Recent Advances In Enhancing

CRISPR/Crispr-associated Proteins for

Genome Editing

Kiran Mishra
009
Sem 3
Msc Part II
Overview
01 Introduction
02 Altering PAM specificity
03 Enhancing Delivery systems

04 Increasing uniformity of edited

cells
05 Conclusion
06 Future Prospects
07 Review
INTRODUCTION
 Timeline
First successful gene
editing case
Ashanthi de Silva SCID – Lack
Discover of Restriction of Adenosine deaminase
Enzymes

1953 1973 2007

1960s 1990
Discovery of double helix Generation of first rDNA
Experiments
molecule
demonstrating role of
CRISPR Cas 9 together
 Genome editing

 Zinc finger nucleases

 Transcription activator like effector Nucleases

 Drawbacks of ZNFs and TALENs

So, how does the CRISPR Cas 9 work??
Ways to
Enhance 01
Altering PAM
Specificity of Cas

the 9

CRISPR- Enhancing delivery

Cas9
systems 02
activity
Increasing
03 uniformity of
edited cells
- SpCas9 recognizes the 5′-NGG-3′ PAM

- SpG recognizes 5’ –NGN- 3’ PAM (N - A, C, G, or

T). Altering
- SpRY – 5’ – NRN- 3’ preferable to 5’ –NYN- 3’,
where R are purines; Y are pyrimidines
PAM
- Observed almost negligible off target editing. specificity of
CAS9

SpGCas9      

NGH PAM Sites PAM Sequence No. of sites tested Editing Efficiency

  NGA 3 41.0–76.7%

  NGT 3 35.7–83.7%

  NGC 3 17.3–40.67%
N – A/C/G/T SpRY      

NGN PAM Sites NAN 8/12 15.50–80.67%

  NGN 12/12 15.67–65.67%

  NTN 6/12 4.0–50.3%

  NCN 5/12 6.0 - 42.0%

Initial Delivery Methods –
Plasmid based expression
Viral Vectors – Adeno associated Viral vectors

• Adv - serological compatibility with humans

Problem with plasmids – no • Disadv - small cargo size, thus the Cas systems and sgRNA
targeted delivery, poor control must be encoded on separate vectors
over Cas9.

Enhancing
delivery
systems

Lipid Nanoparticles
Ideal traits of delivery
• Cationic ionizable lipids - effective nucleic acid
methods –
encapsulation, cellular transport, and endosomal release.
• High editing efficiency • Light triggered Liposome – Verteporfin (photosensitive)
• Low immunogenicity
• Targeted delivery
 Increasing uniformity of edited cells
- Hei tags for short
- Delivers Cas9 directly to the
nucleus
High Efficiency - hei-tag, a myc-tag coupled to
Tags an optimized NLS via a flexible
linker, to Cas9
- heiCas9 displayed a 70%
increase in bi-allelic targeting
efficiency
- Difference between HDR and NHEJ - Done in zebrafish as well, 17
pathways wrt to CRISPR editing . fold more results
- CtIP protein
- Addition of small N terminal of CtIP to
Cas9 Stimulating HDR
- Limitation – guides play some instead of NHEJ
unidentified role, therefore larger pool
of guides need to be tested to enhance
the efficiency to max.
Conclusion:
- CRISPR remains one of the best genome
editing tools.

- Specified modifications still need to be

optimized for use in cell lines, and then
hopefully use in humans as well.
Future Prospects:

- Ethical issues of using genome

editing in humans

- Efficiency of these models in in-

vivo systems.
References:
1. Ma D, Liu F. 2015. Genome Editing and Its Applications in Model Organisms. Genomics Proteomics Bioinformatics 13:336–344.

2. Yang H, Ren S, Yu S, Pan H, Li T, Ge S, Zhang J, Xia N. 2020. Methods Favoring Homology-Directed Repair Choice in Response to
CRISPR/Cas9 Induced-Double Strand Breaks. Int J Mol Sci 21:6461.

3. WareJoncas Z, Campbell JM, Martínez-Gálvez G, Gendron WAC, Barry MA, Harris PC, Sussman CR, Ekker SC. 2018. Precision
gene editing technology and applications in nephrology. Nat Rev Nephrol 14:663–677.

4. Barrangou R, Doudna JA. 2016. Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34:933–941.

5. Varshney GK, Sood R, Burgess SM. 2015. Understanding and Editing the Zebrafish Genome, p. 1–52. In Advances in Genetics.
Elsevier.

6. 2020. CRISPR/Cas9 Technology in Translational Biomedicine. Cell Physiol Biochem 54:354–370.

7. Sharma P, Sharma BS, Verma RJ. 2021. CRISPR-based genome editing of zebrafish, p. 69–84. In Progress in Molecular Biology and
Translational Science. Elsevier.
8. Huck S, Bock J, Girardello J, Gauert M, Pul Ü. 2019. Marker-free genome editing in Ustilago trichophora with the
CRISPR-Cas9 technology. RNA Biol 16:397–403.

9. Auer TO, Del Bene F. 2014. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods 69:142–
150.

10. Yang Y, Xu J, Ge S, Lai L. 2021. CRISPR/Cas: Advances, Limitations, and Applications for Precision Cancer Research.
Front Med 8:649896.

11. Liang F, Zhang Y, Li L, Yang Y, Fei J-F, Liu Y, Qin W. 2022. SpG and SpRY variants expand the CRISPR toolbox for
genome editing in zebrafish. Nat Commun 13:3421.

12. Rosenblum D, Gutkin A, Kedmi R, Ramishetti S, Veiga N, Jacobi AM, Schubert MS, Friedmann-Morvinski D, Cohen
ZR, Behlke MA, Lieberman J, Peer D. 2020. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer
therapy. Sci Adv 6:eabc9450.

13. Aksoy YA, Yang B, Chen W, Hung T, Kuchel RP, Zammit NW, Grey ST, Goldys EM, Deng W. 2020. Spatial and
Temporal Control of CRISPR-Cas9-Mediated Gene Editing Delivered via a Light-Triggered Liposome System. ACS Appl
Mater Interfaces 12:52433–52444.
14. Thumberger T, Tavhelidse-Suck T, Gutierrez-Triana JA, Cornean A, Medert R, Welz B, Freichel M, Wittbrodt J. 2022.
Boosting targeted genome editing using the hei-tag. eLife 11:e70558.

15. Shams F, Bayat H, Mohammadian O, Mahboudi S, Vahidnezhad H, Soosanabadi M, Rahimpour A. 2022. Advance
trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified
CRISPR-Cas9 systems. BioImpacts 12:371–391.

16. Charpentier M, Khedher AHY, Menoret S, Brion A, Lamribet K, Dardillac E, Boix C, Perrouault L, Tesson L, Geny S,
De Cian A, Itier JM, Anegon I, Lopez B, Giovannangeli C, Concordet JP. 2018. CtIP fusion to Cas9 enhances transgene
integration by homology-dependent repair. Nat Commun 9:1133.
Thank You