the birth of children affected with chromosome imbalances. Preimplantation genetic diagnosis techniques have also been applied to

  • increase implantation rates,
  • reduce the incidence of spontaneous abortion, and
  • prevent trisomic offspring in women of advanced maternal age undergoing fertility treatment.

A third group of patients receiving preimplantation genetic diagnosis are those at risk of transmitting a single gene disorder to their offspring. The number of monogenic disorders that have been diagnosed in preimplantation embryos has increased each year. So far, at least 700 healthy babies have been born worldwide after undergoing the procedure, and the number is growing rapidly.

Prenatal diagnosis is used to diagnose a genetic disease or condition in a developing fetus.

The techniques currently in use or under investigation for prenatal diagnosis include

  • (1) fetal tissue sampling through amniocentesis, chorionic villi sampling (CVS), percutaneous umbilical blood sampling, percutaneous skin biopsy, and other organ biopsies, including muscle and liver biopsy;
  • (2) fetal visualization through ultrasound, fetal echocardiography, embryoscopy, fetoscopy, magnetic resonance imaging, and radiography;
  • (3) screening for neural tube defects by measuring maternal serum alpha-fetoprotein (MSAFP);
  • (4) screening for fetal Down Syndrome by measuring MSAFP, unconjugated estriol, and human chorionic gonadotropin;
  • (5) separation of fetal cells from the mother’s blood; and
  • (6) preimplantation biopsy of blastocysts obtained by in vitro fertilization.

The more common techniques are amniocentesis, performed at the 14th to 20th week of gestation, and CVS, performed between the 9th and 13th week of gestation. If the fetus is found to be affected with a disorder, the couple can plan for the birth of an affected child or opt for elective abortion.

Newborn screening is performed in newborns on a public health basis by the states to detect certain genetic diseases for which early diagnosis and treatment are available. Newborn screening is one of the largest public health activities in the United States. It is aimed at the early identification of infants who are affected by certain genetic, metabolic or infectious conditions, reaching approximately 4 million children born each year. According to the Centers for Disease Control and Prevention (CDC), approximately 3,000 babies each year in the United States are found to have severe disorders detected through screening. States test blood spots collected from newborns for 2 to over 30 metabolic and genetic diseases, such as

  • phenylketonuria,
  • hypothyroidism,
  • galactosemia,
  • sickle cell disease, and
  • medium chain acyl CoA dehyrogenase deficiency.

The goal of this screening is to identify affected newborns quickly in order to provide treatment that can prevent mental retardation, severe illness or death.

It is possible that somatic cell nuclear transfer (cloning) techniques could eventually be employed for the purposes of reproductive genetic testing. In addition, germline gene transfer is a technique that could be used to test and then alter the genetic makeup of the embryo. To date, however, these techniques have not been used in human studies.

Ethical Issues

Any procedure that provides information that could lead to a decision to terminate a pregnancy is not without controversy. Although prenatal diagnosis has been routine for nearly 20 years, some ethicists remain concerned that the ability to eliminate potential offspring with genetic defects contributes to making society overall less tolerant of disability. Others have argued that prenatal diagnosis is sometimes driven by economic concerns because as a society we have chosen not to provide affordable and accessible health care to everyone. Thus, prenatal diagnosis can save money by preventing the birth of defective and costly children. For reproductive genetic procedures that involve greater risk to the fetus, e.g., preimplantation diagnosis, concerns remain about whether the diseases being averted warrant the risks involved in the procedures themselves. These concerns are likely to escalate should

  • cloning or
  • germline gene transfer

be undertaken as a way to genetically test and select healthy offspring.



Read Full Post »